Catalytic activity of human guanylate-binding protein 1 coupled to the release of structural restraints imposed by the C-terminal domain

Catalytic activity of human guanylate-binding protein 1

coupled to the release of structural restraints imposed by

the C-terminal domain

1

2

1

Semra Ince , Ping Zhang

, Miriam Kutsch

, Oktavian Krenczyk , Sergii Shydlovskyi and

1

Christian Herrmann

1

2

Physical Chemistry I, Ruhr-University, Bochum, Germany

Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA

Keywords

Human guanylate-binding protein 1 (hGBP-1) shows a dimer-induced

acceleration of the GTPase activity yielding GDP as well as GMP. While

the head-to-head dimerization of the large GTPase (LG) domain is well

understood, the role of the rest of the protein, particularly of the GTPase

effector domain (GED), in dimerization and GTP hydrolysis is still

obscure. In this study, with truncations and point mutations on hGBP-1

and by means of biochemical and biophysical methods, we demonstrate

that the intramolecular communication between the LG domain and the

GED (LG:GED) is crucial for protein dimerization and dimer-stimulated

GTP hydrolysis. In the course of GTP binding and c-phosphate cleavage,

conformational changes within hGBP-1 are controlled by a chain of amino

acids ranging from the region near the nucleotide-binding pocket to the

distant LG:GED interface and lead to the release of the GED from the

LG domain. This opening of the structure allows the protein to form

GED:GED contacts within the dimer, in addition to the established LG:

LG interface. After releasing the cleaved c-phosphate, the dimer either dis-

sociates yielding GDP as the ﬁnal product or it stays dimeric to further

cleave the b-phosphate yielding GMP. The second phosphate cleavage step,

that is, the formation of GMP, is even more strongly coupled to structural

changes and thus more sensitive to structural restraints imposed by the

GED. Altogether, we depict a comprehensive mechanism of GTP hydroly-

sis catalyzed by hGBP-1, which provides a detailed molecular understand-

ing of the enzymatic activity connected to large structural rearrangements

of the protein.

aluminum ﬂuoride; guanylate-binding

protein; intramolecular interactions; large

GTPase; protein–protein interaction

Correspondence

C. Herrmann, Physical Chemistry I, Ruhr-

University Bochum, 44780 Bochum,

Germany

Tel: +49 234 3224173

E-mail: chr.herrmann@rub.de

Semra Ince and Ping Zhang contributed

equally to this work

(Received 5 February 2020, revised 10 April

2020, accepted 27 April 2020)

doi:10.1111/febs.15348

Database

Structural data are available in RCSB Protein Data Bank under the accession numbers: 1F5N ,

1

DG3 , 2B92 .

Abbreviations

AlF

X

, aluminum ﬂuoride; DSC, differential scanning calorimetry; DSP, dynamin superfamily protein; FRET, F o€ rster resonance energy transfer;

0

GDP, guanosine diphosphate; GED, GTPase effector domain; GMP, guanosine monophosphate; GppNHp, 5 -guanylyl imidodiphosphate

0

(nonhydrolysable GTP analog); GTP, guanosine triphosphate; GTPcS, guanosine 5 -O-(gamma-thio)triphosphate; hGBP, human guanylate-

binding protein; LG, large GTPase domain; LP, labeled protein; MD, middle domain; NLP, nonlabeled protein; SEC, size-exclusion

chromatography.

The FEBS Journal (2020) ª 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of

Federation of European Biochemical Societies

1

This is an open access article under the terms of the Creative Commons Attribution License, which permits use,

distribution and reproduction in any medium, provided the original work is properly cited.

Functional coupling of hGBP-1’s N- and C termini

S. Ince et al.

Introduction

Human guanylate-binding proteins (hGBPs) with

seven paralogs (hGBP-1 to hGBP-7) comprise a sub-

group of interferon (IFN)-inducible large GTPases

within the dynamin superfamily proteins (DSPs) [1–3].

Most abundantly induced by IFN-c and other pro-in-

ﬂammatory cytokines, hGBPs are found to mediate

innate immune functions against a diverse array of

intracellular pathogens including viruses, bacteria, and

protozoa [3–14]. Their cellular activities further enclose

antiproliferative effects on endothelial cells [15,16]. In

colorectal cancer cells, particularly hGBP-1 is estab-

lished as reliable marker, which inhibits tumor growth

through both strong anti-angiogenic activity and its

direct antitumorigenic effects [17–19]. However,

hGBP-1 is also shown to render some cancer cell lines

resistant to chemotherapeutics or radiation [20,21],

which altogether indicate a cell- and disease-speciﬁc

activity of hGBPs awaiting proper characterization of

the underlying molecular mechanisms.

Human GBP-1 is the ﬁrst identiﬁed and hitherto

best characterized hGBP in terms of biochemical and

structural properties [3,22]. As a member of the DSPs,

hGBP-1 has a typical multidomain architecture and

exhibits characteristic features such as low binding

afﬁnity for guanine nucleotides (micromolar range),

nucleotide-dependent homo oligomerization, and a

dimer-induced acceleration of the GTPase activity

Fig. 1. Structure and enzymatic activity of hGBP-1. (A) Ribbon plot

of hGBP-1 in the GppNHp-bound form (PDB ID: 1F5N) is colored

according to the subdomains: the N-terminal large GTPase domain

(

LG domain) in blue, the middle domain (MD) in yellow, and the C-

[2,23]. The 68-kDa protein consists of three subdo-

terminal GTPase effector domain (GED) in orange. GppNHp is

depicted in stick presentation, and the C-terminal amino acids

including the CaaX motif not being solved in the structure are

indicated by a dotted line. The schematic above will be used

hereafter to indicate mutational alterations. (B) Schematic

illustration of the unique GTPase reaction catalyzed by dimeric

hGBP-1 is adopted and modiﬁed from Refs [29,39], according to

which two competitive steps are proposed: After binding to GTP

and the ﬁrst step of phosphate cleavage, the GDP-bound dimer

either remains and catalyzes the second step of phosphate

cleavage yielding GMP as ﬁnal product, or, the GDP-bound dimer

dissociates irreversibly and releases GDP as ﬁnal product.

mains (Fig. 1A), a globular large GTPase domain (re-

ferred to as LG domain from here on) at the N

terminus, followed by a purely a-helical domain, which

is subdivided into a middle domain (MD, a7-11), and

an elongated C-terminal domain termed GTPase effec-

tor domain (GED, a12-13). Overall, the LG domain

and MD are entirely ﬂanked by the GED within which

the a13 helix is positioned next to the LG domain by

a coiled-coil type of interaction with helix a12 [24,25].

The LG domain of hGBP-1 represents the catalytic

unit, which harbors all sequence elements for guanine

nucleotide binding and hydrolysis. Binding of the sub-

strate GTP induces dimerization of the LG domains in

a protein concentration-dependent fashion [24,26].

Those head-to-head dimers serve to mutually reposi-

tion their internal catalytic residues into the active

sites, and thereby to stabilize the transition state for

efﬁcient cleavage of the GTP c-phosphate [26]. The

self-stimulated GTPase activity of hGBP-1 achieved by

successive cleavage of the c- and b-phosphate groups

[26–28]. Mechanistically, this is achieved by a shift of

the intermediate product GDP to the active site subse-

quent to the cleavage and release of the c-phosphate.

Thus, GDP’s b-phosphate occupies the position where

the c-phosphate was located previously, and the same

set of residues acts again to catalyze the GDP hydroly-

sis [26]. Importantly, this sequence of events relies on

the GTP-induced hGBP-1 dimers (Fig. 1B), while

monomeric hGBP-1 can catalyze very slowly the ﬁrst

but not the second step of GTP hydrolysis yielding

ꢀ

1

dimerization reaches up to 100 min under physiolog-

ical and substrate-saturated conditions [2]. Above that,

hGBP-1 has the unifying feature to hydrolyze GTP to

GDP and GMP in two steps accomplished through

2

The FEBS Journal (2020) ª 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of

Federation of European Biochemical Societies

S. Ince et al.

Functional coupling of hGBP-1’s N- and C termini

only GDP as product [26,29,30]. With its release from

the active site, GDP becomes a dead-end product,

which—upon re-association to hGBP-1—does not

induce dimers which are required for catalysis of the

b-phosphate cleavage. Likewise, GDP from bulk solu-

tion is bound by hGBP-1 but in contrast to GTP can-

not serve as direct substrate for hGBP-1 [24,27].

Consequently, GMP emerges only in the course of

GTP hydrolysis, that is, the second step of b-phos-

phate cleavage occurs only in continuation of the ﬁrst

step of c-phosphate cleavage, provided that the reac-

tion is catalyzed by LG domain-mediated dimers of

hGBP-1.

available farnesyl tail enables membrane attachment

and, alternatively, polymer formation of hGBP-1.

The present study highlights the special role of the

GED in the enzymatic machinery of hGBP-1, espe-

cially with regard to the second hydrolysis step that

provides GMP. To address this, we generated an array

of suitable point and truncation mutants of hGBP-1

and characterized their enzymatic activity as well as

their capability to undergo nucleotide-dependent struc-

tural changes and dimerization by means of reversed-

phase HPLC, F o€ rster resonance energy transfer

(FRET), and size-exclusion chromatography (SEC).

We performed all measurements at 25 °C where the

hGBP-1-catalyzed GTPase reaction yields an almost

balanced ratio of the products GDP and GMP. This

condition is in particular suitable to demonstrate the

contributions of individual amino acids and subdo-

mains on the product share. To obtain valuable mech-

anistic insights into the enzymatic machinery of

hGBP-1, we studied here exclusively the nonfarnesy-

lated form, which on the one hand is sufﬁcient to form

the catalytic competent unit of a dimer and on the

other hand is incapable of forming higher ordered oli-

gomers, potent to distort the investigations.

Interestingly, several mutants of hGBP-1 have been

described that cause notable differences in the ﬁnal

product GMP share obtained from GTP hydrolysis

[31–33]. These encompass not only positions along the

nucleotide-binding pocket but also along the distant

position on the LG domain adjacent to the GED. At

the latter position, an intramolecular network of salt

bridges is established in the LG domain and GED

interface by individual contacts from both a12 and

a13 to the LG domain [32]. Those salt bridges main-

tain a compactly ‘closed’, safety-pin-like conformation

of hGBP-1 in the absence of nucleotide [24,32]. In the

course of GTP binding and/or hydrolysis, conforma-

tional changes within the LG domain occur that lead

to disruption of the salt bridge contacts, thereby

releasing the GED to promote intermolecular interac-

tions with the GED of another hGBP-1 molecule

Results

Retaining GTPase activity, the isolated LG

domain of hGBP-1 favors GMP production and

gains additional GDPase activity compared to the

full-length protein

(GED:GED) [32]. A structure of a full-length hGBP-1

dimer has not been solved yet, and it was once

reported that through GED:GED interactions, hGBP-

GTP-dependent dimerization of hGBP-1, which in

turn stimulates the successive hydrolysis of GTP to a

mixture of GDP and GMP, is mediated by head-to-

head dimerization of the catalytic LG domains [26].

Since the earlier studies were performed under low salt

conditions [26,34,40], we ﬁrst conﬁrmed and character-

ized the differences in enzymatic properties between

full-length hGBP-1 and the isolated LG domain under

physiological salt conditions in detail.

1

builds tetramers from LG domain-mediated dimers

33]. However, the new ﬁnding that tetramers do not

[

occur implicates that hGBP-1 GED:GED interactions

occur within a bulky dimer [34].

The role of the LG domain within the GTPase cycle

is well understood [26,35]. However, the roles for the

remaining subdomains and in particular for the C-ter-

minal GED remain elusive. Besides the putative rele-

vance to the ﬁnal product share of GTP hydrolysis,

there is a growing list of different functions mediated

by the GED, suggesting a multifaceted importance of

this subdomain. For instance, once the protein carries

a farnesyl moiety at the C-terminal CaaX-box, two

additional features are added as compared to the non-

farnesylated form: One is the ability to interact with

membranes and the other to assemble to polymers,

which are notably higher ordered than dimers [36,37].

The idea is that GTP binding leads to the release of

the farnesyl tail previously embedded in a hydrophobic

pocket between MD and GED [36,38]. The now

Experimentally, dimerization-dependent acceleration

of the GTPase activity can be obtained by a typical

GTPase assay, where rates of GTP hydrolysis cat-

alyzed by varying protein concentrations are measured

and further processed according to the multiturnover

assay [28,35]. In this assay, dividing the rate of GTP

hydrolysis (Fig. 2A,B, blue squares) by the respective

protein concentration yields catalytic GTPase activity

of hGBP-1 (k^G_c_a^T_t^P). An k_c^G_a^T_t^Pincrease with increasing

protein concentration indicates that the acceleration of

GTPase activity of hGBP-1 depends on dimerization

(Fig. 2C,D, blue squares). Furthermore, ﬁtting the

The FEBS Journal (2020) ª 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of

Federation of European Biochemical Societies

3

Functional coupling of hGBP-1’s N- and C termini

S. Ince et al.

A

B

Fig. 2. Comparison of the GTPase reaction

catalyzed by hGBP-1 (wt) and its isolated

LG domain. (A-D) The GTPase activity of wt

(

A, C) and the LG domain (B, D) was

determined by a typical GTPase assay

where the GTPase reaction with 500 µM

GTP was monitored at varying protein

concentrations. Linear ﬁts to time courses

of GTP consumption (blue squares and

lines) as well as GDP (orange triangles and

lines) and GMP formation (black circle and

lines), as exemplary shown at 2 µM of wt

Time (min)

C

D

(

A) and LG domain (B), yielded initial rates.

These in turn divided by the corresponding

protein concentration gave the catalytic

activity for each protein concentration as

plotted in (C) and (D), respectively. The

E

F

maximum catalytic GTPase activities

GTP;max

cat

(

k

) and dimer dissociation constants

K

d

) were obtained according to a ﬁt model

solid lines) that considers slow activity

provided by the monomer and fast activity

provided by the dimer as described in the

text. (E, F) The long-term hydrolysis assay

performed with 500 µM GTP and varying

concentrations of wt (E) and LG domain (F)

depicts the nucleotide composition (same

symbols as in A, B) at either concentration

after 24 h of incubation.

data accordingly [28] yields two characteristic parame-

ters, namely the maximum catalytic GTPase activity

Obtained maximum activities of GDP and GMP

ꢀ

1

formation for wt were 12.2 and 6.9 min , respec-

GTP;max

(

k

), which is achieved by the dimeric hGBP-1,

and the dimer dissociation constant (K ), which gives

tively. Notably, the sum of those two values equals

k

cat

GTP;max

cat

and this illustrates two important facts.

d

a measure for the dimer afﬁnity.

We performed this assay at 25 °C in buffer maintain-

ing physiological salt conditions and determined a

Firstly, GDP and GMP are formed successively by

cleaving primarily the c-phosphate and then the b-

phosphate. Secondly, there is no additional GDPase

reaction under the GTP-saturated condition, meaning

the produced GDP was not reused as substrate. Thus,

dividing each of the maximum activities for product

formation by the maximum activity of GTP consump-

tion yields the share of ﬁnal products being roughly

60% GDP and 40% GMP, which is in line with our

previous data [32,35].

GTP;max

cat

ꢀ1

k

value of 19.1 min and a K value of 0.2 µM

d

for full-length hGBP-1 (wt) (Fig. 2C, blue squares and

solid line). Within this experiment, we also monitored

formation of products GDP and GMP. Similar to the

GTP consumption, both GDP and GMP formation

followed linear initial reactions, the rates of which were

derived from linear ﬁts (Fig. 2A, orange triangles,

black circles, and solid lines). Catalytic activities were

obtained dividing the initial rates by the respective pro-

tein concentrations, and maximum catalytic activities

The same experiment was performed on the isolated

LG domain of hGBP-1 (LG). We conﬁrmed that the

LG domain-catalyzed GTPase reaction is two times

GTP;max

ꢀ1

of GDP and GMP formation were acquired from the

GTP;max

faster (k_c_a_t

one, while having

(K = 0.3 µM) (Fig. 2D, blue squares). However, the

= 47.8 min ) than the wt-catalyzed

same quadratic ﬁt used to determine k_c_a_t

orange triangles, black circles, and solid lines).

(Fig. 2C,

a

similar dimer afﬁnity

d

4

The FEBS Journal (2020) ª 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of

Federation of European Biochemical Societies

S. Ince et al.

Functional coupling of hGBP-1’s N- and C termini

product development unraveled one major difference

between the two GTPase reactions: The LG domain,

other than wt, catalyzed the formation of GMP

more than 10-fold slower than the GTPase activity

GTP;max

cat

ꢀ1

GDP;max

cat

ꢀ1

(k

= 43.8 min

versus

k

= 3.3 min ).

However, this is still a 1000-fold higher activity as

compared to the full-length protein, which achieves

ꢀ1

(

35.1 min

)

12.8 min ), consequently yielding GMP as predomi-

prominently

faster

than

GDP

ꢀ1

(

roughly 0.0015 min at the highest protein concentra-

tion measured (20 µM, Fig. 3A).

nant product, which accounted for roughly 70% of the

total product (Fig. 2D, orange triangles and black cir-

cles). Here, also the LG domain did not reuse the pro-

duct GDP as substrate in the presence of a saturated

amount of GTP.

In summary, we show that the LG domain catalyzes

GTP hydrolysis two times faster than wt and that in

contrast to wt, the deletion of the C-terminal helical

domains rather facilitates the successive b-phosphate

cleavage to occur. This is likely achieved through

maintaining a larger fraction of GDP-bound interme-

diate dimers (see Fig. 1B). Furthermore, we showed

that the gained GDPase activity is a result of GDP-in-

duced dimerization. Altogether, these two signiﬁcant

features of the isolated LG domain of hGBP-1 indicate

that the rest of the protein, namely the MD and GED

domains, has impact on hGBP-1’s enzymatic activity.

To have an alternative approach for determining the

product ratio, we newly established the long-term

hydrolysis assay. The setup is similar to the GTPase

assay (Fig. 2C,D) with the only difference that we

investigate not the kinetics of the enzymatic reaction

but the ﬁnal nucleotide composition after 24 h of incu-

bation (Fig. 2E,F). Despite the long incubation time,

the observation that GTP was almost not hydrolyzed

at low protein concentrations below the K_dvalue

emphasizes the very slow activity of monomeric pro-

tein. However, at higher protein concentrations where

GTP was completely hydrolyzed due to the high activ-

ity of the dimer, the percentages of GDP and GMP

gave a direct read-out for the product share. By this

approach, approximately 60% GDP and 40% GMP

were obtained from the wt-catalyzed GTPase reaction

The C-terminal GED of hGBP-1 inﬂuences the

hydrolysis of GDP catalyzed in the N-terminal LG

domain

Motivated by the enzymatic differences between wt

and the LG domain, we investigated the inﬂuence of

the C-terminal part of the hGBP-1 on its enzymatic

properties. We generated two truncated mutants on

the basis of full-length hGBP-1, a mutant Da13, which

lacks helix a13 and a mutant DGED, which lacks the

entire GED (both a12 and a13, see Figs 4A and 5B).

In order to distinguish the effects of a13 from the

effects of the whole GED on GTPase and GDPase

activity of hGBP-1, we compared the generated

mutants with wt protein by means of the GMP forma-

tion during GTP hydrolysis (Fig. 4C) and the ability

to utilize GDP as substrate (Fig. 4B). For this com-

parison, we used 5 µM of protein catalyzing hydrolysis

of 500 µM GTP and 10 µM of protein catalyzing

hydrolysis of 500 µM GDP.

(Fig. 2E), which is in good agreement with the kineti-

cally obtained values. The same is also true for the LG

domain, although the values of approximately 30%

GDP and 70% GMP were detected only within a lim-

ited protein concentration range between 0.1 and

0

.5 µM (Fig. 2F). At concentrations above, another

reaction set in as reﬂected by an additional increase of

GMP up to 100% accompanied by the decrease of

GDP to 0% (Fig. 2F). This assigns the second major

difference of the LG domain’s enzymatic activity as

compared to wt, namely the ability to utilize GDP as

substrate.

By offering GDP instead of GTP in the long-term

hydrolysis assay, we conﬁrmed both the GDPase activ-

ity of the LG domain (Fig. 3D) and the inability of wt

to utilize GDP as substrate in the tested concentration

range of the proteins (Fig. 3C). Former studies identi-

ﬁed GDP-induced dimerization of LG domains as the

prerequisite for efﬁcient and self-stimulated hydrolysis

of GDP similar to hydrolysis of GTP (Fig. 3B) [26].

Accordingly, we further quantiﬁed a protein concen-

tration-dependent increase of the GDPase activity

using the same assay as for the GTPase activity, yield-

ing an almost 30-fold weaker afﬁnity of the GDP-

Both mutants Da13 and DGED catalyzed GTP

hydrolysis to yield ~ 80% GMP and 20% GDP

(Fig. 4B,C), suggesting that helix a13 has a critical

impact on the b-phosphate cleavage within the GTPase

reaction. Moreover, when offering GDP instead of

GTP as substrate, a direct correlation was revealed

GDP

between catalytic GDPase activity (k_c_a_t) and the

truncation steps. Starting with a very low GDPase

GDP

ꢀ1

activity of wt (k_c_a_t= 0.00075 min ), a roughly 10-

fold higher activity was obtained with each truncation

GDP

ꢀ1

step. With k_c_a_t= 0.0071 min , the GDPase activity

of Da13 is still slow but 10-fold higher than wt. More-

over, DGED was capable to hydrolyze GDP more

bound dimers (K = 8.5 µM) (Fig. 3B) as compared to

d

the GTP-bound ones (K = 0.3 µM) (Fig. 2D). Also,

d

GDP;max

GDP

ꢀ1

the maximum catalytic GDPase activity (k_c_a_t

) was

than 100-fold faster than wt (k_c_a_t= 0.11 min ). And

The FEBS Journal (2020) ª 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of

Federation of European Biochemical Societies

5

Functional coupling of hGBP-1’s N- and C termini

S. Ince et al.

A

B

Fig. 3. Comparison of the GDPase reaction

catalyzed by hGBP-1 and its isolated LG

domain. The GDPase activity of wt (A) and

the isolated LG domain (B) was assayed

analogously to the GTPase activity (see

Fig. 2C,D) using 500 µM GDP instead of

GTP. Long-term hydrolysis assays were

performed with 500 µM GDP and varying

concentrations of wt (C) and LG domain (D).

The share of nucleotides (GDP, orange

triangles; GMP, black circles) was analyzed

at either concentration after 24 h of

incubation.

C

D

ﬁnally, deletion of both the GED and the MD, which

corresponds to the isolated LG domain, shows a more

than 1000-fold higher GDPase activity than wt

(DSC) (Fig. 4D, Table 1). The highest T_mwas

obtained for hGBP-1 (wt), indicating that the highest

stability was given in the absence of any mutation. In

comparison, a signiﬁcant decrease in stability by at

least 3 °C was observed with each truncation, which

gives evidence for mutual stabilization of the subdo-

mains as established through intramolecular interac-

tions. The rather high T_mof the LG domain among

all truncation mutants was assumed to be caused by

its compact globular shape. Most interestingly, the

loose GED mutant, in which only two amino acids are

mutated (R227E/K228E) in a4´ helix of the full-length

construct, showed a similar destabilization as the trun-

cation mutants. This underscores a key role of the

amino acids R227/K228 in establishing the intramolec-

ular LG:GED interactions.

GDP

ꢀ1

(

k

= 1.4 min , Fig. 4B).

Considering that the GED within a full-length con-

cat

struct may affect GTP/GDP hydrolysis through

intramolecular interactions with the LG domain, we

performed the same experiments with mutants gener-

ated previously affecting these intramolecular interac-

tions [32] (see Fig. 5). We observed that artiﬁcial

coupling of the GED to the LG domain by a chemical

cross-linker, resulting in a tight GED conformation,

did not affect the catalytic GTPase activity but almost

abolished GMP formation as well as utilization of

GDP as substrate (Fig. 4B,C). On the contrary, the

mutant (R227E/K228E), which abolishes intramolecu-

lar salt bridges between the GED and the LG domain

and presumably has a loose GED, performed nucleo-

tide hydrolysis activities similar to DGED. In addition

to predominating GMP formation in the GTPase reac-

In sum, stepwise deletion of the C-terminal helices

(Da13, DGED, and DMD-GED) or loosening the con-

tact between the LG domain and the GED (R227E/

K228E) leads to an enhanced production of GMP dur-

ing GTP hydrolysis and the ability to utilize GDP as

substrate. Disruption of the salt bridge network

between the LG domain and the GED and therefore

release of the GEDs within the hGBP-1 dimer enables

hGBP-1 to perform the second hydrolysis step.

tion (Fig. 4C), the R227E/K228E mutant efﬁciently

ꢀ1

hydrolyzed GDP as substrate (k^G^D^P= 0.29 min

Fig. 4B).

,

cat

These data suggest that the b-phosphate cleavage

capability of hGBP-1 strongly correlates with the

strength of the LG:GED intramolecular interactions.

We hypothesized that weakening of this intramolecular

interaction by our mutations and truncations results in

reduced thermal stability. Therefore, we performed

temperature-induced unfolding studies in the absence

of nucleotide using differential scanning calorimetry

A cluster of LG domain phenylalanines is critical

for intramolecular GED arrangement

Given the described impact of the GED on the cat-

alytic activities, we further characterized newly

6

The FEBS Journal (2020) ª 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of

Federation of European Biochemical Societies

S. Ince et al.

Functional coupling of hGBP-1’s N- and C termini

A

D

5

4

3

0

5

0

5

0

B

1

0

1

0

.1

.01

.001

0

D

wt

ΔG

13

LG

E

loose

GED

9

9%

60%

40%

19%

81%

17%

83%

11%

89%

29%

71%

Δα

C

1

%

Fig. 4. Alterations in hGBP-1’s enzymatic activity and stability depending on C-terminal contributions. (A) Schematics of point mutants and

truncation mutants of hGBP-1 depict altering arrangements of the GED as compared to wt (see text and Fig. 5; colored according to the

subdomains in Fig. 1A). (B) GTPase (blue columns) and GDPase activities (gray columns) of respective proteins were determined separately

using 5 µM of protein and 500 µM of GTP or 10 µM of protein and 500 µM of GDP. (C) Pie charts depict the share of nucleotide products

GMP (red) and GDP (gray) resulting from GTP hydrolysis catalyzed by the respective mutant. (D) Thermal stability of wt and selected

mutants is given by their melting temperatures T

m

derived from differential scanning calorimetry. Error bars represent mean ꢁ SD of three

independent experiments (n = 3). All values from the ﬁgure are summarized in Table 1.

generated point mutants for their conformational state,

that is, the arrangements of the GED relative to the

rest of the protein. We hypothesized that the GED,

particularly through intramolecular interactions to the

LG domain, impacts the unique b-phosphate cleavage

step of hGBP-1. Moreover, as the loose GED mutant

enzymatic activity of wt (Table 1), which is why we

conceived these residues not important for LG:GED

interactions.

In close proximity to that loop, we further identiﬁed

a cluster of four phenylalanine residues, F229 from

helix a4´ next to the two critical residues R227/K228

as well as F171, F174, and F175 from helix a3´. By

structural analysis of different nucleotide-bound states,

these four residues were found to experience confor-

mational changes along the GTPase cycle, and thus,

we marked them as candidates to inﬂuence the

intramolecular GED arrangement (Figs 5 and 6A). We

replaced these residues by alanines, either through sin-

gle mutations (F229A, F171A, and F175A) or a triple

(R227E/K228E), being similar to the DGED, has an

inﬂuence on the GDPase activity, we suggest that loos-

ening or opening the GED is the reason for the

increased GDPase activity. To conﬁrm this, we sought

further residues, which are responsible for the contact

between the LG domain and the GED.

Within the LG domain and in close proximity to the

GED, we initially investigated a nonstructured loop

region, which precedes helix a3´. This loop region has

several negatively charged residues, which might inter-

act with a set of positively charged residues from the

very C terminus of hGBP-1 (Fig. 5C). However, nei-

ther neutralization nor charge reversal of these resi-

dues (see mutants LoopAAA and LoopRRR in

Fig. 5B) led to notable changes as compared to the

0

mutation of all a3 -located phenylalanines (F171A/

F174A/F175A, termed TripleF). While having a simi-

lar high catalytic GTPase activity as wt, all four

mutants showed a remarkable shift of the product

ratio in favor of GMP (70–75%), as also conﬁrmed by

long-term hydrolysis studies (Fig. 6B, pie charts and

Table 1). Since we have observed similar results for

The FEBS Journal (2020) ª 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of

Federation of European Biochemical Societies

7

Functional coupling of hGBP-1’s N- and C termini

S. Ince et al.

Fig. 5. Mutational analysis of critical regions for intramolecular LG:GED interactions of hGBP-1. (A) Ribbon plot of full-length hGBP-1 (wt)

bound to GppNHp (sticks) (PDB ID: 1F5N) depicted in top and side view and colored according to the subdomains (large GTPase (LG)

domain: light blue; middle domain (MD): yellow; and the GTPase effector domain (GED) consisting of a12-13: orange). The nonresolved C

terminus of 10 amino acids including the CaaX motif is indicated by a dotted orange line. Structural elements of the LG domain investigated

0

in this work are highlighted in colors; adjacent to the GED and in dark blue are the two helices a3 and a4 as well as the nonresolved loop

0

region preceding a3 (dotted line); distant from the GED and in magenta is the switch I loop. (B) Point and C-terminal truncation mutants of

hGBP-1 visualized by its domain architecture (colored according to the subdomains and structural elements presented in panel A; previously

generated mutants [31,32] are marked with an asterisk; aa: amino acid). (C) Schematic excerpt of the LG domain and GED highlights

charged residues with shown and potential effects on intramolecular interactions (blue and red), the cluster of phenylalanines (gray), and the

switch I positions (black) investigated in this work (PDB ID: 1F5N). The tight GED mutant grounds on the mutational positions E217C and

K567C (green) used for covalent coupling of the LG domain and GED by a chemical cross-linker (green dotted line). To ensure speciﬁc

positioning of the cross-linker, noteworthy, the tight GED mutant has all natural and solvent-accessible cysteines removed [32]. Salt bridge

interactions between residues R227/K228 from the LG domain and glutamate residues from the GED are depicted as gray dotted lines.

Δa13 (Fig. 4B,C and Table 1), this suggests that all

positions have at least some impact on the C-terminal

a13 arrangement. Above that, all mutants signiﬁcantly

gained the ability to utilize GDP as substrate, which

indicates a wider impact of the GED arrangement.

Among the four mutants, the mutant F229A presented

GDPase activity was even twofold faster than that of

the LG domain (Fig. 6C) (Table 1). By measuring a

protein concentration-dependent increase of the

GDPase activity, we determined the maximum cat-

alytic GDPase activity of the TripleF mutant to be

GDP;max

ꢀ1

k

= 4.1 min

hydrolysis is also stimulated by protein dimers, we

(Fig. 6D). Assuming the GDP

cat

ꢀ1

the slowest catalytic GDPase activity (0.11 min ),

although this GDPase activity still remained in the

range of what had been obtained for the ΔGED or the

loose GED (R227E/K228E) (Table 1). The mutants

F171A and F175A showed roughly 10–20 times faster

GDP hydrolysis as compared to the mutant F229A,

and both catalytic GDPase activities were similar to

the activity obtained for the LG domain (Fig. 6C)

obtained a dimer dissociation constant (K ) of 0.1 µM

d

for the TripleF mutant, which indicates a more than

100-fold higher afﬁnity compared to the GDP-induced

LG domain dimer (see Fig. 3B, K = 8.9 µM).

d

Based on these enzymatic properties and decreased

thermal stabilities of these mutants (Table 1), we con-

cluded that besides the intramolecular salt bridge net-

work, particularly the a3´ phenylalanines play a

(Table 1). For the TripleF mutant, the catalytic

8

The FEBS Journal (2020) ª 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of

Federation of European Biochemical Societies

S. Ince et al.

Functional coupling of hGBP-1’s N- and C termini

Table 1. Catalytic activity and thermal stability of hGBP-1 mutants.

Turnover numbers of GTP hydrolysis (k^G^T^P) were derived from a

monomeric when bound to GMP and dimeric when

cat

bound to GDPꢂAlF (an analog mimicking the inter-

x

reaction with 1–5 µM of protein and 500 µM GTP. The share of

GMP on total nucleotide product from GTP hydrolysis was either

derived from the same experiment using the ratio k_c^G_a^M_t^P/k_c^G_a^T_t^Por

mediate state of c-phosphate cleavage during GTP

hydrolysis). The latter conﬁrms the GTP hydrolysis-in-

duced dimerization of all constructs, which was

expected because of the self-stimulated GTPase activity

(Table 1). Interestingly, with GTP analogs (GppNHp

and GTPcS), which mimic the state of GTP binding,

we observed notable deviations among the constructs

in correlation with the availability of the C-terminal

GED. While the wt and the tight GED remained

mainly in the monomeric state, both the ‘open’ type of

mutant (here represented by F171A) and the mutants

having the GED truncated (DGED, LG domain)

shifted mainly to the dimeric state. Here, the mutant

Da13, which has a partly truncated GED, shifted only

partly to the dimeric state. As the dimeric protein con-

formation in the presence of GTPcS exclusively

occurred with the released or truncated GED mutants,

we suspected that the LG:GED intramolecular interac-

tion might affect the GTP binding-induced dimeriza-

tion of hGBP-1.

from long-term hydrolysis studies. Turnover numbers of GDP

ꢃGDP

hydrolysis (k

) were determined with 10 µM protein and 500 µM

) were obtained from

cat

GDP. Melting temperatures of proteins (T

m

the DSC measurements using 5–15 µM of protein. N.d.: not

determined. Actual mutations for either abbreviation are

summarized in Fig. 5.

GTP

cat

ꢀ1

GDP

cat

ꢀ1

(min )

Protein

k

(min

)

GMP (%)

k

m

T (°C)

wt

11

23

43

40

34

39

14

15

12

19

15

5.4

20

16

22

4.1

60

20

40

81

83

71

1

0.00075

0.0071

0.1076

1.4

49

Δa13

45

ΔGED

41

LG

45

Tight GED

Loose GED

LoopAAA

LoopRRR

F229A

≤ wt

0.2881

≤ wt

0.11

1.9

n.d

42

89

43

44

69

70

72

75

3

n.d

45

F171A

44

F175A

0.94

3.8

n.d

44

TripleF

FL-H74A

ΔGED-H74A

LG-H74A

TripleF-H74A

FL-K76A

ΔGED-K76A

LG-K76A

≤ wt

n.d

49

To explore this, we generated double-labeled wt and

F171A and monitored intramolecular structural

changes in the presence of different nucleotides by

F o€ rster resonance energy transfer (FRET) according

to the previously described method [34]. Due to differ-

ent accessibilities of cysteines, the acceptor ﬂuorophore

locates on the single cysteine of the GED (the very C-

terminal CaaX cysteine, see Fig. 5), and the donor ﬂu-

orophore locates on another cysteine on a subdomain

of the same protein molecule (MD or LG domain). By

addition of nucleotide, a decrease of the FRET signal

reports release of the GED from the rest of the pro-

tein, that is, an intramolecular opening [34]. As

observed, a maximum opening of wt was achieved

60

40

87

3

n.d

0.90

≤ wt

n.d

1

n.d

3

n.d

critical role in establishing intramolecular interactions

between the LG domain and the GED, which upon

mutation render an ‘open’ type of conformation.

Intramolecular LG:GED interaction regulates the

dimer formation of hGBP-1

only in the presence of GDPꢂAlF , which was also the

x

only analog that successfully induced dimerization in

the SEC method (Fig. 7B,A). In contrast, the GTP-

binding analogs GTPcS and GppNHp, which on SEC

retained wt in the monomeric state (Fig. 7A), induced

minor or no intramolecular opening similar to GMP.

For the mutant F171A, except for GMP, all the tested

nucleotides induced intramolecular opening to the

same maximal extent as marked by the trace of

Dimerization of hGBP-1 has been found to be an

essential prerequisite to catalyze its unique b-phos-

phate hydrolysis step yielding GMP [29,30]. Since the

demonstrated mutants deviate from the wild-type pro-

tein in exactly this enzymatic step, we further investi-

gated putative alterations in their capability to

dimerize.

According to our previous studies, we analyzed dif-

ferent protein constructs in the presence of different

nucleotides with size-exclusion chromatography (SEC)

where dimerization of proteins was indicated by a shift

to lower elution volumes (Fig. 7A) [34,40]. In addition

to wt, we investigated all C-terminal truncation

mutants as well as the mutants tight GED and F171A.

As shown in Fig. 7A, all constructs appeared

GDPꢂAlF (Fig. 7B), and all these nucleotides also

x

induced dimerization observed by SEC (Fig. 7A).

Overall, this demonstrates that the intramolecular

release of the GED is tightly related to the dimeriza-

tion of protein.

Given the intramolecular release of the GED, we

wanted to investigate further the GED:GED interac-

tion, which was suggested in our previous study

The FEBS Journal (2020) ª 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of

Federation of European Biochemical Societies

9

Functional coupling of hGBP-1’s N- and C termini

S. Ince et al.

Fig. 6. Regulation of hGBP-1’s GDPase activity by a cluster of phenylalanines. (A) Superimposition of the nucleotide-free and monomeric

full-length structure of hGBP-1 (PDB ID: 1DG3; blue: LG domain; yellow: GED) with the GDPꢂAlF -bound structure of the isolated LG domain

PDB ID: 2B92; pink) highlights a cluster of phenylalanines, which in close proximity to the GED are localized in the LG domain. The cluster

x

(

0

is formed by four residues: F229 from a4 and F171, F174, F175 from a3 , altogether pointing away from the GED helix a12 (yellow) in the

nucleotide-free state (blue sticks). A deviating orientation of the residues in the GDPꢂAlF -bound state (pink sticks) indicates potential

x

rearrangements and in particularly steric clashes with the GED in the course of GTP binding and/or hydrolysis. (B) GTPase activity and

product formation of the phenylalanine mutants measured with 1 µM of protein and 500 µM of GTP. Obtained catalytic activities of GTP

hydrolysis (blue columns), GDP formation (gray columns), and GMP formation (red column) are compared to wt. The pie charts depict

resulting product shares giving (GDP: gray; GMP: red). (C) GDPase activity of the same mutants in comparison with wt and the LG domain

measured with 10 µM of protein and 500 µM of GDP. (D) Concentration-dependent GDPase activity of TripleF assayed with 500 µM of GDP.

[

33,34]. Therefore, we generated single-labeled proteins

exclusively subsisted in a heterodimer with the nonla-

beled protein constructs. Thus, alterations in the

release of GED of wt, which was monitored in this

setup, could be assigned to the heterodimer partner of

wt. As shown in Fig. 7D, when the ‘open’ mutant

F171A was offered as a dimerization partner, the

intramolecular opening of labeled wt reached a similar

level as offering the nonlabeled wt instead. Besides the

F171A mutant, the mutant Da13 also induced similar

intramolecular opening of labeled wt. However,

intramolecular opening was strikingly restricted when

the dimerization partner was disabled to provide its

a12 for the GED:GED interaction, for instance, when

the GED was absent (LG, DGED) or artiﬁcially

locked (tight GED) (Fig. 7D). These observations

illustrate that the intramolecular opening of hGBP-1

and the intermolecular GED:GED interactions are

highly interdependent processes.

having either a donor or an acceptor ﬂuorophore

localized to the single cysteine of the GED, which

allowed us to monitor GED:GED interactions by an

increase of the FRET signal [34]. For wt or F171A, all

nucleotides, which induced intramolecular release of

the GED and dimerization observed with SEC, also

consistently induced a signiﬁcant increase of the inter-

molecular FRET signal (Fig. 7C). This indicates that

GED:GED interactions indeed occur during dimeriza-

tion of the tested hGBP-1 constructs.

Finally, as suggested by similar kinetics of the

respective intra- and intermolecular FRET traces

(

Fig. 7B,C), we asked whether intramolecular release

of the GED and intermolecular GED:GED interac-

tions are two associated events. To answer this, we

used

a modiﬁed intramolecular FRET approach,

where the GDPꢂAlF -induced GED release of wt (dou-

x

ble-labeled) was monitored in the presence of different

mutants (nonlabeled) (Fig. 7D) [40]. A 10-fold molar

excess of nonlabeled over labeled protein was applied

to maintain a pool of dimers where labeled wt almost

Overall, we demonstrate that the LG:GED

intramolecular interaction controls the intermolecular

GED:GED interaction on the one hand and inﬂuences

the catalytic activity on the other hand.

1

0

The FEBS Journal (2020) ª 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of

Federation of European Biochemical Societies

S. Ince et al.

Functional coupling of hGBP-1’s N- and C termini

A

wt

tight GED

F171A

*

1

0

*

**

1

0

1.0

1.5

V mL)

2.0 1.0

1.5

V (mL)

1.0

1.5

e

2.0

V (mL)

e

C-terminal truncation mutants

Fig. 7. Nucleotide-dependent inter- and

intramolecular interactions of hGBP-1

*

**

1

mutants. (A) Size-exclusion chromatograms

of representative hGBP-1 mutants (denoted

and schematically illustrated above) in the

presence of different nucleotides (orange:

GDPꢂAlF

x

; cyan: GTPcS; blue: GppNHp;

0

gray: GMP). In an enlarged chromatogram

of wt, monomeric (arrowhead) and dimeric

fractions (asterisks) are additionally denoted

which for the following are assigned by the

symbols only. Chromatograms accessed in

1.0

1.5

1.0

1.5

1.0

1.5

2.0

V (mL)

e

B

1

.0

.9

.8

.7

D

0

the presence of GMP and GDPꢂAlF

x

mark

the respective monomeric and dimeric

fraction of each construct. Nucleotide-

dependent (B) intramolecular and (C)

intermolecular FRET traces of wt and

F171A; color code of nucleotides as in A.

Mutant tight GED has by construction no

accessible cysteines (see Ref. [32] and

Fig. 5) for ﬂuorophore labeling; thus, FRET

data are not available. (D) Intramolecular

FRET traces reporting structural

wt

F171A

0.6

0

200 400 600

Time (s)

0

200 400 600 800

Time (s)

1.0

C

0

.9

.8

3.0

2.5

2.0

0

0.7

0.6

0.5

wt

F171A

1

.5

.0

0

.4

.3

rearrangement of wt upon GDPꢂAlF

x

-

200 400 600

Time (s)

0

200 400 600 800

Time (s)

0

100

Time (s)

200 300

induced heterodimerization with the

indicated mutants.

Notably, considering that the LG domain dimerizes

upon GTP binding, it appears surprisingly that wt was

unable to do so (Fig. 7A,C in the presence of

GppNHp and GTPcS), as both constructs have the

LG:LG dimer interface in common [26]. In fact, our

previous study did illustrate a growth of dimeric wt

peak in the presence of GppNHp and GTPcS with

increasing protein concentration [34]. Together with

the detected molecular mass, this suggested that

GppNHp- and GTPcS-bound hGBP-1 are in a rapid

exchange between the monomeric and the dimeric

forms [34]. In the intra-FRET measurements, we

observed a slight release of the GED only induced by

GTPcS but not by GppNHp (Fig. 7B wt). This may

be explained by a slight difference of the two nucleo-

tides. GppNHp is not hydrolysable, whereas GTPcS is

shown to be slowly turned over by hGBP-1 [40]. How-

ever, GppNHp induced no release of the GED, and

both GppNHp and GTPcS failed to induce the GED:

GED interaction for the wt (Fig. 7C). Therefore, we

conclude that the GTP binding-induced wt dimers,

which are in rapid equilibrium with monomers, are

only formed using the LG:LG interactions.

Intramolecular opening between the LG domain

and GED is triggered by the distant switch

position H74

The so-called switch regions, switch I and switch II, are

conserved structural elements nearby the nucleotide-

binding pocket of GTP-binding proteins. These switch

regions, through nucleotide-dependent conformational

The FEBS Journal (2020) ª 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of

Federation of European Biochemical Societies

11

Functional coupling of hGBP-1’s N- and C termini

S. Ince et al.

changes, allow the proteins to interact with their down-

stream effector proteins [41]. In hGBP-1, intriguingly,

replacement of several amino acids within these regions

by alanine turned out to almost abolish the product

GMP formation during GTP hydrolysis [31]. Therefore,

those mutants were awarded as interesting candidates

for the present study.

This was further supported by a prominent monomeric

fraction of GDPꢂAlF -bound H74A monitored by

x

SEC (Fig. 8G; left panel). These properties suggest

that intramolecular conformational changes between

the LG domain and the GED are notably attenuated

by the H74A mutation, that is, the GED release of

H74A is restricted throughout the cycle of GTP bind-

ing and hydrolysis when compared to wt.

As representatives, we selected two mutants from

switch I, H74A and K76A, and performed the protein

concentration-dependent catalytic GTPase activity

assays (Fig. 8A). Albeit dimerization of the mutants

appeared to be attenuated by approximately 15-fold

when compared to wt (Fig. 8B), their maximum cat-

alytic GTPase activity was affected by two to three

times only (Fig. 8C) though yielding almost no GMP

as product (Fig. 8D). Since we observed a visible

increase of the catalytic GTPase activity with increas-

ing protein concentration, we excluded that the lack of

GMP production results from disability of dimeriza-

tion. Noticing that the tight GED mutant had the

same deﬁciency in producing GMP (Fig. 4C), we sus-

pected that the mutants H74A and K76A are also

trapped in a closed conformation. In this conforma-

tion, the GED is locked and disables the protein to

form a tight dimer which under wild-type condition is

established in order to further produce GMP.

Consequently, in TripleF-H74A—wherein the con-

tribution of a3´ phenylalanines to the LG:GED con-

tacts is abolished—these restrictions were reversed

(Fig. 8G–I, right panels). The observed pattern of

nucleotide-dependent inter- and intramolecular interac-

tions moreover resembled all characteristics of an open

mutant (see Fig. 7A–C, F171A). Overall, the open

conformation of TripleF-H74A turned out capable to

bypass the intramolecular restrictions created by

H74A, in turn yielding tight dimers producing GMP

in GTP hydrolysis.

Altogether, we conclude that the residue H74, yet

located at distant position from the LG:GED inter-

face, has still a critical role in the intramolecular inter-

action. As to its particular localization within the LG

domain, it is conceivable that H74 mediates a chain of

conformational changes toward the GED. These con-

formational changes are induced by nucleotide binding

to initiate the GED release for further GED:GED

interactions which is necessary to build tight dimers,

which is important for GMP production.

To test our hypothesis, the GEDs of the mutants

H74A and K76A were truncated to overcome the

closed conformations of the full-length proteins. For

K76A, the truncated mutant DGED-K76A did not

improve the GMP formation in GTP hydrolysis,

although the maximum catalytic GTPase activity was

signiﬁcantly higher than for the DGED construct

Discussion

We found that the GED affects two features of GTP

hydrolysis. Firstly, the GED affects GMP production

during the GTP hydrolysis. We identiﬁed the presence

of the C-terminal end of the GED (a13 helix) to inﬂu-

ence the ratio of the products GDP and GMP. When

this region was deleted from the wt, a signiﬁcant

increase in the GMP production was observed. Sec-

ondly, we discovered that the GED contributes to the

substrate speciﬁcity of hGBP-1. While the wt only

employs GTP as substrate from solution but not

GDP, the GED-truncated mutant gains the feature to

effectively utilize also GDP as substrate.

(Fig. 8E,F). In contrast, the deﬁciency in GMP forma-

tion caused by H74A was notably restored when the

GED was truncated (DGED-H74A), yielding GMP

even as predominating product (Fig. 8F). Similar

results were obtained when H74A was introduced into

an open type of full-length hGBP-1 mutant (TripleF-

H74A) (Fig. 8F). These results therefore indicate dis-

tinct roles of the residues H74 and K76.

The results above suggest that H74 plays a crucial

role in opening; therefore, the alanine mutation hin-

ders the formation of the GED:GED contact. We fur-

ther investigated these properties with intra- and

intermolecular FRET studies. We detected that

intramolecular opening and dimerization of H74A, as

The catalytic properties of hGBP-1 depend highly

on dimerization of the protein [24,26]. Our previous

crystallography studies revealed LG:LG interactions as

the initial dimeric interface [24,26]. However, in a

more recent study, intra- and intermolecular FRET

measurements identiﬁed the GED to participate in the

dimerization of hGBP-1 [34]. Therefore, both the LG:

LG and GED:GED interactions contribute to the for-

mation of a catalytically efﬁcient dimer. In this work,

exclusively induced by GDPꢂAlF , were notably slower

x

than that of wt (Fig. 8H,I; left and middle panels).

Also, the observed minor intramolecular rearrange-

ments of wt induced by GTPcS were not visible for

H74A (Fig. 8I; left and middle panels), indicating a

somehow restricted ﬂexibility of the mutant’s GED.

1

2

The FEBS Journal (2020) ª 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of

Federation of European Biochemical Societies

S. Ince et al.

Functional coupling of hGBP-1’s N- and C termini

A

B

C

4

3

20

1

6

2

8

4

0

1

6

2

8

4

0

1

2

1

0

5

10 15 20

protein] (μM)

[

D

E

F

1

00

0

100

80

60

40

20

0

6

0

8

6

4

2

50

40

30

20

10

0

ED-H74A

Fig. 8. GMP formation deﬁciency of the

switch I mutant H74A and its recovery. (A)

Protein concentration-dependent GTPase

activity of hGBP-1 wt (squares) and the

switch I mutants H74A (circles) and K76A

ΔG

ΔGED-H74A

G

H74A

wt

TripleF-H74A

1

(triangles) measured at given protein

concentrations and 500 µM GTP. From a

quadratic ﬁt to the data (solid blue), dimer

dissociation constants (B) and maximum

catalytic activities (C) were obtained. (D)

Share of GTP hydrolysis products derived

from the same experiments in A and

0

1

.0

1.5

1.0

1.5

1.0

1.5

2.0

V (ml)

e

conﬁrmed with long-term GTP hydrolysis

studies; red: GMP; gray: GDP. (E) Catalytic

GTPase activities and (F) resulting shares of

GTP hydrolysis products (red: GMP; gray

GDP) obtained from reactions with 5 µM of

indicated mutants and 500 µM of GTP. (G)

Size-exclusion chromatograms of indicated

mutants in the presence of GMP (dark

gray), GTPcS (cyan), GppNHp (blue), and

H

H74A

wt

TripleF-H74A

3

2

1.5

1.0

.0

.5

.0

0

1,200 2,400

Time (s)

0 200 400 600 800 0 200 400 600 800

GDPꢂAlF

x

(orange) showing monomeric and

) protein fractions. (H)

Time (s)

dimeric (lower V

e

Time traces of intermolecular FRET

H74A

wt

TripleF-H74A

I

between 1 µM of donor-labeled and 1 µM of

acceptor labeled protein after the addition

of 250 µM nucleotide at t = 0 s, except for

GppNHp where 500 µM was used. (I) Time

traces of intramolecular FRET between

1

0

0.8

.0

.9

0

.7

.6

0.1 µM of labeled and 1.9 µM of acceptor

labeled protein after the addition of 250 µM

nucleotide at t = 0 s, except for GppNHp

where 500 µM was used.

0

1,200 2,400

Time (s)

0 200 400 600 800 0 200 400 600 800

Time (s)

by applying SEC and FRET methods to a panel of

suitable hGBP-1 mutants, we showed that nucleotide

hydrolysis-driven opening of the GED is a prerequisite

for the formation of the GED:GED contact.

We depict a detailed mechanism of GTP hydrolysis

by hGBP-1 in Fig. 9. (i) Binding of GTP induces the

formation of a hGBP-1 dimer with the LG:LG contact

but is not sufﬁcient to release the GED. Here, a

The FEBS Journal (2020) ª 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of

Federation of European Biochemical Societies

13

Functional coupling of hGBP-1’s N- and C termini

S. Ince et al.

structural restraint evoked by the LG:GED contact is

illustrated by ﬂattened areas of the LG domains, and

the GTP-bound dimer is in a rapid exchange with the

monomers (Fig. 9). (ii) Upon catalyzing c-phosphate

cleavage of the bound GTP, an intramolecular opening

of the GED is triggered to allow establishment of the

GED:GED interface that reversely stabilizes the

intramolecular opening. More importantly, this separa-

tion of the GED from the LG domain removes the

structural restraint evoked by the LG:GED contact

and may consequently add structural changes in the

LG:LG interface including the nucleotide-binding

pocket. In Fig. 9, these structural changes are symbol-

ized by an increase of the area at the LG:LG interface

when comparing the two GDP-bound dimers. (iii)

GDP-bound dimers with the rearranged LG:LG inter-

face and catalytic center are able to catalyze the sec-

ond phosphate cleavage step, that is, the formation of

GMP, while the GDP-bound dimers without the rear-

ranged LG:LG interface and catalytic center directly

dissociate to monomers yielding GDP as the ﬁnal pro-

duct (Fig. 9).

located on the LG:GED interface together with the

residues R227 and K228 directly contribute to the

intramolecular interaction, while the H74 located on

the switch region triggers a chain of conformational

changes to transfer the nucleotide binding and hydrol-

ysis signals to the distant LG:GED contact. In total,

we revealed the roles of the GED to provide more

comprehensive understandings of the unique GTP

hydrolysis cycle of hGBP-1.

Most probably, the guanine nucleotide hydrolysis of

hGBP-1 is designed to trigger further biological func-

tions of hGBP-1, which virtually can be dictated by a

timed sequence of events: the GTP binding, the c-

phosphate cleavage, as well as the following b-phos-

phate cleavage. As for almost all GTP-binding pro-

teins, GTP binding activates the protein—for example,

enables effector binding—or in the case of hGBP-1

triggers dimerization and thereby positioning of the

catalytic machinery [41,26]. Notably, these actions are

not achieved when hGBP-1 binds GDP instead of

Of note, in the ﬁrst step of the hydrolysis cycle,

GTP binding-induced dimers of the GED-truncated

mutants (LG domain and DGED) were visible in SEC.

However, for the wt, GTP binding-induced protein

dimers were shown to be in fast exchange with mono-

mers. Thus, we deduce that existence of restraint

GEDs on the wt might even weaken the LG:LG con-

tacts. The scheme in Fig. 9 also reﬂects why the ‘open’

mutants yielded prominently GMP as the product of

GTP hydrolysis and even gain substrate GDPase activ-

ity. When the GED is deleted or released by mutations

such as the R227E/K228E, the structural restraint

evoked by the LG:GED contact is eliminated. There-

fore, compared to the wt, these mutants favor the for-

mation of the dimers with the enhanced LG:LG

interface (as demonstrated by the GDP-bound dimer

with the increased area at the LG:LG interface in

Fig. 9) that are active for intermediate or even sub-

strate GDP cleavage. Quite the contrary, when the

GED is locked to the rest of the protein (tight GED

and H74A), the restraint on the LG:GED contact is

not or hard to overcome. Consequently, dimers cannot

further adapt their conformational changes after the c-

phosphate cleavage and thus yield GDP as the ﬁnal

product.

GTP

GTP GTP

GDPGDP

GDP GDP

Fig. 9. Extended GTPase cycle of hGBP-1 considering the GED’s

contribution. (i) Binding of GTP induces the LG:LG contact between

two hGBP-1 molecules, which results in a rapid exchange between

the monomeric and the dimeric forms.

A structural restraint

evoked by LG:GED contact is illustrated by ﬂattened areas of the

LG domains. (ii) The ﬁrst hydrolysis step (c-phosphate cleavage)

triggers intramolecular opening of the GED, which removes the

structural restraint evoked by the LG:GED contact and promotes

the GED:GED interaction. With the removal of the LG:GED

structural restraint, conformational changes may take place in the

LG:LG interface including the nucleotide-binding pocket (illustrated

by the GDP-bound dimer with an increased area at the LG:LG

interface) to catalyze the second phosphate cleavage. (iii) Dimers

with the rearranged LG:LG interface and catalytic center are able

to catalyze the second phosphate cleavage producing GMP, while

dimers without the rearranged LG:LG interface and catalytic center

directly dissociate to monomers yielding GDP as the ﬁnal product.

This model, which is based on the observations

described in the results, explains contributions of the

GED to the catalytic activity of hGBP-1. We further

identiﬁed amino acids located on the LG domain,

which lead to the dynamic motion of the GED in the

GTP hydrolysis. For instance, the a3´ phenylalanines

1

4

The FEBS Journal (2020) ª 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of

Federation of European Biochemical Societies

S. Ince et al.

Functional coupling of hGBP-1’s N- and C termini

GTP from bulk solution. Nevertheless, the intermedi-

ate product GDP is immediately hydrolyzed following

GTP hydrolysis, and this tight control of GMP pro-

duction seems to be crucial for hGBP-1’s biological

function. As typical for GTPases, nucleotide hydrolysis

is employed to make hGBP-1 cycle through different

structural and functional states in order to evoke

effects such as polymerization and membrane binding.

When considering membrane binding, for instance,

the isolated GED carrying a farnesyl moiety was used

and identiﬁed to be sufﬁcient and responsible for tar-

geting membranous compartments of the cell

of mutants (e.g., F171A, looseGED, or TripleF), buffer C

was supplemented with 2 mM DTT in the second step of

puriﬁcation.

Protein concentrations were determined according to Ref.

[

34] using the molar absorption coefﬁcients of e280

=

ꢀ

1

ꢀ1

4

3 240 M ꢂcm forallfull-lengthconstructsandDa13,e280

1 960 M ꢂcm for ΔGED, and e280 = 34 280 M ꢂcm^ꢀ¹for

ꢀ1 ꢀ1 ꢀ1

all LG constructs.

Catalytic GTPase/GDPase activity assay

Protein and substrate (either 500 µM GTP or GDP) diluted

in buffer C (50 mM Tris/HCl, pH 7.9, 150 mM NaCl, and

[

42,43,37,16]. However, the GED underlies more elab-

5

2

mM MgCl ) were mixed and incubated at 25 °C. At dif-

orate mechanisms when it needs to accomplish this

task within the entire protein. Especially, the

intramolecular release of the GED triggered by hGBP-

ferent time points, nucleotide composition was analyzed by

reversed-phase HPLC (Chromolith RP18e HPLC column;

Merck, Darmstadt, Germany) in HPLC buffer (100 mM

potassium phosphate, 10 mM tetrabutylammonium bro-

mide, and 4% (v⁄v) acetonitrile), detecting the absorption

of nucleotides at 254 nm with a MD-2015 diode array

detector (Jasco, Gross-Umstadt, Germany). Initial rates of

substrate (GTP or GDP) turnover or product (GDP or

GMP) formation were obtained from linear ﬁts to nucleo-

tides’ concentration change over time in steady-state

hydrolysis (remaining GTP > 60%). Dividing the initial

rates by protein concentrations, the catalytic activities were

obtained and further plotted against the protein concentra-

tions. Data were further analyzed as described previously,

with a quadratic binding equation that gives two parame-

1

’s enzymatic activity and dimerization is important,

as the interactive regions of GED and the attached

farnesyl moiety are not available for these structural

and functional states when the GED is attached to the

rest of the protein [36,42,14,44].

Materials and methods

Molecular cloning

Bacterial expression vectors pQE-80L (Qiagen, Hilden,

Germany) coding for full-length and C-terminally truncated

constructs of hGBP-1 were generated by molecular sub-

cloning via the restriction enzymes BamHI and SalI (Fer-

mentas, Sankt Leon-Rot, Germany). Point mutations were

introduced by site-directed mutagenesis using KOD Hot

Start DNA Polymerase (Novagen Merck, Darmstadt, Ger-

many), and appropriate primer pairs and template DNA

ters: the apparent dissociation constant of hGBP-1 dimers

GXP;max

(K ), and the maximum catalytic activity (k

d

) [28,35].

cat

Long-term GTP/GDP hydrolysis

As for the GTPase/GDPase assay, varying concentrations

of protein and 500 µM of the respective substrate, either

GTP or GDP, were mixed in buffer C and incubated at

(see Appendix S1 for the list of primers pairs and their

sequences). All mutations were veriﬁed by DNA sequencing

with a 3130xl sequencer (Applied Biosystems, Foster City,

CA, USA).

2

5 °C for 24 h. The resulting nucleotide composition was

analyzed by reversed-phase HPLC as for the catalytic

GTPase/GDPase activity assay.

Expression and puriﬁcation

Differential scanning calorimetry

6

Expression and puriﬁcation of N-terminally (His) -tagged

constructs of hGBP-1 was performed according to the pro-

tocol described for wild-type hGBP-1 [34] using the E. coli

strain BL21 CodonPlus (DE3) RIL and a two-step puriﬁ-

Melting temperatures of different hGBP-1 constructs were

obtained by differential scanning calorimetry (MicroCal

VP-Capillary DSC, Malvern Panalytical, Worcestershire,

UK). Protein samples were diluted to a concentration of

ꢀ

cation procedure with afﬁnity chromatography (HisPur

ꢀ

1

Cobalt Resin, Thermo Scientiﬁc) followed by preparative

size-exclusion chromatography (Superdex 200 26/60,

0

.5 gꢂL in buffer P and measured against the same buffer

in the reference cell during a temperature ramp-up from 30

to 55 °C, yielding a difference in the amount of heat

required to increase the temperature of the sample and ref-

erence. The heat capacity was plotted against the increasing

temperature. Fit of the peak value according to the manu-

3

20 mL, GE Healthcare, Munich, Germany). In the ﬁnal

step, buffer C (50 mM Tris/HCl, pH 7.9, 150 mM NaCl,

and 5 mM MgCl ) was used in which proteins were further

2

stored. To avoid unspeciﬁc oligomerization through disul-

ﬁde bridge formation especially observed for all ‘open’ type

m

facture’s program gave the melting temperature T . [45].

The FEBS Journal (2020) ª 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of

Federation of European Biochemical Societies

15

Functional coupling of hGBP-1’s N- and C termini

S. Ince et al.

Analytical size-exclusion chromatography

Author contributions

Dimerization of hGBP-1 mutants (20 µM) in the presence

of different nucleotides (buffer C supplemented with 250–

SI planned and performed experiments; analyzed data;

discussed results; and wrote paper. PZ planned and

performed experiments; analyzed data; discussed

results; and wrote paper. MK planned experiments,

analyzed data, and discussed results. OK supported

experiments and analyzed data. SS supported experi-

ments and discussed results. CH planned experiments,

analyzed data, discussed results, and wrote paper.

3

20 µM of GMP, GTPcS, or GppNHp; purchased from

Jena Bioscience, Jena, Germany) was analyzed by analyti-

cal size-exclusion chromatography (SEC) using the column

Superdex 200 PC 3.2/30 (2.4 mL, GE Healthcare) as

described previously [34]. Due to almost nonreversible

behavior, exceptionally, SEC of GDPꢂAlF

x

-induced com-

plexes was run with buffer C in the absence of any supple-

mental nucleotide. Prior to that, the protein was diluted to

a total concentration of 20 µM in buffer C containing

References

1

0 mM NaF and 300 µM AlCl3. After the addition of

50 µM GDP, the mixture was incubated for 10–30 min

and injected to the column.

1

Olszewski MA, Gray J & Vestal DJ (2006) In silico

genomic analysis of the human and murine guanylate-

binding protein (GBP) gene clusters. J Interferon

Cytokine Res 26, 328–352.

2

Praefcke GJK & McMahon HT (2004) The dynamin

superfamily: universal membrane tubulation and ﬁssion

molecules? Nat Rev 5, 133–147.

Fluorophore labeling of proteins

Following the labeling strategy described previously [34],

proteins were labeled with the donor and acceptor ﬂuo-

rophores Alexa Fluor 488-C5-maleimide and Alexa Fluor

3 Cheng YS, Colonno RJ & Yin FH (1983) Interferon

induction of ﬁbroblast proteins with guanylate binding

activity. J Biol Chem 258, 7746–7750.

6

47-C2-maleimide (Thermo Scientiﬁc), respectively. Both

single-labeled proteins and double-labeled proteins were

generated: The single-labeled proteins carried maximal one

ﬂuorophore, either a donor or an acceptor dye, which in

combination were used for intermolecular FRET measure-

ments. The double-labeled proteins carried both a donor

and an acceptor dye within one molecule and served to

measure intramolecular FRET.

4 Tripal P, Bauer M, Naschberger E, M o€ rtinger T,

Hohenadl C, Cornali E, Thurau M & Sturzl M (2007)

Unique features of different members of the human

guanylate-binding protein family. J Interferon Cytokine

Res 27, 44–52.

€

5 Lubeseder-Martellato C, Guenzi E, J o€ rg A, T o€ polt K,

Naschberger E, Kremmer E, Zietz C, Tschachler E,

Hutzler P, Schwemmle M et al. (2002) Guanylate-

binding protein-1 expression is selectively induced by

inﬂammatory cytokines and is an activation marker of

endothelial cells during inﬂammatory diseases. Am J

Pathol 161, 1749–1759.

F o€ rster resonance energy transfer

Inter- and intramolecular FRET studies were carried out

according to Ref. [34] in buffer C and at 25 °C. FRET was

monitored at 664 nm while exciting the donor at 498 nm.

If not indicated otherwise, for intermolecular FRET mea-

surements 1 µM donor-labeled and 1 µM acceptor labeled

protein were mixed and incubated for 2–5 min before

nucleotide addition. In analogy, also for intramolecular

FRET measurements protein concentration was adjusted to

6

Krapp C, Hotter D, Gawanbacht A, McLaren PJ,

Kluge SF, St u€ rzel CM, Mack K, Reith E, Engelhart S,

Ciufﬁ A et al. (2016) Guanylate Binding Protein (GBP)

5

is an interferon-inducible inhibitor of HIV-1

infectivity. Cell Host Microbe 19, 504–514.

7

Itsui Y, Sakamoto N, Kakinuma S, Nakagawa M,

Sekine-Osajima Y, Tasaka-Fujita M, Nishimura-Sakurai

Y, Suda G, Karakama Y, Mishima K et al. (2009)

Antiviral effects of the interferon-induced protein

guanylate binding protein 1 and its interaction with the

hepatitis C virus NS5B protein. Hepatology 50, 1727–

1737.

2

1

µM, but containing 0.1 µM of double-labeled protein and

.9 µM of nonlabeled protein to abolish intermolecular

effects.

Acknowledgement

8

9

Nordmann A, Wixler L, Boergeling Y, Wixler V &

Ludwig S (2012) A new splice variant of the human

guanylate-binding protein 3 mediates anti-inﬂuenza

activity through inhibition of viral transcription and

replication. FASEB J 26, 1290–1300.

This work was supported by the Deutsche Forschungs-

gemeinschaft (DFG, HE2679/6-1).

Conﬂicts of interest

Zhu Z, Shi Z, Yan W, Wei J, Shao D, Deng X, Wang

S, Li B, Tong G & Ma Z (2013) Nonstructural protein

The authors declare no conﬂict of interest.

1

6

The FEBS Journal (2020) ª 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of

Federation of European Biochemical Societies

S. Ince et al.

Functional coupling of hGBP-1’s N- and C termini

1

of inﬂuenza A virus interacts with human guanylate-

21 Fukumoto M, Amanuma T, Kuwahara Y, Shimura T,

Suzuki M, Mori S, Kumamoto H, Saito Y, Ohkubo Y,

Duan Z et al. (2014) Guanine nucleotide-binding

protein 1 is one of the key molecules contributing to

cancer cell radioresistance. Cancer Sci 105, 1351–1359.

22 Praefcke GJK (2018) Regulation of innate immune

functions by guanylate-binding proteins. Int J Med

Microbiol 308, 237–245.

binding protein 1 to antagonize antiviral activity. PLoS

ONE 8, e55920.

1

0 Pan W, Zuo X, Feng T, Shi X & Dai J (2012)

Guanylate-binding protein 1 participates in cellular

antiviral response to dengue virus. Virol J 9, 292.

1 Al-Zeer MA, Al-Younes HM, Lauster D, Abu Lubad M

1

&

Meyer TF (2013) Autophagy restricts Chlamydia

trachomatis growth in human macrophages via IFNG-

inducible guanylate binding proteins. Autophagy 9, 50–62.

2 Tietzel I, El-Haibi C & Carabeo RA (2009) Human

guanylate binding proteins potentiate the anti-

chlamydia effects of interferon-gamma. PLoS ONE 4,

e6499.

23 Daumke O & Praefcke GJK (2016) Invited review:

mechanisms of GTP hydrolysis and conformational

transitions in the dynamin superfamily. Biopolymers

105, 580–593.

24 Prakash B, Praefcke GJ, Renault L, Wittinghofer A &

Herrmann C (2000) Structure of human guanylate-

binding protein 1 representing a unique class of GTP-

binding proteins. Nature 403, 567–571.

25 Prakash B, Renault L, Praefcke GJK, Herrmann C &

Wittinghofer A (2000) Triphosphate structure of

guanylate-binding protein 1 and implications for

nucleotide binding and GTPase mechanism. EMBO J

19, 4555–4564.

26 Ghosh A, Praefcke GJK, Renault L, Wittinghofer A &

Herrmann C (2006) How guanylate-binding proteins

achieve assembly-stimulated processive cleavage of GTP

to GMP. Nature 440, 101–104.

27 Schwemmle M & Staeheli P (1994) The interferon-

induced 67-kDa guanylate-binding protein (hGBP1) is a

GTPase that converts GTP to GMP. J Biol Chem 269,

11299–11305.

28 Kunzelmann S, Praefcke GJK & Herrmann C (2005)

Nucleotide binding and self-stimulated GTPase activity

of human guanylate-binding protein 1 (hGBP1).

Methods Enzymol 404, 512–527.

1

3 Wandel MP, Pathe C, Werner EI, Ellison CJ, Boyle

KB, von der Malsburg A, Rohde J & Randow F (2017)

GBPs inhibit motility of Shigella ﬂexneri but are

targeted for degradation by the bacterial ubiquitin

ligase IpaH9.8. Cell Host Microbe 22, 507–518.e5.

4 Piro AS, Hernandez D, Luoma S, Feeley EM, Finethy

R, Yirga A, Frickel EM, Lesser CF & Coers J (2017)

Detection of cytosolic Shigella ﬂexneri via a C-terminal

triple-arginine motif of GBP1 inhibits actin-based

motility. MBio 8, e01979– e01917.

1

5 Shenoy AR, Wellington DA, Kumar P, Kassa H,

Booth CJ, Cresswell P & MacMicking JD (2012) GBP5

promotes NLRP3 inﬂammasome assembly and

immunity in mammals. Science 336, 481–485.

6 Guenzi E, T o€ polt K, Cornali E, Lubeseder-Martellato

C, J o€ rg A, Matzen K, Zietz C, Kremmer E, Nappi F,

Schwemmle M et al. (2001) The helical domain of

GBP-1 mediates the inhibition of endothelial cell

proliferation by inﬂammatory cytokines. EMBO J 20,

1

5

568–5577.

29 Kunzelmann S, Praefcke GJK & Herrmann C (2006)

Transient kinetic investigation of GTP hydrolysis

catalyzed by interferon-gamma-induced hGBP1 (human

guanylate binding protein 1). J Biol Chem 281, 28627–

28635.

1

7 Guenzi E, T o€ polt K, Lubeseder-Martellato C, J o€ rg A,

Naschberger E, Benelli R, Albini A & St u€ rzl M (2003)

The guanylate binding protein-1 GTPase controls the

invasive and angiogenic capability of endothelial cells

through inhibition of MMP-1 expression. EMBO J 22,

30 Abdullah N, Balakumari M & Sau AK (2010)

Dimerization and its role in GMP formation by

human guanylate binding proteins. Biophys J 99, 2235–

2244.

3

772–3782.

1

8 Britzen-Laurent N, Herrmann C, Naschberger E,

Croner RS & St u€ rzl M (2016) Pathophysiological role

of guanylate-binding proteins in gastrointestinal

diseases. World J Gastroenterol 22, 6434–6443.

9 Britzen-Laurent N, Lipnik K, Ocker M, Naschberger E,

Schellerer VS, Croner RS, Vieth M, Waldner M,

Steinberg P, Hohenadl C & et al. (2013) GBP-1 acts as

a tumor suppressor in colorectal cancer cells.

Carcinogenesis 34, 153–162.

0 Tipton AR, Nyabuto GO, Trendel JA, Mazur TM,

Wilson JP, Wadi S, Justinger JS, Moore GL, Nguyen

PT & Vestal DJ (2016) Guanylate-Binding Protein-1

protects ovarian cancer cell lines but not breast cancer

cell lines from killing by paclitaxel. Biochem Biophys

Res Commun 478, 1617–1623.

31 Praefcke GJK, Kloep S, Benscheid U, Lilie H, Prakash

B & Herrmann C (2004) Identiﬁcation of residues in

the human guanylate-binding protein 1 critical for

nucleotide binding and cooperative GTP hydrolysis. J

Mol Biol 344, 257–269.

32 V o€ pel T, Syguda A, Britzen-Laurent N, Kunzelmann S,

L u€ demann M-B, Dovengerds C, St u€ rzl M & Herrmann

C (2010) Mechanism of GTPase-activity-induced self-

assembly of human guanylate binding protein 1. J Mol

Biol 400, 63–70.

1

2

33 Syguda A, Bauer M, Benscheid U, Ostler N,

Naschberger E, Ince S, St u€ rzl M & Herrmann C (2012)

Tetramerization of human guanylate-binding protein 1

The FEBS Journal (2020) ª 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of

Federation of European Biochemical Societies

17

Functional coupling of hGBP-1’s N- and C termini

S. Ince et al.

is mediated by coiled-coil formation of the C-terminal

41 Wittinghofer A & Vetter IR (2011) Structure-function

relationships of the G domain, a canonical switch

motif. Annu Rev Biochem 80, 943–971.

42 Britzen-Laurent N, Bauer M, Berton V, Fischer N,

Syguda A, Reipschl €a ger S, Naschberger E, Herrmann C

& St u€ rzl M (2010) Intracellular trafﬁcking of guanylate-

binding proteins is regulated by heterodimerization in a

hierarchical manner. PLoS ONE 5, e14246.

a-helices. FEBS J 279, 2544–2554.

3

4 Ince S, Kutsch M, Shydlovskyi S & Herrmann C (2017)

The human guanylate-binding proteins hGBP-1 and

hGBP-5 cycle between monomers and dimers only.

FEBS J 284, 2284–2301.

5 Wehner M, Kunzelmann S & Herrmann C (2012) The

guanine cap of human guanylate-binding protein 1 is

responsible for dimerization and self-activation of GTP

hydrolysis. FEBS J 279, 203–210.

6 Shydlovskyi S, Zienert AY, Ince S, Dovengerds C,

Hohendahl A, Dargazanli JM, Blum A, G u€ nther SD,

Kladt N, St u€ rzl M et al. (2017) Nucleotide-dependent

farnesyl switch orchestrates polymerization and

membrane binding of human guanylate-binding protein

43 Naschberger E, Bauer M & St u€ rzl M (2005) Human

guanylate binding protein-1 (hGBP-1) characterizes and

establishes a non-angiogenic endothelial cell activation

phenotype in inﬂammatory diseases. Adv Enzyme Regul

45, 215–227.

44 Sistemich L, Kutsch M, H €a misch B, Zhang P,

Shydlovskyi S, Britzen-Laurent N, St u€ rzl M, Huber K

& Herrmann C (2020) The molecular mechanism of

polymer formation of farnesylated human guanylate-

binding protein 1. J Mol Biol. 432 :2164–2185.

45 Grek S, Davis J & Blaber M (2001) An efﬁcient,

ﬂexible-model program for the analysis of differential

scanning calorimetry protein denaturation data. Protein

Pept Lett 8, 429–436.

1

. Proc Natl Acad Sci USA 114, E5559–E5568.

3

4

7 Modiano N, Lu YE & Cresswell P (2005) Golgi targeting

of human guanylate-binding protein-1 requires nucleotide

binding, isoprenylation, and an IFN-gamma-inducible

cofactor. Proc Natl Acad Sci USA 102, 8680–8685.

8 Ji C, Du S, Li P, Zhu Q, Yang X, Long C & Yu J

(2019) Structural mechanism for guanylate-binding

proteins (GBPs) targeting by the Shigella E3 ligase

IpaH9.8. PLoS Pathog 15, e1007876.

Supporting information

9 Rani A, Pandita E, Rahman S, Deep S & Sau AK

(2012) Insight into temperature dependence of GTPase

Additional supporting information may be found

online in the Supporting Information section at the end

of the article.

Appendix S1. Oligonucleotide sequences for hGBP-1

mutants.

activity in human guanylate binding protein-1. PLoS

ONE 7, e40487.

0 Kutsch M, Ince S & Herrmann C (2018) Homo and

hetero dimerisation of the human guanylate-binding

proteins hGBP-1 and hGBP-5 characterised by afﬁnities

and kinetics. FEBS J 285, 2019–2036.

1

8

The FEBS Journal (2020) ª 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of

Federation of European Biochemical Societies

Article Doi

Catalytic activity of human guanylate-binding protein 1 coupled to the release of structural restraints imposed by the C-terminal domain

DOI: 10.1111/febs.15348

Source and publish data:

Authors:

Article abstract of DOI:10.1111/febs.15348

Full text of DOI:10.1111/febs.15348

Products guided by the article

R&D Labs maybe for 146-91-8

Relevant to this article

Hot Product