Molecular basis of the 14-3-3 protein-dependent activation of yeast neutral trehalase Nth1

Article abstract of DOI:10.1073/pnas.1714491114

The 14-3-3 proteins, a family of highly conserved scaffolding proteins ubiquitously expressed in all eukaryotic cells, interact with and regulate the function of several hundreds of partner proteins. Yeast neutral trehalases (Nth), enzymes responsible for the hydrolysis of trehalose to glucose, compared with trehalases from other organisms, possess distinct structure and regulation involving phosphorylation at multiple sites followed by binding to the 14-3-3 protein. Here we report the crystal structures of yeast Nth1 and its complex with Bmh1 (yeast 14-3-3 isoform), which, together with mutational and fluorescence studies, indicate that the binding of Nth1 by 14-3-3 triggers Nth1’s activity by enabling the proper 3D configuration of Nth1’s catalytic and calcium-binding domains relative to each other, thus stabilizing the flexible part of the active site required for catalysis. The presented structure of the Bmh1:Nth1 complex highlights the ability of 14-3-3 to modulate the structure of a multidomain binding partner and to function as an allosteric effector. Furthermore, comparison of the Bmh1:Nth1 complex structure with those of 14-3-3:serotonin N-acetyltransferase and 14-3-3:heat shock protein beta-6 complexes revealed similarities in the 3D structures of bound partner proteins, suggesting the highly conserved nature of 14-3-3 affects the structures of many client proteins.

Full text of DOI:10.1073/pnas.1714491114

Molecular basis of the 14-3-3 protein-dependent
activation of yeast neutral trehalase Nth1
a
a,b
c
c
d
a,1
Miroslava Alblova , Aneta Smidova , Vojtech Docekal , Jan Vesely , Petr Herman , Veronika Obsilova
and Tomas Obsil
,
a,b,1
a
Department of Structural Biology of Signaling Proteins, Division Biotechnology and Biomedicine Center of the Academy of Sciences and Charles University
b
in Vestec (BIOCEV), Institute of Physiology, The Czech Academy of Sciences, Prague 14220, Czech Republic; Department of Physical and Macromolecular
Chemistry, Faculty of Science, Charles University, Prague 12843, Czech Republic; Department of Organic Chemistry, Faculty of Science, Charles University,
Prague 12843, Czech Republic; and Institute of Physics, Faculty of Mathematics and Physics, Charles University, Prague 12116, Czech Republic
c
d
Edited by Fred Dyda, National Institutes of Health, and accepted by Editorial Board Member Kiyoshi Mizuuchi September 29, 2017 (received for review August
1
6, 2017)
The 14-3-3 proteins, a family of highly conserved scaffolding proteins
ubiquitously expressed in all eukaryotic cells, interact with and
regulate the function of several hundreds of partner proteins. Yeast
neutral trehalases (Nth), enzymes responsible for the hydrolysis of
trehalose to glucose, compared with trehalases from other organ-
isms, possess distinct structure and regulation involving phosphor-
ylation at multiple sites followed by binding to the 14-3-3 protein.
Here we report the crystal structures of yeast Nth1 and its complex
with Bmh1 (yeast 14-3-3 isoform), which, together with mutational
and fluorescence studies, indicate that the binding of Nth1 by 14-3-
One of the enzymes regulated in the 14-3-3-dependent
manner is yeast neutral trehalase 1 (Nth1). Trehalases (EC
3
.2.1.28) are glycoside hydrolases that catalyze the hydrolysis of
the nonreducing disaccharide trehalose (α-D-glucopyranosyl α-D-
glucopyranoside) as the sole substrate. Trehalose is found in
many organisms ranging from bacteria and fungi to invertebrates
and higher plants, where it fulfills a number of physiological roles
(
14, 15). In yeast, trehalose is a major storage carbohydrate,
carbon source, and stress protectant, and its hydrolysis is cata-
lyzed mainly by neutral trehalase with a pH optimum of about 7,
encoded by two genes, NTH1 and NTH2, with NTH1 being more
active (16). Compared with trehalases from prokaryotic and
higher eukaryotic organisms, yeast neutral trehalases possess an
N-terminal extension that contains four cAMP-dependent pro-
tein kinase (PKA) phosphorylation sites (S20, S21, S60, and
S83 in Saccharomyces cerevisiae Nth1) and the calcium-binding
EF-hand-like motif (Fig. 1A), indicating significantly different
regulation (17–19). It was subsequently shown that S. cerevisiae
Nth1 is indeed activated in a PKA- and calcium-dependent
manner in a process that requires interaction with the 14-3-3
protein through motifs containing phosphoserines pS60 and
pS83 (20–23).
3
triggers Nth1’s activity by enabling the proper 3D configuration of
Nth1’s catalytic and calcium-binding domains relative to each other,
thus stabilizing the flexible part of the active site required for catal-
ysis. The presented structure of the Bmh1:Nth1 complex highlights
the ability of 14-3-3 to modulate the structure of a multidomain
binding partner and to function as an allosteric effector. Further-
more, comparison of the Bmh1:Nth1 complex structure with those
of 14-3-3:serotonin N-acetyltransferase and 14-3-3:heat shock pro-
tein beta-6 complexes revealed similarities in the 3D structures of
bound partner proteins, suggesting the highly conserved nature of
14-3-3 affects the structures of many client proteins.
14-3-3 protein
| trehalase | crystal structure | enzyme | allostery
he 14-3-3 proteins, a family of highly conserved dimeric
proteins ubiquitously expressed in all eukaryotic cells, interact
with and regulate the function of several hundred partner pro-
teins by recognizing phosphoserine- (pS) or phosphothreonine
pT)-containing motifs (1, 2). Mechanistically, 14-3-3 proteins act
Significance
T
14-3-3 proteins are conserved scaffolding proteins expressed in
all eukaryotic cells, where they regulate the function of several
hundreds of partner proteins by constraining their conforma-
tion. Yeast neutral trehalases (Nth), enzymes responsible for the
hydrolysis of trehalose, compared with trehalases from other
organisms, possess distinct structure and regulation involving
phosphorylation followed by binding to 14-3-3. Here we pre-
sent the crystal structures of yeast Nth1 and its complex with
the 14-3-3 protein and propose a molecular mechanism in which
(
as allosteric regulators and/or molecular scaffolds that constrain
the conformation of the binding partner; if the target protein is an
enzyme, this can affect its catalytic activity. Many enzymes in-
cluding c-Raf protein kinase (3), serotonin N-acetyltransferase
(
AANAT) (4), tyrosine and tryptophan hydroxylases (5, 6), apo-
ptosis signal-regulated protein kinase 1 (7), and Cdc25 phospha-
tases (8) have been shown to be regulated in such a manner.
Nonetheless, the underlying molecular mechanisms are only par-
tially identified, mainly as a result of the lack of structural data.
Only one high-resolution structure of the complex between 14-3-3
and a fully active enzyme, AANAT, has been reported so far (9).
This structure showed that regions of AANAT that interact with
14-3-3 activates Nth1 by stabilizing the flexible part of its active
site. Comparison of the 14-3-3:Nth1 complex structure with
those of other 14-3-3 complexes suggests the highly conserved
nature of 14-3-3 affects the structures of many client proteins.
Author contributions: V.O. and T.O. designed research; M.A., A.S., V.D., J.V., P.H., V.O.,
and T.O. performed research; M.A., A.S., V.D., J.V., P.H., V.O., and T.O. analyzed data; and
V.O. and T.O. wrote the paper.
1
1
4-3-3 include those involved in substrate binding, suggesting that
4-3-3 structurally modulates the active site of bound AANAT,
and thus increases its substrate binding affinity with an accompa-
nying increase in catalytic activity. Besides the direct structural
modulation, 14-3-3 proteins can also stabilize the tertiary and/or
quaternary structure of the bound enzyme (10, 11) or change its
subcellular localization (8, 12). In addition, 14-3-3 can also mediate
the interaction between two different proteins, as has been dem-
onstrated for the florigen activation complex composed of florigen
homolog Hd3a, plant 14-3-3 protein GF14c, and a peptide com-
prising the nine carboxyl-terminal amino acids of phosphorylated
transcription factor FD1 (13).
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. F.D. is a guest editor invited by the Editorial
Board.
Published under the PNAS license.
Data deposition: The atomic coordinates and structure factors have been deposited in the
Protein Data Bank, www.wwpdb.org (PDB ID codes 5N6N, 5JTA, 5M4A, and 5NIS).
1
To whom correspondence may be addressed. Email: veronika.obsilova@fgu.cas.cz or
obsil@natur.cuni.cz.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1714491114/-/DCSupplemental.
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Fig. 1. Overview of pNth11–751:Bmh1 structure. (A) Domain structure of yeast Nth1. The N-terminal extension and the calcium-binding domain (Nth1-CaBD)
are shown in cyan, the catalytic domain (Nth1-CD) in dark cyan. The positions of PKA phosphorylation sites which are also 14-3-3 binding motifs are indicated
with red arrows. The position of the EF-hand-like motif is indicated with its sequence. Residues involved in calcium ion coordination are shown in orange.
Numbering is according to Nth1 from S. cerevisiae. (B) Structure of the pNth11–751:Bmh1 complex. The protomers of the Bmh1 homodimer are shown in
yellow and brown. The N-terminal extension and the calcium-binding domain (Nth1-CaBD) are shown in cyan, the catalytic domain (Nth1-CD) in dark cyan.
The phosphorylated S60 and S83 are shown in red. The calcium ion is shown in orange. (C) Close-up view of calcium ion-binding site.
To understand how the ability to interact with the 14-3-3 protein teraction with the Bmh1 dimer, which simultaneously interacts with
changed the structure and regulation of yeast neutral trehalase, we both 14-3-3 binding motifs of pNth11–751 (Fig. 1B; the Greek letters
determined the crystal structures of phosphorylated full-length
S. cerevisiae Nth1 (pNth11–751) bound to yeast 14-3-3 protein
Bmh1 and the catalytic domain of Nth1 (Nth1153–751) alone. The
crystallographic analysis, together with mutational analysis and
identify the helices of Nth1 and capital letters those of Bmh1). The
main-chain conformation of the first 14-3-3 binding motif of
60
pNth11–751 (sequence RTRTMpS VFDN), the pS60 coordination,
and other contacts in the 14-3-3 binding groove are similar to those
activity- and time-resolved fluorescence measurements, provides a previously seen in other complexes of 14-3-3 proteins (9, 25). The
useful clue for elucidating the molecular mechanism of the 14-3-3- Bmh1 side chains coordinating the pS60 moiety of pNth11–751 in-
dependent regulation of yeast Nth1. In addition, comparison of clude R58, R132, and Y133 (Fig. 2A). An Arg residue of pNth11–751
the complex structure with other 14-3-3 complexes suggests that at the −5 position relative to the phosphorylated residue pS60 is
the highly conserved nature of 14-3-3 impacts the 3D structures of salt-bridged to E136 and E185 of Bmh1 and the phosphate group
many of their binding partners.
of pS60, and thus similarly to the Arg residue at positions −4 and
−
2 in complexes of the phosphopeptide with the “mode 2” consensus
Results
sequence and AANAT, respectively (9, 25) (Fig. 3). Interactions
between pNth11–751 residues D63 and N64 at the +3 and +4
positions and Bmh1 residues K51 and N52 appear to force a
change in the direction of the polypeptide chain, mimicking the
role of the Pro residue, which is frequently found in 14-3-3 binding
motifs at the +2 position relative to the phosphorylated residue (9,
Overall Structure of the pNth11–751:Bmh1 Complex. Previous work dem-
onstrated that a dimer of Bmh1 binds one molecule of pNth11–751
(23). Thus, for crystallization trials, pNth11–751 and Bmh1 were mixed
in a 1:2 molar ratio. The structure of the complex was solved at a
resolution of 2.29 Å by molecular replacement, using the structures
of the N-terminally truncated Nth1 (Nth1153–751) and 14-3-3e (24) as
search models (SI Appendix, Table S1). The crystal structure of the
Bmh1:pNth11–751 complex shows pNth11–751 in a bidentate in-
25–28). The second 14-3-3 binding motif of pNth1
1
–751
(sequence
83
RRGpS ED) also does not contain a Pro residue at the
+2 position relative to the phosphorylated residue (Fig. 2B).
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Fig. 2. Contacts between Bmh1 and the 14-3-3 binding motifs of pNth11–751. (A) Contacts between Bmh1 and the first 14-3-3 binding motif of pNth11–751
containing pS60. The Nth1 residues are labeled in cyan, the Bmh1 residues are labeled in black. (B) Contacts between Bmh1 and the second 14-3-3 binding
motif of pNth11–751 containing pS83. The Nth1 residues are labeled in cyan, the Bmh1 residues are labeled in black. (C) Contacts between Bmh1 and the
intervening linker sequence between the two 14-3-3 binding motifs of pNth11–751. The omit 2F − Fc electron density map is contoured at 1σ. The
0
Nth1 residues are labeled in cyan, the Bmh1 residues are labeled in black.
Whereas the pS83 moiety is coordinated similar to that of pS60 in a loop between α3 and β2 (SI Appendix, Fig. S1). The calcium
by the side-chains of Bmh1 residues K51, R58, R132, and Y133, ion is coordinated in a pentagonal bipyramidal configuration by
residue R81 of pNth11–751 at the −2 position relative to pS83 is salt-
bridged to D85 at the +2 position, and R80 at the −3 position in-
teracts with the side-chain of Bmh1 residue N229. Interestingly, the
salt bridge between pNth11–751 residues R81 and D85 is responsible
the side-chains of residues D114, D116, N118, and D125; the
main-chain carbonyl group of Q120; and a water molecule (Fig.
1C). The side chains of D114, D116, N118, and D125 and the
main chain of Q120 are 2.24, 2.29, 2.38, 2.27, and 2.29 Å, re-
2+
for an abrupt change in the direction of the polypeptide chain (Figs. spectively, from the Ca ion. Nth1-CaBD is embedded within the
2
B and 3B), thus again mimicking the role of the Pro residue at the
central channel of the Bmh1 dimer, where it borders both phos-
+
2 position (9, 25, 27, 28). The intervening linker sequence between phorylated motifs and interacts with Bmh1 helices A1, A3, and
the two 14-3-3 binding motifs of pNth11–751 (residues 64−79) is or-
dered compared with known structures of 14-3-3 protein complexes,
most likely a result of numerous interactions with helices A1 and
A3 of Bmh1 (Fig. 2C).
A9, as well as the loop between A1 and A2 through numerous
direct and water-mediated contacts. In terms of Nth1-CaBD,
major contributions to this interface arise from the surface of
helix α2, strands β2 and β3, and loops between β2 and β3 and
The structure of the calcium-binding domain of pNth11–751 between α4 and β4 (Fig. 4A).
Nth1-CaBD, residues 96−176) is composed of three α helices and The catalytic domain of pNth11–751 (Nth1-CD, residues 180−751)
(
two antiparallel β-sheets, each composed of two strands with the is positioned on the top of the Bmh1 central channel, where it in-
1
14
125
EF-hand-like motif (sequence D TDKNYQITIED ) located teracts with helices A8 and A9 from the C terminus of one
Fig. 3. Comparison of the Bmh1:pNth11–751, 14-3-3ζ:phosphopeptide, 14-3-3ζ:AANAT, and 14-3-3σ:heat shock protein beta-6 complexes. (A) Comparison of
60
the main-chain conformation of the first 14-3-3 binding motif of pNth1 (sequence RTRTMpS VFDN, shown in cyan) with a “mode 2” 14-3-3 peptide (se-
quence RLYHpSLPA, PDB ID 1QJA, shown in green) (25) and the 14-3-3 binding motifs of AANAT (sequence QRRHpTLPA, PDB ID 1IB1, shown in orange) (9)
and heat shock protein beta-6 (sequence LRRApSAPL, PDB ID 5LTW, shown in red) (27). The C-terminal portion of the 14-3-3 binding motifs is indicated by
83
black ellipse. (B) Comparison of the main-chain conformation of the second 14-3-3 binding motif of pNth1 (sequence QTRRGpS EDDT, shown in cyan) with
83
the same complexes as in A. (C and D) Comparison of the first 14-3-3 binding motif of pNth1 (sequence QTRRGpS EDDT, shown in cyan) with a “mode 2” 14-
-3 peptide [sequence RLYHpSLPA, PDB ID 1QJA (25), shown in green] and the 14-3-3 binding motif of AANAT [sequence QRRHpTLPA, PDB ID 1IB1 (9), shown
3
in orange]. The numbers describe the position relative to pS/pT.
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Bmh1 protomer and the helix α4 and the EF-hand-like motif of
suggesting it is not caused by the absence of a bound ligand (Fig.
Nth1-CaBD. Nth1-CD contributes to this region of the interface via 5B). The “lid” loop of Bmh1-bound pNth11–751 is structured likely as
the loop preceding the helix α6, the C-terminal end of helix α28, the
loop region between α28 and α29, and helices α30 and α31 from the
C terminus of the domain (Figs. 1B and 4B). The total solvent-
excluded surface area between the Bmh1 dimer and pNth11–751 is
,955 Å , of which ∼81% (2,384 Å ) is contributed by the phos- actions appear to stabilize the bent conformation of this loop, thus
phorylated N-terminal segment and Nth1-CaBD (residues 54−176), placing its tip onto the active site pocket. To investigate whether these
whereas the rest is contributed by Nth1-CD. Although just ∼19% of interactions are also present in the absence of Bmh1, we crystallized
a result of interactions with the EF-hand-like motif-containing region
of Nth1-CaBD, including the salt bridge between E124 and R686 and
hydrogen bonds between the side-chain of Q120 and main-chain
carbonyl groups of residues A696 and F698 (Fig. 5C). These inter-
2
2
2
the interface between pNth11–751 and Bmh1 arises from Nth1-CD,
the extensive interactions between Nth1-CD and the rest of the
complex enable a stable and well-defined 3D configuration of this
domain relative to Nth1-CaBD and Bmh1.
an Nth1 construct composed of residues 100−751, thus containing
both Nth1-CaBD and Nth1-CD (SI Appendix, Table S1). However,
this structure only exhibited a continuous electron density for resi-
dues 180−682 and 703−751 of Nth1-CD, and no density was seen for
Nth1-CaBD and residues 683−702 from the “lid” loop. This suggests
that in the absence of Bmh1, the calcium-binding and catalytic do-
mains of Nth1 are not engaged in a stable protein–protein in-
teraction, and as a result, the “lid” loop is disordered similarly as
observed in the structures of Nth1153–751 and Nth1153–751:TRE.
The superimposition of Nth1-CD of the Bmh1 bound pNth11–751
on the crystal structure of the periplasmic trehalase Tre37A from
Escherichia coli (Tre37A) with the bound inhibitor validoxylamine
A (VDM) (30), which consists of only the catalytic domain and
lacks the N-terminal extension, yielded a r.m.s. deviation value of
The structure of the Bmh1 dimer in the complex is very similar
to the recently reported structure of Bmh1 from Lachancea
thermotolerans (29); the two structures can be aligned over 393
Cα atoms with a r.m.s. deviation of 1.26 Å. The most significant
differences involve the C-terminal helices A7–A9, most likely
because of the interaction of helices A7 and A9 with pNth11–751
.
Structure of the Catalytic Domain of Nth1. The structure of Nth1-CD
consists of an (α/α) barrel found in all enzymes belonging to
the six-hairpin glycosidases superfamily of an α/α-toroidal fold. The
6
active site is located in a deep pocket formed by the loops of the 1.25 Å over 337 Cα atoms and revealed a similar arrangement of
α/α)
and II, which are inserted within the N-terminal half of the (α/α)
(
6
barrel (shown in yellow in Fig. 5A) and two subdomains, I helices of the (α/α) barrel (SI Appendix, Fig. S2A). In contrast,
6
6
significant differences exist in both N-terminal subdomain struc-
tures as well as the C-terminal domain, which consists of three
barrel (shown in cyan and green, respectively, in Fig. 5A), and
contains a molecule of sucrose (SUC), which was present in the α-helices in Nth1-CD (α30−32, shown in blue in Fig. 5A), whereas
crystallization solution as an additive. The structure of Nth1-CD Tre37A possesses no helices in this region (SI Appendix, Figs.
of Bmh1-bound pNth11–751 is very similar to the structure of the S1 and S2A). Interestingly, the “lid” loop of Tre37A is ordered
N-terminally truncated Nth1153–751, which is composed of only the and adopts a similar conformation to that only observed in Bmh1-
catalytic domain (Fig. 5B); the two structures can be aligned over bound pNth11–751 (SI Appendix, Fig. S2B). The different behav-
5
26 Cα atoms with an r.m.s. deviation of 0.54 Å. The only dif- iors of the “lid” loops of Tre37A and yeast Nth1 likely result from
ference between these two structures involves the long loop region their distinct sequences and lengths (Fig. 5D). The “lid” loop of
located between α28 and α29, which in complexed pNth11–751
folds back onto the active site and covers it as a “lid” (hereafter
referred to as the “lid” loop). In contrast, in the Nth1153–751
structure, the C-terminal portion of this “lid” loop (residues
Tre37A is significantly shorter and contains a Gly-rich sequence,
and its position is mainly stabilized by contacts with bordering
loop regions (SI Appendix, Fig. S2C).
6
85−700) is disordered, presumably because of its flexibility.
Substrate Binding and the Active Site Structure. The cocrystal structure
Furthermore, the same part of the “lid” loop is also disordered in of the N-terminally truncated, and thus catalytically inactive,
the structure of Nth1153–751 with bound trehalose (Nth1153–751:TRE),
Nth1153–751 with trehalose showed a clear and interpretable electron
Fig. 4. Other Bmh1:pNth11–751 interactions. (A) Close-up view of interactions between Nth1-CaBD (light cyan) and Bmh1 (yellow and brown). (B) Close-up
view of interactions between the C-terminal helices A8 and A9 of Bmh1 (brown) with Nth1-CaBD (light cyan) and Nth1-CD (dark cyan).
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Fig. 5. Structure of catalytic domain of Nth1. (A) Structure of catalytic domain of Bmh1-bound pNth11–751. The (α/α)
C-terminal helices are shown in yellow, cyan, green, and blue, respectively. SUC is shown in gray. The C-terminal portion of the “lid” loop is shown in red.
The rest of the structure is not shown. (B) Superimposition of the catalytic domain of Bmh1-bound pNth11–751 (blue), Nth1153–751 (green), and Nth1153–751
6
barrel, subdomains I and II, and the
:
TRE (orange). The C-terminal portion of the “lid” loop of pNth11–751 is shown in red. (C) Close-up view of interactions between the “lid” loop and the EF-
hand-like motif of Nth1-CaBD. (D) Sequence alignment of “lid” loops of yeast (S. cerevisiae) Nth1 and trehalase Tre37A from E. coli. The C-terminal
portions of the “lid” loops are shown in red. (E) Close-up view of Nth1153–751 active site with bound trehalose (TRE). (F) Close-up view of active site of
trehalase Tre37A from E. coli with bound inhibitor VDM (30).
density for the bound ligand (TRE) in the −1 and +1 subsites of the
active site (Fig. 5E). The glucose moieties in the catalytic −1 and
Tre37A with bound VDM, this residue (E496) is hydrogen bonded
to a water molecule that is responsible for a nucleophilic attack of
the glycoside (Fig. 5F). Although the resolution of the Nth1153–751:
4
1
the leaving-group +1 subsites adopt C
1
and C
4
chair conforma-
tions, respectively, and form contacts that are similar to those
observed between Tre37A and VDM (Fig. 5F), with trehalose
occupying the same position as VDM (30). Comparison of the
Nth1153–751:TRE structure with that of Tre37A suggests that
TRE structure did not enable us to assign water molecules, the
sequence and structural similarities between Nth1-CD and Tre37A
(SI Appendix, Figs. S1 and S2A) strongly suggest that E674 in
Nth1 plays the same role as E496 in Tre37A. This was further
Nth1 residues D478 (D312 in Tre37A) and E674 (E496 in corroborated by site-directed mutagenesis, which revealed that
Tre37A) function as the catalytic acid and base, respectively. Their
both D478A and E674A mutants of pNth11–751 bound to Bmh1 are
side-chains are located 13 Å apart, with D478 being a hydrogen catalytically inactive (SI Appendix, Fig. S3). The structures of apo
bond distance (3.7 Å) from the glycosidic oxygen atom of treha- and TRE-bound forms of Nth1153–751 are highly similar and can be
lose, whereas E674 is located 7.8 Å from the C1 atom of the
glucose moiety in the −1 subsite (Fig. 5E). In the structure of
aligned with an overall r.m.s. deviation at 542 Cα positions of
0.72 Å. The only structural difference between the two forms of
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Nth1153–751 occurs in the vicinity of the active site, where the Although the glucose moiety of SUC occupies a similar position as
substrate binding induces a shift of the surrounding loop regions
toward the active site (Fig. 5B). The rest of the catalytic domain
exhibited a nearly identical conformation.
the glucose moiety of TRE in the catalytic −1 subsite, the fructose
moiety of SUC is located above the leaving-group +1 subsite where
the second glucose moiety of TRE is bound, thus forcing the tip of
the “lid” loop to shift further away from the active site (SI Ap-
pendix, Fig. S2B). The functional importance of Nth1 residues
Implications for Activation. The previous structural work on Tre37A
from E. coli indicated that residues E511 and Y512 from the tip of E690 and Y691 was confirmed by a mutational analysis of pNth11–751
,
the “lid” loop might be directly involved in substrate binding and
which revealed that the E690A mutant exhibits significantly re-
duced activity and the Y691A mutant is catalytically inactive (SI
bonded to the pseudosugar ring of VDM in the +1 subsite, Appendix, Fig. S3).
catalysis, respectively (30). The side-chain of E511 is hydrogen
whereas the side-chain of Y512 was suggested to impede the
attacking water molecule, thus directly participating in catalysis
The structure of pNth11–751 bound to Bmh1 suggested that the
“lid” loop conformation is stabilized through interactions with
the EF-hand-like motif of Nth1-CaBD (Fig. 5C). Moreover, the
(Fig. 5F). Furthermore, this pair of residues (EY) is totally con-
served among 101 trehalase sequences available in the UniProt structure of Nth1100–751 indicated that in the absence of Bmh1,
database (SI Appendix, Fig. S4A), further supporting their func-
the calcium-binding and catalytic domains of Nth1 are not en-
tional importance. The corresponding residues of yeast Nth1 gaged in a stable protein–protein interaction. This indicates that
(
E690 and Y691) are, in contrast to the structure of Tre37A,
contacts between the “lid” loop and Nth1-CaBD, which appear
to be mediated by Bmh1 binding, might be important for the
activation of pNth1. To investigate this hypothesis, we performed
site-directed mutagenesis of residues Q120, E124, and R686
hydrogen-bonded to subdomain II residues T479 and E426, re-
spectively (Fig. 5C). This difference is likely because the active site
of pNth11–751 contains SUC instead of its natural substrate TRE.
Fig. 6. Time-resolved fluorescence experiments with DANS-TRE. (A) DANS-TRE. (B) Four tryptophan residues (W309, W624, W630, and W708) are located in
close proximity to the substrate binding site of yeast Nth1. The molecule of trehalose (TRE) was modeled by superimposing the structure of Nth1153–751:TRE
with Nth1-CD of Bmh1-bound pNth11–751. (C and D) Statistical confidence-interval analysis of intensity fraction of long fluorescence decay component cor-
responding to bound DANS-TRE in the presence of pNth11–751(E674A) (white), Bmh1 (light gray), and the pNth11–751(E674A):Bmh1complex (dark gray), with
DANS-TRE excited at 290 (C) and 355 nm (D). Histograms represent the probability of recovering a particular intensity fraction from measured data as a result
of 2,000 bootstrap fitting cycles (52). Dashed lines border 68% confidence intervals (1 SD). The histograms reveal that the intensity fraction of bound DANS-
TRE is significantly higher in the presence of the pNth11–751(E674A):Bmh1complex compared with the control samples. The significantly increased bound-
intensity fraction on excitation at 290 nm compared with excitation at 355 nm is likely a result of a combined effect of DANS-TRE FRET facilitated by Trp–
DANS-TRE proximity in the binding pocket of the pNth11–751(E674A):Bmh1complex and blue shift of the absorption spectrum of bound DANS-TRE.
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involved in these interactions. Indeed, the enzyme activity
measurements showed that mutations, especially Q120A and
R686A, significantly reduced the catalytic activity of the Bmh1-
bound pNth11–751 (SI Appendix, Fig. S3). In addition, the fact
that in the presence of calcium, the Bmh1-mediated activation of
pNth1 is significantly enhanced further supports this hypothesis, as
the calcium binding likely facilitates these interactions by inducing
folding of the EF-hand-like motif. Taken together, these results
indicate that residues E690 and Y691 from the tip of the “lid” loop,
as well as interactions between the “lid” loop and Nth1-CaBD, are
surrounding the binding site. The effect is also visible from the raw
decays presented in SI Appendix, Fig. S5A. The formation of the
Bmh1:pNth11–751(E674A) complex is accompanied by a significant
increase in the long-decaying component that is visibly above the
noise level. The decay of DANS-TRE alone is omitted for clarity,
as it is almost indistinguishable from the decay in the presence of
pNth11–751(E674A) (SI Appendix, Fig. S5, green curve).
Fluorescence decays of DANS-TRE excited at 355 nm are pre-
sented in SI Appendix, Fig. S5B and Table S3. The observed effects
qualitatively agree with the results for 290 nm excitation de-
scribed earlier. As expected, the measured effect of Bmh1 is smaller
without FRET sensitization, and its significance is also lower.
Finally, we rigorously examined the statistical confidence limits of
changes extracted for the 14.3-ns intensity fraction with 290 nm (Fig.
6C) and 355 nm excitation (Fig. 6D). Histograms represent the
probability of recovering a particular intensity fraction from the
measured data resulting from 2,000 bootstrap fitting cycles (36).
Dashed lines denote 68.2% confidence intervals (±1 SD). The histo-
grams reveal that for both excitations, the intensity fraction of DANS-
TRE bound to the Bmh1:pNth11–751(E674A) complex is significantly
higher than in control samples. This further supports our conclusion
that the binding of Bmh1 induces conformational changes of
pNth11–751(E674A) involving the region close to the substrate
binding site.
essential for the catalytic activity of Bmh1-bound Nth11–751
.
Bmh1 Binding Affects the Conformation of the Active Site of pNth1.
To further investigate the conformational behavior of the “lid”
loop of pNth11–751 in the absence and the presence of Bmh1, we
synthetized 6-[5′-(dimethylamino)-1′-naphthalenesulfonamido]-
6
-deoxy-trehalose (DANS-TRE; Fig. 6A), consisting of a dansyl
group attached at position 6 of one glucose moiety of TRE, and
performed time-resolved fluorescence measurements. We de-
cided to modify the hydroxyl group at position 6 of TRE as it faces
outward from the active site pocket toward the “lid” loop (Fig. 6B).
The dansyl moiety is highly sensitive to solvent polarity and serves
as an excellent fluorescent probe for sensing subtle changes in the
local microenvironment by significantly increased quantum yield
and emission lifetime in a less polar environment (31). To avoid
changes in the substrate concentration during experiments, an
Discussion
enzymatically inactive mutant of pNth11–751(E674A) was chosen The yeast Nth1, compared with trehalases from prokaryotic and
for these experiments. A series of time-resolved experiments was
performed with excitation at 355 and 290 nm, which correspond to
the DANS-TRE and Trp absorption bands, respectively. The latter
experiment explored the possibility of sensitized emission from
higher eukaryotic organisms, exhibits distinct domain structure and
regulation, as its activity is triggered by association with the 14-3-3
protein, which recognizes two phosphorylated motifs within the
N-terminal extension of Nth1 (20–23). Previous studies have sug-
bound DANS-TRE facilitated by Förster resonance energy trans- gested an allosteric mechanism, whereby 14-3-3 protein binding
fer (FRET) from W309, W624, W630, and W708, located near the
affects the structure of the catalytic domain of pNth11–751, with
Nth1-CaBD (the EF-hand-like motif-containing domain) being the
intermediary between 14-3-3 and the catalytic domain (37, 38).
binding site (Fig. 6B). These Trp donors are located within a
0
sphere with a radius of 2R = 42 Å (32) (R0, critical Förster dis-
tance) from the bound DANS-TRE, which makes the FRET Indeed, the crystal structure of the Bmh1:pNth11–751 complex
feasible. Because FRET efficiency is highly sensitive to the distance reported in this study revealed that Bmh1 extensively interacts with
and mutual orientation of the donor-acceptor pair (33), Bmh1-
both Nth1-CaBD and Nth1-CD and enables their proper 3D
induced conformational changes of pNth11–751(E674A) involving configuration relative to each other (Fig. 1). This, in turn, appears
the region surrounding the binding site should affect FRET effi-
ciency and DANS-TRE emission.
to facilitate interactions between the EF-hand-like motif of Nth1-
CaBD and the flexible “lid” loop, which gets stabilized and com-
pletes the active site of pNth1 by providing side-chains important
for catalysis. The suggested mechanism of Nth1 activation was
corroborated by mutational analysis, which confirmed that residues
involved in contacts between the EF-hand-like motif of Nth1-
CaBD and the “lid” loop, as well as residues from the tip of the
“lid” loop, are required for the activation of pNth1 (Fig. 5C and SI
Appendix, Fig. S3). Moreover, time-resolved fluorescence mea-
surements with DANS-TRE further corroborated the suggested
mechanism by indicating conformational changes in the vicinity of
the active site of pNth1 induced by Bmh1 binding (Fig. 6).
Fluorescence decays of DANS-TRE in the presence of
pNth11–751(E674A), Bmh1, and the Bmh1:pNth11–751(E674A)
complex excited at 290 nm can be found in SI Appendix, Fig. S5A
and Table S2. As shown in SI Appendix, Table S2, DANS-TRE
alone decays close to a single exponential with a lifetime near
3
.3 ns with only minor contamination supposedly caused by im-
purities. After binding to the Bmh1:pNth11–751(E674A) complex,
DANS-TRE decay becomes heterogeneous. Apart from the
emission from free sugar, a new lifetime component of 14.3 ns
appears. This could be attributed to a bound fraction of DANS-
TRE sensing lower solvation and decreased microenvironmental
polarity in the binding pocket of the complex. The value is
The interaction between pNth11–751 and Bmh1 was previously
studied using hydrogen-deuterium exchange coupled to MS,
consistent with values commonly found for the dansyl moiety co- chemical cross-linking, and small-angle X-ray scattering (SAXS)
valently linked to globular proteins (34, 35). The fractional in-
tensity of the 14.3-ns component in the Bmh1:pNth11–751(E674A)
complex (4.2%) is significantly higher than in the presence of
pNth11–751(E674A) (0.5%) or Bmh1 alone (1.3%), where the
presence of the long component likely indicates some non-
specific interaction of DANS-TRE with Bmh1. Importantly, the
intensity fraction from DANS-TRE bound to the complex is 2.3×
higher than would result from the simple addition of corre-
sponding intensities from pNth11–751(E674A) and Bmh1 that
constitute the complex. Because both pNth11–751(E674A) and
Bmh1:pNth11–751(E674A) bind TRE with comparable affinities (SI
Appendix, Fig. S6), the observed increase is not caused by higher
saturation of the complex and might be assigned to a Bmh1-
(37, 38). Although the observed changes in deuteration profiles of
both pNth11–751 and Bmh1 are in a very good agreement with the
crystal structure of the Bmh1:pNth11–751 complex (SI Appendix,
Fig. S7A), the previously identified intermolecular cross-links ap-
pear to be incompatible with the conformation of the complex in
the crystal (SI Appendix, Fig. S7B). However, these intermolecular
cross-links were preferentially formed in the absence of calcium
and involved Bmh1 residues K76, K127, and K145 and pNth1
residues K214, K393, and K563. In contrast, in the presence of
calcium, the mentioned lysine residues of pNth1 preferentially
formed intramolecular cross-links within the catalytic domain
(cross-links K214–K563 and K258–K393). This might indicate that
in the presence of calcium, the EF-hand-like motif of pNth1 is
induced conformational change of the pNth11–751(E674A) region folded (Figs. 1B and 5C), thus providing optimal interactions with
Alblova et al.
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the catalytic domain with an accompanying increase in catalytic
activity (SI Appendix, Fig. S3). In contrast, in the absence of calcium,
when the region containing the EF-hand-like motif is likely
unfolded, the interaction between the catalytic domain and the rest
of the complex is less stable and the catalytic domain transiently
interacts with other surfaces of Bmh1, thus bringing lysine resi-
dues to a distance enabling their cross-linking. However, another
possibility for the formation of observed intermolecular cross-
links might be the presence of weak transient interactions that
resemble the crystal-packing contacts, as such interactions would
bring Cα atoms of cross-linked lysine residues of Bmh1 and
Nth1 at a distance of approximately 30 Å (SI Appendix, Fig. S7 C
and D). As a consequence, the previously published structural
model of Bmh1 with the catalytic domain of Nth1 is inaccurate
In conclusion, the structural analysis of yeast Nth1 alone and its
complex with Bmh1, together with mutational analysis and activity
and time-resolved fluorescence measurements, provides a struc-
tural basis for the 14-3-3 protein-mediated activation of Nth1. The
data presented here not only highlight the ability of 14-3-3 to
modulate the structure of a multidomain binding partner and to
function as an allosteric effector of the bound enzyme but also
suggest that the highly conserved nature of 14-3-3 proteins affects
the structures of many client proteins.
Experimental Procedures
Protein Preparation and Purification. DNA encoding full-length and two
N-terminally truncated versions of S. cerevisiae Nth1 (residues 1−751, 100−751,
and 153−751, respectively) were ligated into the pRSFDuet-1 vector (Novagen),
using the NcoI, BamHI, and NotI sites. Nth11–751, Nth1100–751, and Nth1153–751 were
expressed as N-terminal 6×His-tagged fusion proteins in E. coli BL21(DE3). Protein
expressions were induced by isopropyl β-D-1-thiogalactopyranoside for 18 h at
(38). The model, which was based on homology models of
Bmh1 and Nth1-CD (residues 295–721), distance restraints de-
rived from chemical cross-links discussed earlier and the fitting
into the low-resolution SAXS envelope, suggested a clearly in-
correct orientation of Nth1-CD with respect to Bmh1. The su-
perimposition of the Bmh1:pNth11–751 crystal structure with the
previously published SAXS envelope is shown in SI Appendix,
Fig. S8A. Because this SAXS envelope was obtained by refining
the averaged bead model, and thus it might not represent a
consensus model, we also compared the crystal structure of the
complex with the averaged filtered SAXS envelope representing
common features of all bead models used in shape reconstruc-
tion (39) (SI Appendix, Fig. S8B). The reasonable agreement of
the averaged filtered SAXS envelope with the overall shape of
the complex structure indicates similarity of the solution and
crystal structures of the Bmh1:pNth11–751 complex.
25 °C, and the proteins were purified using Chelating Sepharose Fast Flow (GE
Healthcare) according to a standard protocol. Proteins were dialyzed against
buffer 1 [20 mM Tris·HCl, 150 mM NaCl, 10 mM EDTA, 2 mM β-mercaptoethanol,
10% (wt/vol) glycerol at pH 7.5]. The affinity tag and GB1 domain were removed
by tobacco etch virus (TEV) protease cleavage for 2 h at 30 °C and overnight at
4
°C (250 U TEV/mg recombinant protein). After the cleavage, Nth11–751 was
purified by cation-exchange chromatography (HiTrap SP column; GE Healthcare).
The protein was eluted using a 50−1,000-mM gradient of NaCl in 50 mM citric
buffer (pH 6.0), 2 mM DTT. Four hours before phosphorylation, fractions con-
taining Nth11–751 were dialyzed against buffer 2 [20 mM Tris·HCl, 150 mM NaCl,
5
mM DTT, 10% (wt/vol) glycerol at pH 7.5]. Purified Nth11–751 was phosphory-
lated by incubation at 30 °C for 2 h and then overnight at 4 °C with 80 units PKA
(Promega) per milligram protein in the presence of 0.75 mM ATP and 20 mM
MgCl . The final purification step was size-exclusion chromatography (Superdex
2
200; GE Healthcare) in buffer 2. With the Nth1100–751 and Nth1153–751 variants, the
final purification step was size-exclusion chromatography (HiLoad Superdex 75;
GE Healthcare) in buffer containing 1 mM ammonium acetate, 150 mM NaCl,
Sequence alignment of neutral trehalases from four different
yeast species with trehalases from selected prokaryotic and higher
eukaryotic organisms revealed that yeast neutral trehalases possess
distinct “lid” loop sequences that are longer and do not contain a
Gly-rich region (SI Appendix, Fig. S4C, marked with the red box).
Furthermore, only yeast sequences possess the N-terminal extension
containing conserved PKA phosphorylation sites, which are also the
5
mM DTT, and 10% (wt/vol) glycerol at pH 7.3. Selenomethionine-substituted
Nth1153–751 was expressed using SelenoMet Medium Base and SelenoMet Nu-
trient Mix (Molecular Dimensions) and purified according to the same protocol.
All mutants were generated using a QuikChange site-directed mutagenesis kit
(Stratagene), and mutations were confirmed by sequencing. The stability of
prepared Nth1 mutants was checked by measuring the thermally induced protein
denaturation, using differential scanning fluorimetry. No significant differences
14-3-3 binding motifs (23), and the calcium-binding EF-hand-like
motif (SI Appendix, Fig. S4B, marked with green, blue and orange
boxes). In addition, residues involved in contacts between Nth1-
CaBD and the “lid” loop (Q120, E124, R686, marked with red
circles) and residues whose side-chains are involved in interactions
with Bmh1 (SI Appendix, Fig. S4 B and C, marked with blue circles)
are also highly conserved among yeast sequences. This indicates
that all yeast neutral trehalases are regulated in the 14-3-3 protein-
dependent manner.
m
in the temperature of the unfolding transition (T ) were observed for prepared
Nth1 mutants (SI Appendix, Table S4).
Bmh1 (C-terminally truncated version composed of residues 1−236) from S.
cerevisiae was expressed and purified as described previously (42). The N-terminal
6×His-tag was removed using TEV protease, and final size-exclusion chroma-
tography was performed in a HiLoad Superdex 75 column (GE Healthcare).
Crystallization and X-Ray Data Collection. All crystallizations were performed
by the hanging-drop vapor-diffusion method at 291 K. Crystals of native and
selenomethionine-labeled Nth1153–751 were grown from drops consisting of
2 μL of 100 μM protein and 1 μL of 100 mM sodium citrate (pH 5.6), 1 M
lithium sulfate, and 400 mM ammonium sulfate. Crystals of the Nth1153–751:
TRE complex were prepared by cocrystallization with 200 mM trehalose.
Both 14-3-3 binding motifs of pNth1 do not contain a Pro
residue at the +2 position relative to the phosphorylated residue
(
Fig. 2 A and B). This pSer +2 proline is frequently found in 14-
3
-3 binding motifs (26), and is required for introducing a kink in
Crystals of native Nth1100–751 were grown from drops consisting of 2 μL of
the peptide chain and thus directing the C-terminal portion back
into the central channel (25). In the case of the first pNth1 motif,
the change in the direction of the polypeptide chain is achieved
by interactions between pNth1 residues at the +3 and +4 posi-
tions and Bmh1 (Fig. 2A), whereas in the case of the second
motif, the kink is introduced by the intramolecular salt bridge
between pNth1 residues at the −2 and +2 positions (Fig. 2B). As
a result of these interactions, the C-terminal portions of both
pNth1 motifs leave the binding grooves in similar orientation as
polypeptide chains of other proteins and peptides including, for
example, AANAT (9), the heat shock protein beta-6 (27), and
short phosphopeptides (25, 28), that were cocrystalized with 14-
9
5 μM protein and 1 μL of 100 mM sodium citrate (pH 5.6), 1 M lithium
sulfate, and 400 mM ammonium sulfate. Initial crystals of the pNth11–751
:
Bmh1 complex (pNth11–751 and Bmh1 were mixed in a 1:2 molar ratio) were
obtained in condition #53 of the JCSG III screen (Qiagen) consisting of 0.16 M
calcium acetate, 80 mM sodium cacodylate (pH 6.5), 14.4% (wt/vol) PEG
8
000, and 20% (vol/vol) glycerol. This condition was further optimized by
using the Additive Screen (Hampton Research). Diffraction-quality crystals
were grown from drops consisting of 2 μL of 53 μM complex and 1 μL of
100 mM sodium cacodylate (pH 6.5), 200 mM calcium acetate, 18% (wt/vol)
PEG 8000, and 12% (wt/vol) sucrose.
All crystals were cryoprotected using 30% (vol/vol) glycerol and flash
frozen in liquid nitrogen before data collection in oscillation mode at
beamlines P13 of the DESY synchrotron and 14.1 of the BESSY synchrotron.
Diffraction data processing was carried out with the packages XDS and
XDSAPP (43, 44). The crystal structure of the pNth11–751:Bmh1 complex was
solved by molecular replacement in MOLREP (45), using the structures of
Nth1153–751 (described here) and PDB 2BR9 (24) as search models, and refined
at a resolution of 2.29 Å with PHENIX (46). Data collection and refinement
statistics are summarized in SI Appendix, Table S1. No electron density was
3
-3 and whose 14-3-3 binding motifs contain proline residue at
the +2 position (Fig. 3). The observed similarities in the 3D
structures of these 14-3-3 binding partners suggest that 14-3-3
proteins, which are highly conserved both in sequence and
structure (40, 41), provide a highly conserved evolutionary force
that affects the structures of many client proteins.
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present for residues 1−26, 38−53, and 437−447 of pNth1, and residues 209−
15 of one Bmh1 protomer, and these residues were not included in the
Microscale Thermophoresis. The microscale thermophoresis method was de-
scribed in detail elsewhere (50). The binding affinities of uncomplexed and
complexed pNth11–751(E674A) for trehalose were measured using the
Monolith NT.LabelFree instrument (NanoTemper Technologies). The solu-
tion of trehalose was serially diluted from 250 mM to 8 μM in the presence
of 1 μM Nth11–751(E674A) or 0.5 μM pNth11–751(E674A):Bmh1 complex (molar
ratio 1:2). All samples were loaded into standard treated zero background
capillaries (NanoTemper Technologies) after incubation at 23 °C for 20 min.
Measurements were performed at 23 °C in buffer 3 by using 20% LED power
and 40–60% IR-laser power. Three independent measurements were per-
formed for each sample, and the obtained data were analyzed using Ther-
mophoresis and the T-Jump signal, using the software MO.Affinity Analysis
(version 2.1.2; NanoTemper Technologies).
2
final model. The refined model had a MolProbity (47) score of 1.35.
The crystal structure of Nth1153–751 was solved by single-wavelength anom-
alous dispersion phasing with the data from a selenomethionine-labeled pro-
tein crystal diffracting to a resolution 3.13 Å. Single-wavelength anomalous
dispersion phasing and initial model building were performed with PHENIX
(46). The initial model consisting of residues 180−751 was then used to de-
termine the Nth1153–751 structure by molecular replacement of a higher-
resolution native dataset (SI Appendix, Table S1), using MOLREP (45). The
structure of apo Nth1153–751 was refined at 2.72 Å, using REFMAC (48), and the
refined model had a MolProbity (47) score of 2.13. No electron density was
present for residues 153−178 and 680−701, and these residues were not in-
cluded in the final model.
The Nth1153–751:TRE and Nth1100–751 structures were solved by molecular
replacement in MOLREP (45), using the structure of Nth1153–751 as the search
model and refined at 2.9 and 3.15 Å, respectively, using PHENIX (46) (SI
Appendix, Table S1). The refined models had MolProbity (47) scores of
Differential Scanning Fluorimetry. The differential scanning fluorimetry ex-
periments were performed using a real-time PCR LightCycler 480 II (Roche
Applied Science) according to the standard protocol, as described previously
(
51). The pNth11–751 concentrations were 2 μM in buffer 3.
1
1
1
.55 and 1.99, respectively. No electron density was present for residues 153−
79, 613−620, and 685−700 in the Nth1153–751:TRE structure and residues 100−
79, 617−618, and 683−702 in the Nth1100–751 structure, and these residues
Synthesis of DANS-TRE. The synthesis of DANS-TRE and all corresponding
NMR spectra are described in SI Appendix, SI Material and Methods.
were not included in the final model. All structural figures were prepared
with PyMOL (https://pymol.org/2/).
Time-Resolved Fluorescence Measurements and Data Analysis. A full description
of method for time-resolved fluorescence measurements and data analysis is
available in SI Appendix, SI Material and Methods.
Enzyme Activity Measurements. The trehalase activity of Nth1 and its mutants
were measured by estimating the glucose produced by the hydrolysis of
trehalose, using a stopped assay, as described earlier (23, 49). The assay was
ACKNOWLEDGMENTS. This work was supported by the Czech Science
Foundation (Projects 16-02739S and 17-00726S), the Czech Academy of
Sciences (Research Projects RVO: 67985823 of the Institute of Physiology) and
project BIOCEV (Biotechnology and Biomedicine Center of the Academy of
Sciences and Charles University in Vestec; CZ.1.05/1.1.00.02.0109), a project
from the European Regional Development Fund.
performed at 30 °C in buffer 3 [20 mM Tris·HCl, 150 mM NaCl, 10 mM CaCl
where needed), 10% (wt/vol) glycerol at pH 7.5] and 30 mM trehalose. The
final concentrations of Nth1 and Bmh1 (where needed) were 100 nM and
5 μM, respectively. The specific trehalase activity of Nth1 was determined as
micromol glucose liberated/min/mg protein.
2
(
1
1
2
3
4
5
. Wang W, Shakes DC (1996) Molecular evolution of the 14-3-3 protein family. J Mol
Evol 43:384–398.
. Muslin AJ, Tanner JW, Allen PM, Shaw AS (1996) Interaction of 14-3-3 with signaling
proteins is mediated by the recognition of phosphoserine. Cell 84:889–897.
. Freed E, Symons M, Macdonald SG, McCormick F, Ruggieri R (1994) Binding of 14-3-
21. Panni S, Landgraf C, Volkmer-Engert R, Cesareni G, Castagnoli L (2008) Role of 14-3-
3 proteins in the regulation of neutral trehalase in the yeast Saccharomyces cer-
evisiae. FEMS Yeast Res 8:53–63.
22. Schepers W, Van Zeebroeck G, Pinkse M, Verhaert P, Thevelein JM (2012) In vivo
phosphorylation of Ser21 and Ser83 during nutrient-induced activation of the yeast
protein kinase A (PKA) target trehalase. J Biol Chem 287:44130–44142.
23. Veisova D, et al. (2012) Role of individual phosphorylation sites for the 14-3-3-protein-
dependent activation of yeast neutral trehalase Nth1. Biochem J 443:663–670.
3
proteins to the protein kinase Raf and effects on its activation. Science 265:1713–1716.
. Ganguly S, et al. (2001) Role of a pineal cAMP-operated arylalkylamine N-acetyltransferase/
4-3-3-binding switch in melatonin synthesis. Proc Natl Acad Sci USA 98:8083–8088.
1
. Ichimura T, Isobe T, Okuyama T, Yamauchi T, Fujisawa H (1987) Brain 14-3-3 protein is
an activator protein that activates tryptophan 5-monooxygenase and tyrosine 3-
monooxygenase in the presence of Ca2+, calmodulin-dependent protein kinase II.
FEBS Lett 219:79–82.
2
4. Yang X, et al. (2006) Structural basis for protein-protein interactions in the 14-3-
protein family. Proc Natl Acad Sci USA 103:17237–17242.
3
25. Rittinger K, et al. (1999) Structural analysis of 14-3-3 phosphopeptide complexes
identifies a dual role for the nuclear export signal of 14-3-3 in ligand binding. Mol Cell
6
7
8
9
. Banik U, Wang GA, Wagner PD, Kaufman S (1997) Interaction of phosphorylated
tryptophan hydroxylase with 14-3-3 proteins. J Biol Chem 272:26219–26225.
. Zhang L, Chen J, Fu H (1999) Suppression of apoptosis signal-regulating kinase 1-induced
cell death by 14-3-3 proteins. Proc Natl Acad Sci USA 96:8511–8515.
. Kumagai A, Dunphy WG (1999) Binding of 14-3-3 proteins and nuclear export control the
intracellular localization of the mitotic inducer Cdc25. Genes Dev 13:1067–1072.
. Obsil T, Ghirlando R, Klein DC, Ganguly S, Dyda F (2001) Crystal structure of the 14-3-
4
:153–166.
2
2
2
6. Johnson C, et al. (2010) Bioinformatic and experimental survey of 14-3-3-binding sites.
Biochem J 427:69–78.
7. Sluchanko NN, et al. (2017) Structural basis for the interaction of a human small heat
shock protein with the 14-3-3 universal signaling regulator. Structure 25:305–316.
8. Molzan M, et al. (2010) Impaired binding of 14-3-3 to C-RAF in Noonan syndrome
suggests new approaches in diseases with increased Ras signaling. Mol Cell Biol 30:
3
zeta:serotonin N-acetyltransferase complex. A role for scaffolding in enzyme regu-
4
698–4711.
lation. Cell 105:257–267.
2
3
9. Eisenreichova A, Klima M, Boura E (2016) Crystal structures of a yeast 14-3-3 protein
from Lachancea thermotolerans in the unliganded form and bound to a human lipid
kinase PI4KB-derived peptide reveal high evolutionary conservation. Acta Crystallogr
F Struct Biol Commun 72:799–803.
0. Gibson RP, et al. (2007) Molecular basis for trehalase inhibition revealed by the
structure of trehalase in complex with potent inhibitors. Angew Chem Int Ed Engl 46:
1
0. Ottmann C, et al. (2007) Structure of a 14-3-3 coordinated hexamer of the plant
plasma membrane H+ -ATPase by combining X-ray crystallography and electron
cryomicroscopy. Mol Cell 25:427–440.
1
1
1
1
1
1
1
1. Yip-Schneider MT, et al. (2000) Regulation of the Raf-1 kinase domain by phos-
phorylation and 14-3-3 association. Biochem J 351:151–159.
2. Peng CY, et al. (1997) Mitotic and G2 checkpoint control: Regulation of 14-3-3 protein
binding by phosphorylation of Cdc25C on serine-216. Science 277:1501–1505.
3. Taoka K, et al. (2011) 14-3-3 proteins act as intracellular receptors for rice Hd3a flo-
rigen. Nature 476:332–335.
4. Crowe JH, Crowe LM, Chapman D (1984) Preservation of membranes in anhydrobiotic
organisms: The role of trehalose. Science 223:701–703.
5. Avonce N, Leyman B, Thevelein J, Iturriaga G (2005) Trehalose metabolism and glu-
cose sensing in plants. Biochem Soc Trans 33:276–279.
6. Wilson WA, et al. (2010) Regulation of glycogen metabolism in yeast and bacteria.
FEMS Microbiol Rev 34:952–985.
4
115–4119.
3
3
3
3
1. Lakowicz JR (2006) Principles of Fluorescence Spectroscopy (Springer, New York), 3rd
Ed, p 954.
2. Valeur B (2001) Molecular Fluorescence: Principles and Applications (Wiley-VCH, New
York), p 402.
3. Förster Th (1948) Zwischenmolekulare energiewanderung und fluoreszenz. Ann Phys
4
37:55–75.
4. Silhan J, et al. (2009) 14-3-3 protein masks the DNA binding interface of forkhead
transcription factor FOXO4. J Biol Chem 284:19349–19360.
7. Uno I, Matsumoto K, Adachi K, Ishikawa T (1983) Genetic and biochemical evidence
that trehalase is a substrate of cAMP-dependent protein kinase in yeast. J Biol Chem
35. Rezabkova L, et al. (2011) Structural basis for the 14-3-3 protein-dependent inhibition of
the regulator of G protein signaling 3 (RGS3) function. J Biol Chem 286:43527–43536.
36. Davison AC, Hinkley DV (1997) Bootstrap Methods and Their Application (Cambridge
Univ Press, Cambridge, UK).
37. Macakova E, et al. (2013) Structural basis of the 14-3-3 protein-dependent activation
of yeast neutral trehalase Nth1. Biochim Biophys Acta 1830:4491–4499.
38. Kopecka M, et al. (2014) Role of the EF-hand-like motif in the 14-3-3 protein-
mediated activation of yeast neutral trehalase Nth1. J Biol Chem 289:13948–13961.
39. Volkov VV, Svergun DI (2003) Uniqueness of ab initio shape determination in small-
angle scattering. J Appl Crystallogr 36:860–864.
2
58:10867–10872.
1
1
2
8. Kopp M, Müller H, Holzer H (1993) Molecular analysis of the neutral trehalase gene
from Saccharomyces cerevisiae. J Biol Chem 268:4766–4774.
9. Franco A, et al. (2003) A role for calcium in the regulation of neutral trehalase activity
in the fission yeast Schizosaccharomyces pombe. Biochem J 376:209–217.
0. Wera S, De Schrijver E, Geyskens I, Nwaka S, Thevelein JM (1999) Opposite roles of
trehalase activity in heat-shock recovery and heat-shock survival in Saccharomyces
cerevisiae. Biochem J 343:621–626.
Alblova et al.
PNAS Early Edition
|
9 of 10

4
4
4
0. van Heusden GP (2005) 14-3-3 proteins: Regulators of numerous eukaryotic proteins.
IUBMB Life 57:623–629.
1. Obsil T, Obsilova V (2011) Structural basis of 14-3-3 protein functions. Semin Cell Dev
Biol 22:663–672.
2. Veisova D, et al. (2010) The C-terminal segment of yeast BMH proteins exhibits different
structure compared to other 14-3-3 protein isoforms. Biochemistry 49:3853–3861.
3. Kabsch W (2010) XDS. Acta Crystallogr D Biol Crystallogr 66:125–132.
4. Sparta KM, Krug M, Heinemann U, Mueller U, Weiss MS (2016) XDSAPP2.0. J Appl
Crystallogr 49:1085–1092.
47. Chen VB, et al. (2010) MolProbity: All-atom structure validation for macromolecular
crystallography. Acta Crystallogr D Biol Crystallogr 66:12–21.
48. Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by
the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53:240–255.
49. Pernambuco MB, et al. (1996) Glucose-triggered signalling in Saccharomyces cerevisiae:
Different requirements for sugar phosphorylation between cells grown on glucose and
those grown on non-fermentable carbon sources. Microbiology 142:1775–1782.
50. Jerabek-Willemsen M, et al. (2014) MicroScale thermophoresis: Interaction analysis
and beyond. J Mol Struct 1077:101–113.
4
4
4
5. Vagin A, Teplyakov A (1997) MOLREP: An automated program for molecular re-
placement. J Appl Crystallogr 30:1022–1025.
6. Adams PD, et al. (2010) PHENIX: A comprehensive Python-based system for macro-
molecular structure solution. Acta Crystallogr D Biol Crystallogr 66:213–221.
51. Niesen FH, Berglund H, Vedadi M (2007) The use of differential scanning fluorimetry
to detect ligand interactions that promote protein stability. Nat Protoc 2:2212–2221.
52. Efron B, Tibshirani RJ (1993) An Introduction to the Bootstrap (Chapman and Hall,
New York).
4
1
0 of 10
|
www.pnas.org/cgi/doi/10.1073/pnas.1714491114
Alblova et al.