Campylobacter jejuni adenosine triphosphate phosphoribosyltransferase is an active hexamer that is allosterically controlled by the twisting of a regulatory tail

Article abstract of DOI:10.1002/pro.2948

Adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyzes the first committed step of the histidine biosynthesis in plants and microorganisms. Here, we present the functional and structural characterization of the ATP-PRT from the pathogenic ε-proteobacteria Campylobacter jejuni (CjeATP-PRT). This enzyme is a member of the long form (HisG_L) ATP-PRT and is allosterically inhibited by histidine, which binds to a remote regulatory domain, and competitively inhibited by AMP. In the crystalline form, CjeATP-PRT was found to adopt two distinctly different hexameric conformations, with an open homohexameric structure observed in the presence of substrate ATP, and a more compact closed form present when inhibitor histidine is bound. CjeATP-PRT was observed to adopt only a hexameric quaternary structure in solution, contradicting previous hypotheses favoring an allosteric mechanism driven by an oligomer equilibrium. Instead, this study supports the conclusion that the ATP-PRT long form hexamer is the active species; the tightening of this structure in response to remote histidine binding results in an inhibited enzyme.

Full text of DOI:10.1002/pro.2948

Campylobacter jejuni adenosine
triphosphate phosphoribosyltransferase
is an active hexamer that is allosterically
controlled by the twisting of a
regulatory tail
1,2
1,2
Gerd Mittelstadt, Gert-Jan Moggre, Santosh Panjikar,3,4 Ali Reza Nazmi,5
€
ꢀ
and Emily J. Parker1,2*
1Maurice Wilkins Centre, Biomolecular Interaction Centre, Christchurch 8140, New Zealand
2Department of Chemistry, University of Canterbury, Christchurch 8140, New Zealand
3Australian Synchrotron, Clayton, Melbourne, Victoria 3168, Australia
4Department of Biochemistry and Molecular Biology, Monash University, Clayton, Campus, Melbourne, Victoria 3800, Australia
5Biomolecular Interaction Centre and School of Biological Sciences, University of Canterbury, Christchurch 8140, New Zealand
Received 11 April 2016; Accepted 11 May 2016
DOI: 10.1002/pro.2948
Published online 00 Month 2016 proteinscience.org
Abstract: Adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyzes the first commit-
ted step of the histidine biosynthesis in plants and microorganisms. Here, we present the func-
tional and structural characterization of the ATP-PRT from the pathogenic e-proteobacteria
Campylobacter jejuni (CjeATP-PRT). This enzyme is a member of the long form (HisGL) ATP-PRT
and is allosterically inhibited by histidine, which binds to a remote regulatory domain, and competi-
tively inhibited by AMP. In the crystalline form, CjeATP-PRT was found to adopt two distinctly dif-
ferent hexameric conformations, with an open homohexameric structure observed in the presence
of substrate ATP, and a more compact closed form present when inhibitor histidine is bound.
CjeATP-PRT was observed to adopt only a hexameric quaternary structure in solution, contradict-
ing previous hypotheses favoring an allosteric mechanism driven by an oligomer equilibrium.
Abbreviations: ATP-PRT, adenosine triphosphate phosphoribosyltransferase; BTP, 1,3-bis[tris(hydroxymethyl)methylamino]propane;
CjeATP-PRT, ATP-PRT from Campylobacter jejuni; EcoATP-PRT, Escherichia coli ATP-PRT; His, l-histidine; ITC, isothermal titra-
tion calorimetry; MtuATP-PRT, Mycobacterium tuberculosis ATP-PRT; PDB, Protein Data Bank; PR-ATP, phosphoribosyl ATP;
PRPP, phosphoribosyl pyrophosphate; PRT, phosphoribosyltransferase; RMSD, root-mean-square difference; SenATP-PRT, Sal-
monella enterica subsp. enterica Typhimurium ATP-PRT
Additional Supporting Information may be found in the online version of this article.
Significance Statement: ATP-phosphoribosyltransferase catalyzes the first dedicated step in the biosynthesis of the essential
amino acid histidine in microorganisms. We report the functional characterization of this enzyme from human pathogen Campylo-
bacter jejuni. The enzyme is inhibited by histidine, allowing for tuned production of histidine in response to cellular demands. Our
results reveal how the enzyme structure becomes compressed when histidine binds and exposes the molecular details of how this
enzyme performs its function.
Grant sponsors: Maurice Wilkins Centre and by New Zealand Lottery Health; Grant sponsor: University of Canterbury Doctoral Schol-
arships (G.M. and G-J.M.).
*Correspondence to: Emily J. Parker, Department of Chemistry, University of Canterbury, Private Bag 4800, Christchurch 8140, New
Zealand. E-mail: emily.parker@canterbury.ac.nz
C
V
Published by Wiley-Blackwell. 2016 The Protein Society
PROTEIN SCIENCE 2016 VOL 00:00—00
1

Instead, this study supports the conclusion that the ATP-PRT long form hexamer is the active
species; the tightening of this structure in response to remote histidine binding results in
an inhibited enzyme.
Keywords: ATP-PRT; phosphoribosyltransferase; HisG; allostery; conformational change
Introduction
(HisGL) from Salmonella enterica subsp. enterica
Typhimurium (SenATP-PRT)7–9 according to which
the reaction follows an ordered sequential mecha-
nism with ATP binding the enzyme first and PR-
ATP leaving last. ATP-PRT has been reported to be
inhibited by a variety of compounds. Due to the
equilibrium nature of the reaction, substrates and
products naturally compete for the active site, but
the most potent inhibition is achieved by a combina-
tion of the nucleotide AMP and the pathway end-
product His, which act synergistically.10 Individually
AMP, and to a lesser extent also ADP, inhibit ATP-
PRT competitively with respect to both substrates.
This is strongly supported by the observation that
AMP binds the active site of Escherichia coli ATP-
PRT (Eco ATP-PRT).11 ATP-PRT activity and conse-
quently the metabolite flux into His biosynthesis, is
therefore determined by the energy state of the cell
as reflected by the ATP to AMP ratio.1,12 His on the
other hand acts as an allosteric inhibitor, exhibiting
Histidine biosynthesis, the primary metabolic path-
way responsible for the de novo synthesis of the pro-
teogenic
amino
acid
L-histidine
(His),
is
energetically expensive, requiring 41 equivalents of
ATP to synthesize a single His molecule.1 The path-
way involves a total of 10 reactions and also contrib-
utes metabolites to the de novo purine synthesis via
its branch point at the imidazole-glycerol phosphate
synthase reaction, which helps the cell to replenish
its energy pool (Fig. 1). Adenosine triphosphate
phosphoribosyltransferase (ATP-PRT) catalyzes the
first committed step of His biosynthesis and repre-
sents the major control point for its regulation via
feedback inhibition by the pathway end-product His.
The phosphoribosyltransferase (PRT) enzyme
family, to which ATP-PRT belongs, is characterized
by the ability to catalyze the displacement of pyro-
phosphate from substrate phosphoribosyl pyrophos-
phate (PRPP) by a nitrogen-containing nucleophile.
PRT enzymes are found predominantly in the nucle-
otide metabolism, but also in amino acid biosynthe-
sis, and are divided into four types according to
their overall fold and domain architecture. ATP-PRT
represents its own unique type, denoted type IV
PRT.2 ATP-PRT enzymes can be further subdivided
into two groups based on their sequence length and
their distinctly different quaternary structures. The
long form of ATP-PRT (known as HisGL) has a chain
length of 280–310 amino acids and assembles only
as a homomeric form. The short form (HisGS) con-
sists of only 200–220 amino acid residues per chain
and associates with a second gene product, the
tRNA synthetase paralog HisZ,3 to form a fully func-
tional complex.4 While both forms carry out the
same biochemical role and show clear structural
similarity in their catalytic domains, most organisms
encode for only one type of ATP-PRT, except the
Geobacter clade,5 suggesting an early evolutionary
division.6
The ATP-PRT-catalyzed reaction is the reversi-
ble substitution of PRPP by ATP, releasing pyro-
phosphate. The product phosphoribosyl ATP (PR-
ATP) is then converted irreversibly by the subse-
quent enzymes of the pathway, driving the reaction
in the biosynthetic direction. Mg21 ions are required
for the activation and stabilization of the poly-
phosphorylated substrates and are thus essential for
the reaction to occur.7 Extensive kinetic characteri-
zation was carried out on the ATP-PRT enzyme
Figure 1. Histidine biosynthesis. ATP-PRT catalyzes the first
committed step of the de novo synthesis of His. The forma-
tion of the His precursor imidazole-glycerol phosphate leads
to the side product 5-aminoimidazole-4-carboxamide ribonu-
cleotide (AICAR), which is used in the purine metabolism.
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Campylobacter jejuni ATP-Phosphoribosyltransferase

Table I. Steady-State Kinetic Parameters for ATP-PRT
Parameter
CjeATP-PRTa
SenATP-PRT8
MtuATP-PRT14
KM ATP (lM)
KM PRPP (lM)
97 6 10
15 6 1
1.8 6 0.1
19 6 2
120 6 10
356 6 22
382 6 34
44 6 7
110
15
–
263 6 63
49 6 6
0.31 6 0.01
1.2 6 0.3
6.3 6 0.8
kcat (s21
)
kcat/KM ATP (s21 mM21
cat/KM PRPP (s21 mM21
)
k
)
Ki AMP (ATP) (lM)
Ki AMP (PRPP) (lM)
Ki His (ATP) (lM)
Ki His (PRPP) (lM)
480
550
–
27.9 6 1.9
25.7 6 12.8
40 6 1
–
a
Generated by global fitting, using CjeATP-PRT (280 nM), and EcoPPase (6.4 mM) in 50 mM Tris/HCl, MgCl2 (10 mM),
NaCl (100 mM), KCl (50 mM), pH 8.5 at 25˚C. (-) kcat and KI data for Sen ATP-PRT has not been reported.
non-competitive and uncompetitive inhibition pat-
terns against the substrates in SenATP-PRT13 and
Mycobacterium tuberculosis ATP-PRT (MtuATP-
PRT).14 In HisGL His binding occurs in a remote
regulatory binding site in a discrete domain and the
binding has been observed for both E. coli and M.
tuberculosis enzymes.
enzyme, whereas the active species, without His
bound, was thought to be dimeric.2,15 This is consist-
ent with earlier analysis of the oligomeric state of
EcoATP-PRT, where multiple species have been
detected.21,22 However, more recent results fail to
support the existence of a stable dimeric form of the
MtuATP-PRT enzyme, making an oligomeric equilib-
rium unlikely.
Crystal structures have been determined for both
long form and short form ATP-PRT enzymes. The
long form ATP-PRT, represented by the E. coli11 and
M. tuberculosis15 enzymes, possesses a homohexa-
meric structure with a 2- and 3-fold internal symme-
try, where all active sites are secluded in the interior
of the protein. Each chain consists of three distinct
domains. Domains I and II both have mixed a-b open
twisted sheet folds, with the active site located in the
cleft between them. This bilobal “catalytic core” forms
the basis of ATP-PRT function and is the common
structural characteristic between short and long form
enzymes.16 The C-terminal domain III is exclusive to
the ATP-PRT long form and arranges in two trimeric
units, one on either end of the hexamer. This domain
has been identified to be an ACT domain (named
from aspartokinase, chorismate mutase and
TyrA)17,18 responsible for the feedback regulation.
Three His binding sites have been observed in both
trimeric regulatory regions of the protein in the
MtuATP-PRT (PDB ID: 1NH8) and archaeal Metha-
nothermobacter thermautotrophicus ATP-PRT (PDB
ID: 2VD3) structures. A number of key residues for
His binding have also been identified in Corynebacte-
rium glutamicum ATP-PRT, which lowered the sensi-
tivity of the enzyme towards His upon mutation19
confirming the identified binding site as the func-
tional allosteric site. The remarkably different struc-
ture of the heterooctameric HisGS/Z complex is made
up of four HisZ chains in a central X-shaped unit and
two HisGS dimers on opposite sides. His binding to
this complex has been observed at several places in
the interface between the HisG and HisZ chains.4,20
Based on the involvement of the ACT domains
in His binding at the interfaces between the chains
of the homohexameric form, it was proposed that
the hexamer represents the inhibited form of the
In this study, we examine the biochemical and
regulatory properties of the HisGL ATP-PRT from
Campylobacter jejuni (CjeATP-PRT). Through struc-
tures showing the binding of ligands at both the
active and allosteric sites and complementary solu-
tion state observations, we illuminate an allosteric
mechanism that involves conformational change of
an open homohexameric active protein to a closed
hexameric His-bound inhibited form.
Results
C. Jejuni ATP-PRT is inhibited by AMP and His,
and more potently by an AMP/His-combination
Highly purified CjeATP-PRT enzyme was found to be
active under the described assay conditions and the
kinetic parameters were determined (Table I). Reac-
tion kinetics followed a sequential mechanism and
optimal concentrations for Mg21 and Na1/K1 were
found to be 10 mM and 100–200 mM, respectively
(Supporting Information Figs. S2 and S3). The derived
kinetic parameters also show good agreement with
those of long form ATP-PRTs from other sources.8,10,14
CjeATP-PRT activity was shown to be suscepti-
ble to inhibition by the native inhibitors AMP and
His. Moreover, the two inhibitors show a clear syner-
gistic behavior towards the inhibition of CjeATP-
PRT, a known phenomenon of the long form ATP-
PRT.10 The effect was most prominent for inhibitor
mixtures at concentrations that only marginally
reduce the enzyme activity (5–20% activity loss)
individually (Fig. 2).
While our results depict standard competitive
inhibition for the effect of AMP with respect to both
ATP and PRPP, for CjeATP-PRT the allosteric inhib-
itor His was found to follow a non-competitive model
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Figure 2. Inhibition of C. jejuni ATP-PRT. Relative activity of CjeATP-PRT in the presence of the inhibitors AMP and His. Error
bars represent standard deviations of the measurements at each point. A: Dose response curves and IC50 fits for His and AMP
individually. B: The synergistic effect of both inhibitors shown as columns. The activity was determined in the presence of no
inhibitor (none), 2 mM AMP (AMP), 20 mM His (His), and both 2 mM AMP and 20 mM His (AMP 1 His). For comparison, a theo-
retical value is given representing the simple addition of the individual AMP and His inhibitory effects.
of inhibition with respect to both substrates (Sup-
porting Information Fig. S4). The resulting inhibi-
tion constants for AMP and His are consistent with
kinetically obtained values for SenATP-PRT (AMP)10
and MtuATP-PRT (His)14 (Table I).
ence of 1 mM AMP. In this case the binding followed
a sequential model with two binding events, giving
rise to Kd values that are lower than observed for
His alone, indicating a higher affinity of the enzyme
for His in the presence of AMP. Likewise, the bind-
ing constant of AMP for the enzyme was also shown
to decrease approximately 6-fold in the presence of
1 mM His (Table II).
Complex binding of His and AMP
The strong binding of His to CjeATP-PRT, seen in
the kinetic assay, was confirmed by ITC experi-
In contrast to His, the binding of the active site
inhibitor AMP follows the classic single-site model,
highlighting that all present active sites are equiva-
lent. The resulting Kd value is significantly lower
than the kinetically measured Ki AMP values, which
is consistent with the competitive nature of AMP
towards both substrates and the kinetic determina-
tion of Ki in the presence of saturating concentra-
tions of either substrate. Consequently, AMP-
titration in the presence of 1 mM ATP resulted in a
higher Kd value (83 6 8 lM).
ments, which also show
a clear cooperativity
between multiple binding events for the enzyme (fit-
ted with a sequential binding model). When His
alone was titrated against the CjeATP-PRT a strong
initial exothermic signal was seen that quickly
declined in subsequent injections. Thereafter the
curve has a bi-phasic shape. The binding constants
(Kd) for His range between 15 and 50 lM.
A very differently shaped isotherm was observed
when His was titrated into the enzyme in the pres-
Table II. Isothermal Titration Calorimetry
Ligand
Parameter
Kd (mM)
158 6 13
51 6 4
Stoichiometry
DH (kcal/mol)
22.5 6 0.2
211.3 6 0.6
211.5 6 0.1
214.9 6 0.2
DS (cal/mol)
9.1
ATPa
AMPb
AMPc
Hisd
1.07 6 0.08
1.00 6 0.04
1.14 6 0.01
NDe
218.4
215.2
232.0
8.3 6 0.2
19.3 6 0.2, 14.5 6 0.2,
36 6 1, 50 6 1, 47 6 1
4.8 6 0.7, 2.3 6 0.1
Hisf
0.91 6 0.20
224.2 6 1.0
257.1
Parameters derived from the ATP, AMP and His titrations against 100 lM CjeATP-PRT. Errors are derived from data fits.
a
4 mM, single site model.
3.5 mM, single site model.
2 mM, in the presence of 1 mM His, single site model.
1.5 mM, sequential binding model (five events).
Not determined (ND) due to the complex binding
1 mM, in the presence of 1 mM AMP, sequential binding model (two events).
b
c
d
e
f
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Campylobacter jejuni ATP-Phosphoribosyltransferase

Figure 3. Inhibitor binding to CjeATP-PRT. ITC isotherms resulting from the titration against CjeATP-PRT, subtracted by the
R
corresponding heat of dilution data series, and fitted using OriginLabV version 7.0 (OriginLab Corp.) A: 3.5 mM AMP, B: 1.5 mM
His, C: 2 mM AMP in the presence of 1 mM His, D: 1 mM His in the presence of 1 mM AMP.
Additionally, the presence of His was shown to
have a dramatic influence on the ATP binding abil-
ity of CjeATP-PRT. In the absence of other ligands,
ATP can readily bind to the enzyme, resulting in a
Kd value close to the kinetically determined KM
value of 97 6 10 lM. In contrast, the presence of
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His, and His alone (Table III). ATP-bound CjeATP-
PRT (PDB code: 4YB7) was solved in space group
P1 and the unit cell contains 12 chains forming two
˚
complete hexamers (0.50 A RMSD between hexam-
ers). A single Mg21-bound ATP molecule is found in
the active site of each chain. The two inhibitor-
bound structures were solved in space group P21
and contain six chains each in an asymmetric unit,
arranged in a single hexamer. Both His-bound (PDB
code: 4YB5) and AMP and His-bound (PDB code:
4YB6) structures feature six well-defined His mole-
cules bound to the regulatory sites of the hexamer.
The latter additionally contains one AMP molecule
per active site. Mg21 ions were observed in the
active sites containing both ATP and AMP. Depend-
ing on the individual chains, a small number of resi-
dues (4–11) are disordered and not visible in the
electron density map. The areas of missing residues
include the N-terminus, as well as external loops
(a1-b2, b6-a4, and b13-b14 loops).
Figure 4. Effect of ligand binding on thermostability. DSC
scans of CjeATP-PRT were determined in 50 mM Tris/HCl,
100 mM NaCl, 5 mM MgCl2, pH 8.5 in the presence of no
ligand (green), 1 mM His (blue), 2 mM AMP (red), and the
mixture of the latter two (purple). Dotted lines correspond to
the fit. Midpoint temperatures of the thermal unfolding are
given next to the peaks.
Each individual CjeATP-PRT chain is composed
of 10 a-helices, 16 b-strands and the connecting
loops. Every chain is arranged in three domains,
domains I, II, and III, adopting the same overall
architecture as both MtuATP-PRT and EcoATP-
PRT.11,15 CjeATP-PRT domain I is composed of resi-
dues 1–103 and 191–225, which form a Rossmann
fold, commonly found for nucleotide binding pro-
teins.23 Residues 104–190 form domain II, which is
inserted into domain I between the two b-strands b5
and b11. Together the two domains form a bilobal
core unit that contains the catalytic site in the cleft
between them, where the binding of ATP and AMP
is observed. The last 74 C-terminal residues form
1 mM His led to no measurable ATP binding signal
(Supporting Information Fig. S5).
The binding of AMP and His also affects the sta-
bility of CjeATP-PRT, visible as shifts of the thermal
denaturation temperature determined by DSF and
DSC experiments (Supporting Information Table
S6). The addition of 1 mM His and 2 mM AMP
increased the melting temperature by 2 and 38C,
respectively. This stabilization effect appeared to be
additive with a shift of 58 when a combination of
AMP and His as added (Fig. 4).
CjeATP-PRT is hexameric
regulatory ACT domain.18 The
The quaternary structure of highly pure CjeATP-PRT
in solution was explored using AUC, as well as ana-
lytical SEC and SEC-coupled static light scattering
techniques. A set of three low protein concentrations
(3.0, 1.5, and 0.74 lM) was used for sedimentation
velocity experiments. The resulting normalized size
distribution functions (Fig. 5) show one major species
with a sedimentation coefficient of 7.9 S in all three
preparations. The derived molecular weight of 184
kDa is close to the theoretical mass corresponding to
a hexamer, 202 kDa. The existence of a single hex-
americ species in solution was also confirmed by the
results of SEC (204 kDa) and SEC-coupled static
light scattering (188 kDa) (Supporting Information
Fig. S7). As the lowest measured enzyme concentra-
tions is very close to the kinetic assays concentration
of 0.28–0.44 lM, it is apparent that CjeATP-PRT
mainly exists as a hexamer under assay conditions.
domain III,
a
Figure 5. AUC analysis of CjeATP-PRT. Sedimentation
velocity experiments were performed in 10 mM Tris/HCl,
100 mM NaCl, 5 mM MgCl2, pH 8.5 using 0.1 mg/mL (blue),
0.05 mg/mL (green), and 0.025 mg/mL (red) CjeATP-PRT.
Continuous size distribution functions were generated using
SEDFIT,21 normalized by the total area under the curve and
smoothed with a four point moving average using Prism 6.0
(GraphPad Software).
CjeATP-PRT crystallizes as a homohexamer in
the presence of ATP, AMP, and His
Datasets were collected from three different CjeATP-
PRT co-crystals featuring the ligands ATP, AMP and
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Campylobacter jejuni ATP-Phosphoribosyltransferase

Table III. Crystal Parameters, Data collection, and Refinement Statistics
CjeATP-PRT
ATP
His
His/AMP
A. Data collection
Crystal system, space group
Unit cell parameters
a, b, c (A)
Triclinic, P1
Monoclinic, P2
Monoclinic, P21
˚
91.67, 91.83, 154.90
101.11, 95.21, 118.14
50.00–2.20 (2.24–2.20)
839,825
91.14, 123.22, 95.70
90.00, 110.66, 90.00
50.00–2.24 (2.28–2.24)
472,477
91.87, 124.91, 92.81
90.00, 115.86, 90.00
50.00–1.98 (2.01–1.98)
493,057
a, b, c (ꢀ)
Resolution range (A)
˚
Total reflections
Unique reflections
Redundancy
Completeness (%)
<I/r(I)>
212,958
3.9 (4.0)
98.1 (97.3)
9.8 (1.8)
94,235
5.0 (4.5)
89.9 (89.7)
9.9 (2.4)
127,994
3.9 (3.9)
98.1 (96.9)
14.6 (2.9)
Rmerge
Rmeas
CC1/2
Wilson B-value (A )
0.076 (0.841)
0.108 (1.189)
0.982 (0.568)
36.8
0.093 (0.795)
0.119 (1.027)
0.997 (0.625)
43.8
0.059 (0.422)
0.081 (0.584)
0.998 (0.837)
21.3
2
˚
˚
Matthews coefficient (A/Da)
2.72
2.48
2.37
B. Refinement
Resolution (A)
˚
48.04–2.20
0.236
0.259
48.04–2.24
0.211
0.241
48.04–1.98
0.203
0.220
Rcryst
Rfree
Chain length
300
300
300
Observed number of residues
2x292, 2x293,
3x294, 1x295, 4x296
365
2x290, 1x291,
1x292, 2x295
128
1x289, 1x290,
1x293, 2x295, 1x296
714
6 His, 6 AMP,
9 Mg21, 6 PEG
Water molecules
Other
12 ATP, 12 Mg21
,
6 His, 8 SCN-,
5 K1, 3 PEG
6 PO342, 1 acetate
2
˚
Mean B (A )
Protein
47.13
35.20
55.83
51.27
50.03
38.62
61.30
34.64
24.58
26.94
45.09
Water
Other ligand
13.06, 24.81
RMSD from target values
˚
Bond lengths (A)
0.009
1.470
0.073
0.012
1.465
0.075
0.009
1.372
0.077
Bond angles (8)
Dihedral angles (8)
Ramachandran
Preferred (%)
Allowed (%)
98.22
1.78
98.56
1.44
98.36
1.64
Outliers (%)
0.00
0.00
0.00
PDB entry
4YB7
4YB5
4YB6
allosteric inhibitor His is bound at the interface of
two of these domains (Fig. 6).
interface occurs six times per hexamer and is gener-
ated between the two different faces of neighboring
domain IIIs, so that each domain contributes to two
interfaces. The main point of interaction is the C-
terminal b-strand b16 (residues K296-L298), which is
donated into the b-sheet of the adjacent chain’s ACT
domain. According to the analysis of the structure
with PDBe PISA,24 the trimer interface was classified
as essential for the CjeATP-PRT hexamer, with a
higher complex formation significance score (0.54–
0.62) than the dimer interface (0.33–0.38) in all three
CjeATP-PRT structures, which is consistent with the
solution oligomeric state analysis results. This
directly contrasts with the previous analysis of the
MtuATP-PRT and EcoATP-PRT interfaces,11 which
deemed the dimer interface the more significant of
the two (Supporting Information Table S9).
In all three structures, the CjeATP-PRT chains
assemble into homohexamers via interaction at two
main interfaces. The first of these, the “dimer inter-
face”, is created between the catalytic domains of
two adjacent chains and buries an area of 950–1000
2
2
˚
˚
A
(4YB6 and 4YB7), and 1150–1200 A (4YB5),
corresponding to 6–8% of the overall surface of a sin-
gle chain. The second interface, the “trimer inter-
face,” contributes significantly to the overall
hexameric structure. This interface predominantly
involves interactions of domain III: Domain III from
three neighboring chains each come together to form
two trimeric arrangements at either end of the
formed hexamer, placing all active sites on the inside
of the hollow-centred complex. Accordingly, the trimer
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Figure 6. The CjeATP-PRT structure. Architecture of the CjeATP-PRTstructure as cartoon representation. A: Single chain of the
ATP-bound CjeATP-PRT. Domains I (green), II (yellow), and III (red) are highlighted separately. Secondary structure elements
are labeled according to the sequence. B: View on two chains with bound ATP in their dimeric arrangement, facing the dual
active site cavity in the center of their interface. Second chain highlighted in blue. C-F: The hexameric complex of CjeATP-PRT
with bound ATP (C 1 E) and bound His and AMP (D 1 F) shown from two different angles with two single chains colored
according to B. The ligands ATP (cyan), His (purple), and AMP (pink) are displayed as spheres.
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Campylobacter jejuni ATP-Phosphoribosyltransferase

Figure 7. CjeATP-PRT ligand binding. Real space representation of ATP (A), AMP (B), and His (C) surrounded by the observed
electron density (Fo-Fc map, contoured at 2.0 sigma, gray mesh) and the CjeATP-PRT residues of the corresponding (white)
and adjacent chain (green) as sticks. Water molecules (red) and the Mg21 ion (green) are represented as spheres. Hetero-
atoms are colored according to element: oxygen (red), nitrogen (blue), phosphorous (orange).
Information Fig. S8). The triphosphate moiety of
ATP is flanked by the side chains of Arg54 and
Arg16, which form hydrogen bonds to the g and a-
phosphate, respectively. A distinguishing feature of
the ATP binding site is the presence of a Mg21 ion
in complex with the bound ATP triphosphate. This
ion is coordinated in an octahedral arrangement cre-
ated by the side chains of residues D55 and D56, the
b and g-phosphate of ATP, and two water molecules.
This coordination embeds the Mg21 into the binding
site of CjeATP-PRT and provides strong interaction
with the substrate. The ATP ribose ring is positioned
in a cavity created by the backbone and short side
chains of Leu17, Gly73, Gly102, Ser191, Arg192 and
Ala193 and the ribose oxygen atoms form hydrogen
bonding interactions with the functionally conserved
residue Asn75. The adenine portion of ATP is held
in place by hydrophobic interactions with the side
chain of Arg16 on one side and Leu170 and Cys104
on the other side. This “hydrophobic stack” allows
the adenine to adopt two different orientations. Ade-
nine was observed in either a catalytic relevant posi-
tion, placing the N1 nitrogen close to the expected
ATP binding is consistent with a sequential
mechanism
In the ATP-bound CjeATP-PRT structure all chains
crystallized in the same conformation, previously
described as the “active” conformation.15 The repre-
sentation of an active enzyme complex by this struc-
ture is underlined by its ability to bind substrate
ATP. In the observed binding mode ATP occupies the
majority of the active site, but does not involve the
conserved 14 amino acid PRT signature motif,25
a
fold characteristic to type I and IV PRT enzymes,
including the PRPP loop that is known to specifi-
cally bind phosphate-groups or chemically similar
compounds and plays a key role in PRPP recogni-
tion. This finding is in line with a sequential binding
mechanism, in which ATP can bind first, leaving the
active site open for interaction with PRPP.
To our best knowledge this is the first study of
an ATP-PRT structure describing convincing elec-
tron density for ATP with full occupancy. Here, ATP
is accommodated in the nucleotide binding groove of
domain I, involving interaction with a number of
residues that are in part conserved (Supporting
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Mittelstadt et al.
PROTEIN SCIENCE VOL 00:00—00
9

Figure 8. Changes induced by His binding. A series of superimpositions of the ATP-bound (green) and His-bound (purple) con-
formations of CjeATP-PRT shown as cartoon representations. A: Complete hexamers. B: Trimeric units side by side. C: Single
chains superimposed on the catalytic core domains. D: Dimeric unit superimposed on the catalytic core of the upper chain.
PRPP binding site, or flipped by an 180˚ rotation,
moving N1 away from the possible reaction site [Fig.
7(A)]. The ATP conformers were populated through-
out the 12 chains, nine showing the catalytically rel-
evant orientation and three the non-relevant,
without a distinguishable pattern.
atoms of Ser173 and Thr176. On the other end of the
AMP molecule, the adenine is found stacked with
Leu170, as described for ATP, but with an inverted
orientation. Consequently, AMP and ATP binding
modes overlap in this region of the active site, which
strongly supports the competitive characteristic of
AMP towards both substrates observed in the kinetic
analysis (Supporting Information Fig. S10).
AMP binding mode confirms competitive
behavior towards ATP
Differences between the His-bound and AMP-
and His-bound structures are minimal. The catalytic
subunits of the His-bound and AMP and His-bound
Overall the AMP binding mode found in CjeATP-PRT
confirms the results of the AMP-bound EcoATP-PRT
structure,11 with the main point of interaction being
the PRPP binding loop [Fig. 7(B)]. Residues Ser172-
Thr176 tightly coordinate the phosphate group of
AMP by hydrogen bonding interactions with their
backbone nitrogen atoms and the side chain oxygen
˚
CjeATP-PRT are very similar (RMSD5 0.346 0.2 A)
with the position of the a7 helix being the only signif-
icant difference. This helix is shifted inwards by
˚
approximately 4 A in respect to the hexamer surface
in the AMP and His-bound structure, which is likely
10 PROTEINSCIENCE.ORG
Campylobacter jejuni ATP-Phosphoribosyltransferase

˚
due to the presence of AMP restricting the conforma-
tional freedom of the surrounding PRPP binding loop.
A when His is bound. Moreover in the presence of
His a tight hydrogen bonding network is formed
between all three neighboring ACT domains via the
residues Thr252, Tyr228, and His266, the conserved
water and the bound His molecules, strengthening
the interface.
CjeATP-PRT exists in two distinct
conformational states
As described for MtuATP-PRT previously,15 the pre-
sented crystal structures of CjeATP-PRT represent
two distinctly different conformations. The ATP-
bound structure (4YB7) differs decisively from the
His-bound structures (4YB5 and 4YB6) by its over-
all hexamer conformation, which is apparent by the
differences in the orientation of the individual
chains towards each other and the consequent
Whereas His binding directly and prominently
affects the trimer interface, the catalytic domains
also contribute to the overall changes in the hex-
amer. A large conformational change is not observ-
˚
able (1.02 6 0.05 A RMSD between residues 4–225 of
the AMP and His-bound and ATP-bound CjeATP-
PRT), but changes in the dimer interface are evident
comparing the His-bound and ATP-bound structures.
The two chains of a dimeric unit undergo a rota-
tional flex [Fig. 8(D)], which elongates the structure
changes in their interfaces (Figs. 6 and 8). In com-
˚
parison to the ATP-bound hexamer (117 6 1
A
˚
length, 96 6 1 A width), both inhibitor-bound struc-
˚
˚
tures possess a longer and more ellipsoid overall
(122 6 1 A vs. 117 6 1 A). The majority of the central
dimer interface contacts, involving residues Thr152-
Leu163 (a6 and flanking loops) of the first chain and
Gln37’-Ile40’ (a1-b2 loop), Arg8’ (b1), and Asp57’
(a2) of the second chain, remain intact upon His
binding, but close contacts between residues Thr176-
Asn180 (a7) and Gln37’-Ile40’ (a1-b2 loop) are lost
as the a7 helices are shifted towards the outside of
the hexamer. Additional interactions are formed on
the opposite side of the dimer interface, as Asn148-
Leu151 (b8) and Asp57’-Asp64’ (a2) and the C-
terminal ends of the a3 helices (Leu87 and Leu87’)
are now in close proximity to each other. This move-
ment increases the distance between the two active
˚
˚
shape (122 6 1 A length, 92 6 1 A width). Superimpo-
sition of the two crystallized hexamer conformations
shows these differences as a large discrepancy in the
RMSD value (Supporting Information Table S11).
Individually the conformational change is visible by
the rotational twist of the ACT domain relative to
the catalytic core by approximately 30˚ [Fig. 8(C)].
This change is driven by the binding of His at the
interface of neighboring domain IIIs, which pulls the
complex closer together, significantly increasing the
buried surface area of the trimer interface from
2
2
˚
˚
830 6 20 A (4YB7) to 1220 6 30 A (4YB5/4YB6).
Very similar changes between the apo and His-
bound conformation have also been reported for
MtuATP-PRT, although these changes were not
attributed to activity loss.
˚
sites by approximately 3 A (as measured between
Lys13 and Ser154) and thereby alters the geometry
of the dual-active site cavity.
His binding in both structures occurs at the
interface between two adjacent domains III in their
trimeric arrangement, similar to the His-bound
MtuATP-PRT structure.15 Consequently one His is
bound per chain, but each chain provides two differ-
ent faces for the interaction with His. The His car-
boxyl and amino groups are coordinated via
hydrogen bonding interactions with the backbone
and Thr252 side chain of the highly conserved bind-
ing loop between a9 and b13 of one ACT domain
(consensus sequence: PGXXXPT, CjeATP-PRT resi-
dues Pro246-Thr252; Supporting Information Fig.
S8), whereas the imidazole side chain is inserted
into a small cavity created by the side chains on the
b-sheet of the other ACT domain, involving interac-
tions with His232’, Ser288’, Leu290’, and a con-
served water molecule [Fig. 7(C)].
Although the changes in hexamer conformation
are only observed when His is present and are inde-
pendent from AMP binding to the active site, as
seen for of EcoATP-PRT (PDB ID: 1H3D),11 the
AMP and His-bound structure highlights an impor-
tant inter-subunit contact only occurring at the
active site of the inhibited CjeATP-PRT hexamer:
The a1-b2 loop (residues Gly29-Pro47), which is
involved in the dimer interface formation in the
active hexamer, is released upon His binding and
protrudes into the active side of a neighboring chain,
which brings the side chains of His33’ and His35’ in
close proximity to the PRPP binding loop allowing
for additional interactions with the bound inhibitor
[Fig. 7(B)] thereby altering the active site further in
the presence of His.
Comparison to the unoccupied allosteric site in
the ATP-bound structure revealed clear differences
in the distance and orientation of the two faces of
the binding site relative to each other. Although the
conserved binding loop does not change its confor-
mation, it is significantly displaced from the adja-
cent monomer’s binding face, with an average
Discussion
ATP-PRT acts as the gateway into His biosynthesis
and as such plays an important metabolic role. It
has been the focus of several studies and identified
as a potential target of new antibiotic therapies.26,27
The nature of the active species in solution of the
long form of this enzyme and the details of allostery
have been unclear; with early solution studies
˚
distance of 12 A when His is absent compared to 10
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Mittelstadt et al.
PROTEIN SCIENCE VOL 00:00—00 11

favoring a change in quaternary structure from
active dimer to inactive His-bound oligomer.21,22 The
loose hexameric structure observed in the absence of
His for the MtuATP-PRT and EcoATP-PRT was
interpreted as consistent with an inactive hex-
amer.2,11,15 Our solution and crystal studies with
CjeATP-PRT support an active homohexamer and
illuminate the conformational constriction of this
form of the protein as the mechanism for the deliv-
ery of allosteric control at the gateway to His
biosynthesis.
the active form of MtuATP-PRT has also been sup-
ported in more recent solution experiments.14
Our three structures of the hexameric CjeATP-
PRT provide insight into key active site interactions
and display the changes that are associated with
His binding. The clear observation of ATP for the
first time in the active site provides a model for the
active hexameric form of this enzyme. The binding
site of PRPP can be clearly inferred from this struc-
ture and from the positioning of AMP in the active
site in the AMP/His-bound structure. The reported
binding mode for ATP highlights the importance of
Mg21 for the ATP-PRT reaction, which was observed
to aid in the substrate binding via a double aspar-
tate motif (Asp55-Asp56). Although clearly func-
tional, this metal-binding site along with most other
identified binding interactions of ATP is surprisingly
not conserved, which stands in stark contrast to the
highly conserved PRPP binding motif. Despite the
size of the molecule, it therefore seems sufficient for
enzymatic efficiency to orientate ATP correctly in
the active site, leaving some evolutionary flexibility
as to how the binding of its functional groups is
achieved in detail.
CjeATP-PRT shares many biochemical and
structural properties with the long form ATP-PRT
enzymes that have been studied from other sour-
ces.8,10,11,15,19,28 However, extensive kinetic studies
are rare. The kinetic parameters determined for
CjeATP-PRT are closest to the data reported for Sen-
ATP-PRT,8,10 which shares 65% sequence identity
with CjeATP-PRT (Supporting Information Fig. S8).
Competitive behavior of AMP towards both sub-
strates has been observed for CjeATP-PRT and this
has also been shown for the SenATP-PRT8 and
MtuATP-PRT14 enzymes, and it is consistent with
the active site binding of AMP where it is observed
to be present in both ATP and PRPP binding sites.
Direct binding studies using calorimetry, used for
the first time here on the CjeATP-PRT protein, are
entirely consistent with this interpretation and the
competitive inhibition shown by AMP.
In the ATP-PRT hexamer all active sites are
positioned inside the oligomer, which poses the ques-
tion how the large, charged substrate and product
molecules can enter or leave, if this is the active
form of the protein? The hexameric structures of all
structurally characterized ATP-PRT long form
enzymes possess the same large openings between
neighboring domains, which would likely permit
access to the interior of the complex. Another possi-
ble entrance for substrate molecules is present in
form of a small opening in the dimer interface,
observed in the ATP-bound structure, which leads
directly to the active site and thus may function as
Highly complex His binding was observed by
calorimetry, consistent with communication and co-
operativity between the binding sites. It appears
possible that the initial strong exothermic event is
based on the rearrangement of the enzyme complex
upon His binding, adopting the inhibited state with
close contacts at the ACT domain interfaces. It is
notable that this strong exothermic signal was not
observed when the protein had AMP bound, and
that the binding of His becomes less complex when
AMP is bound. This result suggests that the con-
straints on conformation imposed by the AMP facili-
tate His binding, providing the synergistic response
observed in the kinetic measurements both with
a
substrate channel. In addition, the dynamic
motion of the hexamer may loosen and widen the
protein or provide a “breathing” motion, allowing
substrate access through the weak interfaces
between the catalytic dimer core structure (domains
I and II). Some limited evidence for such free move-
ment comes from the thermal stability experiments
performed on CjeATP-PRT, which shows a signifi-
cantly increased (5˚C) melting temperature for the
inhibitor-bound enzyme. It should also be noted that
all chains observed in the hexameric structures of
CjeATP-PRT differ marginally from each other, but
more so in the ATP-bound crystal resulting in the
loss of symmetry.
CjeATP-PRT and with other long form enzymes.10
A
structural rationale for the synergism can be found
in the a1-b2 loop, which aids in AMP binding at the
neighboring active site in the His-bound hexamer
conformation, providing a visible link between active
and allosteric binding sites.
We employed SEC and AUC techniques at low
concentration of protein to assess the quaternary
structure of the enzyme. Our studies provide no evi-
dence for a dimer, even at low concentrations, close
to those employed in the kinetic assays. This obser-
vation also ruled out quaternary structure change
leading to the unusual complex thermogram
observed for His binding. A hexameric structure as
So what differences in the hexameric form con-
tribute to the low activity of the His-bound form of
this protein? As noted above the thermal shift
observed is consistent with a rigidification of the
structure. His binding to the interfaces of the regu-
latory trimers found at either end of the core clearly
constricts movement of this part of the protein, and
12 PROTEINSCIENCE.ORG
Campylobacter jejuni ATP-Phosphoribosyltransferase

R
these regulatory trimers are observed to rotate rela-
tive to the catalytic hexameric core, which stretches
and flexes in turn. The large active site of this pro-
tein spans from the hinge between domains I and II
to the residues of the dimer interface, and both abso-
lute conformation and conformational flexibility at
these areas of the protein are clearly likely to impact
on catalysis. Thus in addition to the reduced access
to the active site in this closed form of the protein, it
seems likely that the conformational restrictions
impact directly on catalysis.
5 mL HiTrapTM TALONV crude column (GE Health-
care) and eluted by applying a linear gradient to
high-imidazole buffer (50 mM phosphate, 500 mM
NaCl, 5 mM MgCl2, 250 mM imidazole, pH 8.0) over
35 mL. Protein containing fractions were pooled,
mixed with TEV protease in a 1:10 ratio for tag
removal. Incubation was performed with an initial
1-h period at 378C followed by three days at 48C.
The chain retained an N-terminal glycine residue
from the protease site. Highly pure CjeATP-PRT was
acquired by subsequent size exclusion chromatogra-
phy using
a 26/600 Superdex 200 column (GE
Many of the observed conformational and inter-
face changes between His-bound and non His-bound
CjeATP-PRT hexamer are also strikingly similar in
the reported structures of MtuATP-PRT and
EcoATP-PRT.11,15 This suggests a common allosteric
mechanism for all long form ATP-PRT enzymes. In
light of our own work and recent studies on
MtuATP-PRT,14 this mechanism does not appear to
involve the participation of an active dimeric species
and must therefore be mediated by the observed
changes of the homohexameric molecule. Thus our
preferred model of His inhibition to be almost
entirely driven by the rotational movement of the
domain III. This “twisting of the regulatory tail”
alters the overall conformation of the enzyme hex-
amer impacting on the catalytic properties of the
ATP-PRT hexamer.
Healthcare) with 10 mM Tris/HCl, 100 mM NaCl,
5 mM MgCl2, pH 8.0 as running buffer.
Determination of kinetic parameters
CjeATP-PRT activity was measured in vitro using a
continuous enzyme assay, based on the work of
Ames,7 directly following the accumulation of the
product PR-ATP via the absorbance increase at
290 nm. To force the reaction equilibrium completely
onto the product side E. coli pyrophosphatase (Eco
PPase) (Supplementary Material) was added in
excess.
The standard reaction mixture contained ATP
(2 mM), CjeATP-PRT (280 nM), and EcoPPase (6.4
mM) in 50 mM Tris/HCl, MgCl2 (10 mM), NaCl
(100 mM), KCl (50 mM), pH 8.5 at 258C in a total
volume of 1 mL. The reaction was initiated by addi-
tion of PRPP (200–300 mM). All assay components
were prepared in pre-treated water (Chelex100 resin
(BioRad)). ATP and PRPP solutions were mixed in a
1:1 molar ratio with MgCl2.
MATERIAL AND METHODS
Cloning and expression of Cje hisG
The hisG gene (locus tag Cj1597) was amplified from
C. jejuni subsp. jejuni NCTC 11168 genomic DNA in
three successive nested PCR steps (Supporting Infor-
mation Table S1), which incorporated a N-terminal
tobacco edge virus (TEV) protease cleavage site and
Kinetic parameters were obtained by fitting the
data to the Michaelis-Menten equation using GraFit
5.0 (Erithacus Software) or Prism 6.0 (GraphPad
Software). KM and kcat values were determined
using substrate concentrations ranges of 2.5–200 mM
for PRPP and 10–2000 mM for ATP. Ki values for His
and AMP were obtained from competitive assays
against both substrates using concentration ranges
of 0.5–3.0 mM AMP, 0.01–1.0 mM His, 0.08–1.0 mM
ATP and 0.03–0.3 mM PRPP. Excess substrate con-
centrations were held at 2 mM for ATP and 0.3 mM
for PRPP. Optimal concentrations for Mg21, Na1,
and K1 were measured using 0–100 mM Mg21, 0–
500 mM Na1, and 0–600 mM K1.
the attB sites required for subsequent cloning using
R
V
the Invitrogen Gateway system. The sequence veri-
fied Cje hisG was shuttled into expression vector
pDESTTM17 and this construct was transformed into
expression strain E. coli BL21* (DE3) pBB542 1
pBB540 (Chaperone 3).
Growth media (LB-Lennox) containing 100 mg/L
ampicillin, 25 mg/L chloramphenicol and 100 mg/L
spectinomycin was inoculated with a similar over-
night preculture (25 mL per L growth) and left to
grow at 378C and 180 rpm until the logarithmical
growth phase was reached. After addition of IPTG to
a final concentration of 0.5 mM the cells were fur-
ther cultured at 238C and 180 rpm over night until
harvested.
Differential scanning calorimetry
330 lL of degassed 1 mg/mL CjeATP-PRT in 50 mM
Tris/HCl, 100 mM NaCl, 5 mM MgCl2, pH 8.5 were
analyzed using a NanoDSC (TA Instruments) against
the identical buffer. Ligand concentrations used were
2 mM AMP and 1 mM His. The temperature was
increased from 20 to 1008C in increments of 18C per
min under a constant pressure of three atmospheres.
Data analysis was performed with the NanoAnalyze
software version 3.3.0 (TA Instruments).
Purification of CjeATP-PRT
Freshly harvested cells were resuspended in lysis
buffer (50 mM phosphate, 500 mM NaCl, 5 mM
MgCl2, 10 mM imidazole, pH 8.0) and lysed by soni-
cation. The clarified supernatant was loaded onto a
€
Mittelstadt et al.
PROTEIN SCIENCE VOL 00:00—00 13

was 2 or 4 lL and the reservoir solution volume 500
lL. Typically crystals were fully formed after 1–3
days. Crystals were cryoprotected in a mix of 20%
PEG400 and 80% reservoir solution (His co-crystals)
or with 40% glycerol added directly to the drop
before looping. Crystals were flash cooled in liquid
nitrogen and stored in pucks.
Isothermal titration calorimetry (ITC)
ITC experiments were run using a VP-ITC microca-
lorimeter at 298 K (MicroCal, GE Healthcare). Prior
to use all solutions were filtered and degassed under
vacuum. All experiments were conducted in 10 mM
Tris/HCl, 100 mM NaCl, 5 mM MgCl2, pH 8.5. For
Kd measurements ATP (4 mM), AMP (3.5 mM), and
His (1.5 mM) were titrated into 100 lM (150 lM in
case of ATP) CjeATP-PRT. The synergistic ligand
binding effects were investigated using 1 mM His
(1 mM AMP background) and 2 mM AMP (1 mM
His background), respectively. Titrations were per-
formed with 55 injections of ligand: one 2-mL injec-
tion followed by fifty-four 5-mL injections. Heat of
dilution experiments were measured independently
and subtracted from the integrated data before
curve-fitting in Origin 7.0.
Diffraction data was acquired at the MX131 and
MX2 beamlines of the Australian Synchrotron. The
data was processed using XDS32 and scaled using
the program AIMLESS. Initial phases were solved
by a molecular replacement strategy using the CCP4
program suite.33 The initial search model was gener-
ated from E. coli ATP-PRT (PDB ID: 1H3D), con-
taining only domain I and II. A suitable dimeric unit
was identified and used in subsequent runs until a
hexameric arrangement was found. The missing reg-
ulatory domains were added through multiple
rounds of refinement using REFMAC534 and model
building using Coot.35 The ATP-bound (PDB ID:
4YB7) and His-bound (PDB ID: 4YB5) crystal struc-
tures were solved following this strategy, while the
AMP and His-bound structure (PDB ID: 4YB6) was
solved using the His-bound CjeATP-PRT structure.
The structures were validated using the MolProbity
server36 and wwPDB validation server37 before dep-
osition. All RMSD values given are based on Ca
atoms.
Analytical ultracentrifugation (AUC)
Sedimentation velocity experiments were performed
in a Beckman Coulter model XL-I analytical ultra-
centrifuge equipped with UV–visible scanning optics.
Reference (400 mL of 10 mM Tris/HCl, 100 mM
NaCl, 5 mM MgCl2, pH 8.5) and sample (380 mL)
solutions were loaded into 12-mm double-sector cells
with quartz windows, and the cells were then
mounted in an An-50 Ti eight-hole rotor. Proteins
were centrifuged at various rotor speeds at 208C,
and radial absorbance data were collected at 230 nm
wavelengths, due to the relatively low absorbance of
CjeATP-PRT at 280 nm, in continuous mode every 8
min without averaging. Data was fitted to a continu-
ous distribution c(s) model using SEDFIT29 version
14.6. Partial specific volume (0.7482 g/mL), buffer
density (1.00618 cp) and viscosity (0.01033 poise)
were calculated using SEDNTERP.30 The frictional
coefficient ratio calculated from the c(s) fit was 1.29.
Electronic supplementary material: Methods for
preparation of E. coli pyrophosphatase, mass spec-
trometry, differential scanning fluorimetry, and
results showing metal ion concentration optimiza-
tion for assays, full kinetic plots, ITC data for ATP
binding in the presence of His, thermal stability,
size exclusion chromatography, sequences alignment
and analysis of interfaces and RMSD values.
Acknowledgments
X-ray data were collected at the MX beamlines of
the Australian Synchrotron with access provided by
the New Zealand Synchrotron Group.
Protein crystallization and structure
determination
R
After initial screening with a MosquitoV Crystal
robot (TTP Labtech) using the PACT premierTM HT-
96, Clear StrategyTM I HT-96 and Clear StrategyTM
II HT-96 screens (Molecular Dimensions), several
suitable conditions were identified and further
refined. Co-crystallization was achieved in 0.1M
sodium acetate, 0.1M MgCl2, 13–15% w/v PEG 4000,
pH 5.5 (ATP), 0.1M BTP, 0.2M KSCN, 13–14% w/v
PEG 3350, pH 6.5 (His) and 0.1M Tris, 0.1M MgCl2,
13–15% w/v PEG 4000, pH 7.5 (His/AMP). The
required CjeATP-PRT concentration was 2.0–3.5 mg/
mL and ligand concentrations used were 10 mM,
5 mM and 1 mM for ATP, AMP and His, respec-
tively. All crystals were grown using the hanging
drop vapor diffusion method in 24-well VDX plates
(Hampton Research). Protein solutions were mixed
1:1 (v/v) with the reservoir solution. The drop size
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Campylobacter jejuni ATP-Phosphoribosyltransferase

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