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Crystal Structure of the 47-kDa Lipoprotein of Treponema pallidum
Reveals a Novel Penicillin-binding Protein*
Received for publication, July 23, 2002, and in revised form, August 7, 2002
Published, JBC Papers in Press, August 24, 2002, DOI 10.1074/jbc.M207402200
Ranjit K. Deka‡, Mischa Machius§, Michael V. Norgard‡
¶
, and Diana R. Tomchick§
From the Departments of ‡Microbiology and §Biochemistry, University of Texas Southwestern Medical Center,
Dallas, Texas 75390
Syphilis is a complex sexually transmitted disease
caused by the spirochetal bacterium Treponema palli-
dum. T. pallidum has remained exquisitely sensitive to
penicillin, but the mode of action and lethal targets for

-lactams are still unknown. We previously identified
the T. pallidum 47-kDa lipoprotein (Tp47) as a penicil-
lin-binding protein (PBP). Tp47 contains three hypo-
thetical consensus motifs (SVTK, TEN, and KTG) that
typically form the active center of other PBPs. Yet, in
this study, mutations of key amino acids within these
motifs failed to abolish the penicillin binding activity of
Tp47. The crystal structure of Tp47 at a resolution of
1.95 Å revealed a fold different from any other known
PBP; Tp47 is predominantly

-sheet, in contrast to the
␣
/

-fold common to other PBPs. It comprises four dis-
tinct domains: two complex

-sheet-containing N-termi-
nal domains and two C-terminal domains that adopt
immunoglobulin-like folds. The three hypothetical PBP
signature motifs do not come together to form a typical
PBP active site. Furthermore, Tp47 is unusual in that it
displays

-lactamase activity (k
cat
for penicillin ⴝ 271 ⴞ
6s
ⴚ1
), a feature that hindered attempts to identify the
active site in Tp47 by co-crystallization and mass spec-
trometric techniques. Taken together, Tp47 does not fit
the classical structural and mechanistic paradigms for
PBPs, and thus Tp47 appears to represent a new class of
PBP.
Syphilis is a chronic, complex sexually transmitted disease of
humans caused by the spirochetal bacterium Treponema palli-
dum. Humans are the only known reservoir for T. pallidum,
and although syphilis is one of the oldest recognized sexually
transmitted diseases, a major impediment to research on T.
pallidum continues to be the inability to cultivate the organism
in vitro. Consequently, despite decades of intensive efforts,
many features of T. pallidum ultrastructure, physiology, and
membrane biology remain obscure (1).
T. pallidum is exquisitely sensitive to penicillin, which con-
tinues to be the drug of choice for syphilotherapy. Penicillin
and other

-lactams are bactericidal via their ability to inhibit
cytoplasmic membrane-bound enzymes (penicillin-binding pro-
teins (PBPs))
1
involved in peptidoglycan biosynthesis (2). Gen-
erally, bacteria contain several PBPs that are classified within
two categories (high molecular weight or low molecular weight)
(3, 4). In Escherichia coli, the high molecular weight PBPs tend
to be bifunctional (transglycosylase/transpeptidase activities)
and are the lethal targets of

-lactams (5). The low molecular
weight PBPs can be either monofunctional DD-carboxypepti-
dases, bifunctional DD-carboxypeptidases/DD-endopeptidases,
or monofunctional DD-endopeptidase (6). In T. pallidum, the
lethal targets for

-lactams are not known. However, two pre-
vious studies in which T. pallidum was incubated in vitro with
radiolabeled

-lactams implicated polypeptides of 94, 80, 63,
58, 47, and 38 kDa (7) or 180, 89, 80, 68, 61, 41, and 38 kDa (8)
as PBPs. As a follow-up to an earlier study by us (7), we have
shown that the major 47-kDa membrane lipoprotein of T. pal-
lidum (Tp47) is a PBP. More recent genome information (9) has
suggested that T. pallidum encodes at least three theoretical
PBPs of molecular masses of 71 (TP0500, PBP-1; TP0760,
PBP-3) and 98 (TP0705; PBP-2) kDa, but direct biochemical
evidence for these proteins as PBPs are lacking. An additional
protein putatively has been assigned as a serine-type DD-
carboxypeptidase (53-kDa, TP0800), and another as a DD-
carboxypeptidase (29-kDa, TP0221). No

-lactamases have
been predicted to be present in T. pallidum (9).
The notion that Tp47 is a PBP has been paradoxical. First,
Tp47 has no homologies with any other bacterial or eukaryotic
proteins. Second, conventional PBPs contain three conserved
motifs, SXXK, S(Y)XN, and KT(S)G, which comprise the active
site for the covalent binding of

-lactams (10–12). The serine of
the SXXK motif is important for nucleophilic attack on the

-lactam ring. Tp47 contains three such appropriately spaced
hypothetical motifs (SVTK, TEN, KTG) (13). However, prelim-
inary experiments replacing Ser in the SVTK motif of Tp47
with Gly, Ala, Cys, or Thr all yielded mutant enzymes that still
bound

-lactam comparable with wild-type Tp47 (14). Finally,
lipidation of PBPs also is uncommon (15).
The numerous incongruities surrounding Tp47 as a PBP
prompted the current biochemical and biophysical study. Spe-
cifically, it was envisioned that precise structural information
derived from x-ray crystallography could provide strategic in-
formation to guide future biochemical studies on the enzymatic
activity of Tp47. In this study, it was found that Tp47 has a
crystal structure unique to any other known PBP, and thus it
appears to represent an entirely new class of PBP.
* This work was supported by Grant AI-16692 from the NIAID,
National Institutes of Health, and by Grant I-0940 from the Robert A.
Welch Foundation. Use of the Argonne National Laboratory Structural
Biology Center beamline at the Advanced Photon Source was supported
by the United States Department of Energy, Office of Biological and
Environmental Research, under Contract W-31-109-ENG-38. The costs
of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked “advertise-
ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this
fact.
¶
To whom correspondence should be addressed: Dept. of Microbiol-
ogy, University of Texas Southwestern Medical Center, 6000 Harry
Hines Blvd., Dallas, TX 75390. Tel.: 214-648-5900; Fax: 214-648-5905;
E-mail: michael.norgard@utsouthwestern.edu.
1
The abbreviations used are: PBP, penicillin-binding proteins; Dig-
Amp, digoxigenin-labeled ampicillin; MALDI-TOF, matrix-assisted la-
ser desorption ionization time-of-flight; ESI-MS, electrospray ioniza-
tion-mass spectrometry; NAM, N-acetylmuramic acid.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 44, Issue of November 1, pp. 41857–41864, 2002
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org 41857
EXPERIMENTAL PROCEDURES
Construction of Wild-type and Variant Tp47-Streptavidin Fusion
Proteins—The post-translationally modified N-terminal cysteine of na-
tive Tp47 was designated as amino acid 1 (16). To ensure production in
E. coli of a nonlipidated version of Tp47, a DNA fragment encoding
amino acids 2– 415 (residue 415 is the last amino acid before the first
TAG termination codon in tp47) (16) was amplified by PCR using T.
pallidum genomic DNA (17) as template. The PCR primers were 5⬘-
tccCCGCGGCTCGTCTCATCATGAGACGCA-3⬘ and 5⬘-catgCCATG-
GTTACTACTGGGCCACTACCTCGCA-3⬘. The forward primer con-
tained both a tcc overhang and a SacII site (bold); the reverse primer
contained a catg overhang, a NcoI site (bold), and two contiguous stop
codons (TTA, CTA). PCR amplification was performed using Vent DNA
polymerase (New England Biolabs). Amplified fragments were cleaved
with SacII and NcoI and cloned directionally into SacII- and NcoI-
cleaved pASK-IBA7 vector (Sigma). This construct was designated as
wild type, and was verified by DNA sequencing and then transformed
into E. coli DH5
␣
.
Site-directed mutagenesis of tp47 was carried out by a PCR-based
method, using two complementary mutation-harboring oligonucleotides
for each mutant and the QuikChange site-directed mutagenesis kit
(Stratagene). Five different mutant genes were constructed; four en-
coded proteins with single amino acid substitutions and one contained
a double substitution. The mutant proteins expressed were designated
as Tp47S100G, Tp47S100C, Tp47K287Q, Tp47C296A, and Tp47H5S/
H9S, based on the amino acid positions involved. Finally, a fusion
construct in which C-terminal residues 329–415 were deleted (corre-
sponding to Domain D; see crystal structure Fig. 2 (Tp47⌬D)) also was
constructed by PCR subcloning as described above, except that the
reverse primer was 5⬘-catgCCATGGTTACTAATCAGCAACTACGT-
CC-3⬘. All resulting mutants were sequenced to verify the specific
mutation(s) intended. Mutant tp47 genes were expressed in E. coli, and
the cognate proteins were purified as described below for wild-type
Tp47. SDS-PAGE analysis revealed that the mutants expressed quan-
tities of proteins comparable with wild-type Tp47, suggesting that none
of them was unstable (data not shown).
Expression and Purification of Tp47—E. coli DH5
␣
containing the
respective cloned tp47 gene fusion was grown at 37 °C in LB medium
containing 100
g of ampicillin per ml; when the A
600
of the culture
reached 0.6, the culture was shifted to 30 °C and expression of the
Tp47-streptavidin fusion protein was induced (via a tetA promoter) by
the addition of 200
g/liter of anhydrotetracycline. After 3 h, cells were
harvested by centrifugation and solubilized by B-PER II (Pierce). After
centrifugation at 15,000 rpm for 20 min (4 °C) to remove cellular debris,
the supernatant was loaded onto a StrepTactin-Sepharose column. The
fusion protein was then purified according to the Strep-tag II protein
expression and purification system manual (Sigma). The yield of puri-
fied proteins tended to be about 25 mg/liter of bacterial culture. Purified
protein was subjected to buffer exchange with buffer A (20 m
M Hepes
buffer, pH 7.4, 20 mM NaCl) using a PD-10 column (Amersham Bio-
sciences). The protein was then concentrated to about 15 mg/ml using a
Centricon YM-10 device (Amicon). Protein purity was analyzed by SDS-
PAGE (18) and by electrospray ionization-mass spectrometry (ESI-MS).
The concentration of purified protein was estimated spectrophotometri-
cally using a calculated extinction coefficient of
⑀
280
⫽ 54,050 M
⫺1
cm
⫺1
(19).

-Lactam Binding to Tp47—Binding of digoxigenin-labeled ampicil-
lin (Dig-Amp) to Tp47 was determined by a chemiluminescent detection
method (13, 20); the use of Dig-Amp circumvents problems associated
with utilizing radiometric methods for assaying

-lactam binding (20).

-Lactam binding to Tp47 also was examined by ESI-MS; in these
experiments, a typical 100-
l reaction mixture contained 100
gof
protein, 2 m
M ZnCl
2
,and2mM

-lactam (in buffer A) and was incubated
at 37 °C for various times. The reaction was terminated by the addition
of 30
l of 5% formic acid. Excess

-lactam was removed by a Microcon
YM-30 device (Amicon), and samples were recovered in 1% formic acid
for ESI-MS analysis (21). The peak heights of free and acylated Tp47
were measured from the ESI-MS spectra and the percentage of Tp47
acylation was calculated using the equation: % acylation ⫽ [acylated
Tp47/(acylated Tp47 ⫹ free Tp47)] ⫻ 100 (22). In an attempt to identify
the Tp47 amino acid involved in penicillin binding, liganded sample
was digested with trypsin in 100 m
M ammonium bicarbonate (pH 7.8,
37 °C); after digestion for various times, samples were subjected to
MALDI-TOF MS (23).
Kinetic Analysis of

-Lactamase Activity—The hydrolytic activity of
Tp47 on various

-lactams was assessed at 37 °C in buffer A using a
Shimadzu UV-1601PC UV-visible spectrophotometer equipped with a
thermostated multicell transport system. The molar absorption coeffi-
cients used were as follows: penicillin G, ⌬
⑀
235
⫽⫺775 M
⫺1
cm
⫺1
;
ampicillin, ⌬
⑀
235
⫽⫺820 M
⫺1
cm
⫺1
; nitrocefin, ⌬
⑀
486
⫽ 16,000 M
⫺1
cm
⫺1
.

-Lactam solutions were freshly prepared in buffer A. Initial rates were
determined from the first 5–10% of the reactions at various substrate
concentrations. K
m
and V
max
values were determined by fitting all data
to the Lineweaver-Burk equation using the program UV Probe
(Shimadzu).
Tazobactam inhibition of the hydrolytic activity of Tp47 was per-
formed with penicillin as a competitor substrate in buffer A. Tazobac-
tam at various concentrations was preincubated with Tp47 for 5 min at
37 °C before the addition of penicillin. Steady-state rates during the
course of penicillin hydrolysis were used to calculate the remaining
activity. The inhibition constant (K
i
) was deduced from Dixon plots
using the UV Probe software.
Protein Crystallization and Data Collection—Wild-type Tp47 de-
scribed above did not yield crystals in preliminary screening experi-
ments. However, one of the variant versions of Tp47, in which His-5 and
His-9 were replaced with Ser (Tp47H5/H9S; Fig. 1), crystallized readily
and thus was designated as crystallizable Tp47 (cTp47). Of particular
importance, cTp47 retained PBP activity comparable with the wild-type
(Fig. 1). cTp47 was crystallized by the hanging-drop vapor diffusion
method (24) using 24-well Linbro plates (Hampton Research) at room
temperature. Sparse matrix crystallization kits (Hampton Research)
were used to screen preliminary crystallization conditions. Crystals of
average dimension of 50
m appeared within 3–4 weeks. Further
growth of the crystals was hindered because of phase separation/oil
formation, and these crystals diffracted poorly to a Bragg spacing (d
min
)
of 6 Å. Crystallization optimization using dextran sulfate eliminated
the phase separation and yielded substantially larger crystals (about
500
m) within 2– 4 days that diffracted to better than a d
min
of 3 Å.
Crystals were routinely obtained with drops containing 5
l of protein
solution (about 15 mg/ml in buffer A), and 5
l of 32% (w/v) PEG 4000
in 100 m
M sodium citrate, pH 5.6, 200 mM ammonium acetate, 3% (w/v)
dextran sulfate 8000 (Sigma), and ⫾100
M ZnCl
2
, equilibrated against
500
l of the latter solution at room temperature. Prior to data collec-
tion, crystals were transferred sequentially for 5 min to each of 5, 10,
and 15% (v/v) glycerol-enriched reservoir solution for cryogenic condi-
tioning. Diffraction data were collected at 100 K using a Rigaku RU300
rotating copper anode x-ray generator and R-axis IV image plate detec-
tor (Molecular Structures Corp., The Woodlands, TX). The diffraction
data were indexed, integrated, and scaled in the HKL2000 program
package (25).
The cTp47 crystals were found to exhibit the symmetry of space
group P3
2
21 with unit cell dimensions of a ⫽ b ⫽ 129.1 Å, c ⫽ 151.5 Å.
The crystals contained two molecules per asymmetric unit. The crystal
structure of cTp47 was determined by single wavelength anomalous
dispersion using a xenon derivative. The xenon derivative of a cTp47
crystal was prepared by exposing a preconditioned native crystal (in
glycerol-enriched reservoir solution) in a xenon chamber (kindly pro-
vided by Zhenming Wang) at 400 p.s.i. for 15 min at room temperature.
The chamber was then depressurized and the crystal flash-cooled in
liquid propane within 15 s. Diffraction data to a d
min
of 2.28 Å were
recorded. The data were reduced with the program package HKL2000.
Xenon sites were identified and refined to 3.0 Å within the program
package CNS (version 1.0) (26), resulting in an overall figure of merit of
0.35. The phases were further improved by density modification in CNS
including histogram matching, solvent flipping, and phase extension to
a d
min
of 2.28 Å, resulting in a final figure of merit of 0.95 (Table
I).
After the structure was solved, a synchrotron data set on a xenon-
derivatized cTp47 crystal was collected to a d
min
of 1.95 Å at the
Structural Biology 19-ID beamline at the Advanced Photon Source
(Argonne National Laboratory, Argonne, IL). Data collection and single
wavelength anomalous dispersion phasing statistics are provided in
Table I.
Model Building and Structure Refinement—Model building was per-
formed automatically (arp_warp 5.0) (27) and manually with the pro-
gram O (28). Structure refinement using the synchrotron data set was
carried out within CNS employing cycles of simulated annealing, con-
jugate gradient minimization, and calculation of individual atomic dis-
placement parameters. An overall anisotropic atomic displacement pa-
rameter and bulk solvent correction were used throughout the
refinement procedure. Water molecules were added where stereochemi-
cally reasonable after the protein part of the model was complete.
Inspection of the F
obs
⫺ F
calc
difference density map revealed a large
volume of positive difference density extending across the noncrystal-
lographic 2-fold axis, and located in the positively charged cleft between
domains B and C of each monomer. This density was modeled as a
Structure of the 47-kDa PBP of T. pallidum41858
dextran sulfate polysaccharide with an
␣
136 linkage and two sulfate
groups (on O-2 and O-3) per glucose. The final model contains residues
7 to 34 and 44 to 414 of molecule A, and residues 7 to 34 and 40 to 413
of molecule B, 14 residues with alternate conformations, five xenon
atoms, two complete and two partial sugar moieties of a dextran sulfate
polysaccharide, and 407 water molecules. Residues 2 to 6, 35 to 43, and
415 in molecule A, and residues 2 to 6, 35 to 39, and 414 to 415 in
molecule B were disordered in the crystal structure and could not be
traced in the electron density. The final R
free
value is 23.5% and the
R
work
value is 21.2% (Table I).
Analytical Ultracentrifugation—Sedimentation equilibrium studies
were performed in a Beckman XL-1 Optima analytical ultracentrifuge
at 4 °C. Tp47 samples corresponding to absorbancies of 0.1, 0.2, and 0.4
at 280 nm in buffer A were used. Samples were centrifuged at 14,000 ⫻
g to remove aggregates prior to loading. Experiments were conducted at
a rotor speed of 13,000 and 18,000 rpm and the radial scans at 280 nm
were recorded until equilibrium was reached. The sedimentation equi-
librium data were analyzed using the supplied software.
RESULTS AND DISCUSSION
Expression and Purification of Tp47—Bacterial lipoproteins
are membrane proteins by virtue of their three long-chain fatty
acids (post-translationally added to an N-terminal cysteine)
that serve solely as membrane insertion anchors (29). As such,
the long-chain fatty acids do not contribute to the conformation
of the protein. The proteins, in the absence of their acyl chains,
thus tend to be water soluble (consistent with the polypeptides
protruding into the periplasm or extracellular environment). A
cloning strategy therefore was implemented in which the
leader sequence and N-terminal cysteine codon of tp47 were
deleted, ultimately to yield a nonlipidated, water soluble ver-
sion of Tp47. Finally, soluble Tp47 and its variants were cre-
ated as fusion proteins with an N-terminal streptavidin tag,
which is only 18 amino acids long; the streptavidin tag thus
should have minimal, if any, conformational influence on Tp47.
This contention was corroborated by the findings that the fu-
sion proteins performed as predicted in PBP assays (see below).
Properties of Mutant Tp47 Enzymes—Ser-100 of a putative
SVTK tetrad in Tp47 (13) was altered to cysteine (Tp47S100C);
this mutation did not abolish Dig-Amp binding (Fig. 1) or
penicillin binding to Tp47 (ESI-MS data not shown). Similarly,
conversion of Ser-100 to glycine did not abrogate the binding of
penicillin to Tp47, as assessed by ESI-MS (not shown). Thus, as
initially proposed (14), it appears that Tp47 does not employ an
active-site serine to serve as a nucleophile and subsequent
covalent attachment site for

-lactams. This is in sharp con-
trast to what has been observed for other classical PBPs (30,
31). That a mutation of the presumptive active site serine had
no influence on the PBP activity of Tp47 provided the first
compelling evidence that Tp47 might be dissimilar from other
conventional, serine-type PBPs.
The KTG triad also forms a key component of the active site
cleft and is highly conserved within PBPs (10–12). However, if
Tp47 is not a serine-type PBP, it was postulated that the KTG
motif in Tp47 may be coincidental, or may function in some
other unknown manner. For example, the positive charge on
the Lys might interact with the carboxylate group of the
D-Ala-
TABLE I
Data collection and refinement statistics
Data collection values are as defined in the program SCALEPACK (25). Model refinement values are as defined in the program CNS (26).
Data set Laboratory source Synchrotron
A. Data Collection
Wavelength Cu-K
␣
0.97938 Å
Space group P3
2
21
P3
2
21
Cell dimensions a ⫽ 129.1 Å, c ⫽ 151.5 Å a ⫽ 128.9 Å, c ⫽ 151.2 Å
Data range (Å) 30.4–2.17 28.2–1.95
No. of measurements 521,495 478,972
No. of independent reflections 139,645
a
105,601
R
merge
(%)
b
Overall 4.6 5.8
Last shell 53.0 (2.20–2.17 Å) 70.2 (2.02–1.95 Å)
Data completeness (%)
Overall 93.1 99.8
Last shell 82.3 100
I/(
)I
Overall 22.3 21.4
Last shell 2.0 1.9
R. refinement
No. of reflections used in refinement
Working set 94,665 (35.0–1.95 Å)
Test set 5,036
No. of non-H protein atoms 6,356
No. of Xe atoms 5
No. of polysaccharide atoms 39
No. of water molecules 407
R
work
(%)
21.2
R
free
(%)
23.5
R.m.s.d. in bond lengths (Å) 0.010
R.m.s.d. in bond angles (°) 1.45
Mean B value (Å
2
)
Main chain 41.6
Side chain 44.5
Xenon atoms 46.7
Polysaccharide atoms 62.4
Water molecules 41.3
A
-Coordinate error (Å)
0.22
Missing residues Molecule A: 2–6, 35–43, 415
Molecule B: 2–6, 35–39, 414–415
No. of alternate conformations 14
a
This data set was scaled with Bijvoet pairs unmerged.
b
R
merge
⫽ 100兺
h
兺
i
ⱍI
h,i
⫺ 具I
h
典ⱍ/兺
h
兺
i
I
h,i
, where the outer sum (h) is over the unique reflections and the inner sum (i) is over the set of independent
observations of each unique reflection.
Structure of the 47-kDa PBP of T. pallidum 41859
D-Ala, and hence the carboxylate group of penicillin (30, 32).
However, when Lys-287 of the KTG triad in Tp47 was mutated
to Gln (Tp47K287Q), the mutant protein retained its penicillin
binding activity (Fig. 1). Inasmuch as the mutation of Lys in
the KTG motifs of other PBPs typically adversely impacts PBP
activity (30), our results further underscore the atypical char-
acter of Tp47.
cTp47 Structure—Findings that Tp47 seemed not to rely on
either an active site serine (of SVTK) nucleophile or a KTG
motif for PBP activity were anomalous. This prompted a struc-
tural approach to gain further insights into the structure-
function relationships for Tp47 as perhaps a novel PBP. Ini-
tially, crystal growth of cTp47 was hampered by the occurrence
of phase separation, and the resulting crystals were small and
diffracted poorly to a Bragg spacing (d
min
)of6Å. Phase sepa
-
ration could be overcome by the addition of dextran sulfate,
resulting in larger crystals (up to 500
m in the largest dimen-
sion) that diffracted to a d
min
of 3 Å. These crystals exhibited
the symmetry of space group P3
2
21, with two molecules per
asymmetric unit. The crystal structure of cTp47 was deter-
mined via the single wavelength anomalous dispersion tech-
nique using a xenon derivative. Derivatization with xenon not
only provided phase information, but also increased the diffrac-
tion limit of the cTp47 crystals to a d
min
of 1.95 Å using
synchrotron radiation.
The crystal structure of cTp47 revealed four distinct domains
arranged to give the molecule a crab-like appearance (Fig. 2).
The first domain (domain A; residues 7 to 34 and 156 to 204) is
mainly composed of

-strands and is sequentially non-contig-
uous. The core of this domain is formed by a strand-helix-
strand motif (A

2-A
␣
3-A

3) (Fig. 3) in a right-handed super-
helical arrangement. Adjacent to A

2isa

-hairpin (strands
A

4 and A

5) whose tip interacts with the helix to create a
barrel-like structure. The N terminus of cTp47 forms a

-strand (A

1) that inserts between A

2 and A

3 to complete
a five-stranded, highly twisted, mixed

-sheet (order 3, 1, 2, 4,
5). A helix-loop-helix motif (A
␣
1 and A
␣
2) next to the

-hairpin
completes domain A and connects to the adjacent domain B. A
structural comparison of this domain using the program DALI
(33) did not reveal any similarity with proteins in the Protein
Data Bank (highest Z-score of 1.7). The largest recognizable
structural motif within this domain is generated by strand
A

1, helix A
␣
3, and strand A

3 that forms an anti-parallel
two-stranded

-sheet with an opposing helix. This motif also
has been observed in the Lactobacillus casei Hpr kinase (Pro-
tein Data Bank code 1jb1).
Domain B (residues 44 to 155) contains 10

-strands and a
single
␣
-helix (Figs. 2 and 3). Its main structural feature is a
central four-stranded, anti-parallel

-sheet (strands B

1,
B

10, B

2, and B

5). This sheet is opposed by an
␣
-helix (B
␣
1)
resulting in an arrangement that resembles a right hand, with
the strands being the fingers (strand B

1 is the index finger)
and the helix as the thumb. The backside of the sheet forms a
flat outer surface. At the N and C termini of strand B

5 are two
large

-hairpins (strands B

3/B

4 and B

8/B

9) that are ori-
ented perpendicular to the central sheet. These hairpins, to-
gether with large connecting loops and a third

-hairpin
(strands B

5/B

6) in between them, form a second flat outer
surface. The central motif consists of strands B

1, B

10, and
B

3, and helix B
␣
1, which is typical of cysteine proteases (34).
In fact, the topology of domain B in cTp47, except for the
hairpin formed by strands B

3 and B

4, is conserved in the
cysteine protease staphopain from Staphylococcus aureus (Pro-
tein Data Bank code 1cv8). Yet, Tp47 does not appear to be a
cysteine protease as a cysteine is not present in a region equiv-
alent to the active site in cysteine proteases. Furthermore,
mutation of the sole Cys (Cys-296, which is buried in the
hydrophobic core of domain C) to alanine had no effect on PBP
activity (Fig. 1), thereby ruling out involvement of this residue
in catalysis.
Domain C (residues 205 to 332) is the largest domain (Fig. 2).
It is mainly characterized by an immunoglobulin fold with two
opposing

-sheets that form the typical barrel-like structure. In
contrast to the classical immunoglobulin fold, however, domain
C of cTp47 has an additional

-strand inserted after strand 3.
Also, the strands are connected by rather large loops. Helices
are inserted between strands 2 and 3 and between strands 4
and 5.
Domain D (residues 333 to 414) also features an immunoglob-
ulin fold. In contrast to domain C, it contains only the character-
istic seven-stranded barrel and short loops. As in domain C, a
single
␣
-helical turn is inserted between strands 2 and 3.
Dimer Formation—In our crystals of cTp47, a dimer was
formed between two neighboring molecules (Fig. 4). Domains B
and D act as the pincers on a crab that make contact with the
pincers of the opposing molecule. The monomer-monomer in-
terface has an area of about 1,830 Å
2
and features a series of
polar and hydrophobic interactions as well as six ionic interac-
tions. This finding prompted further assessment of Tp47 dimer
formation in free solution by analytical ultracentrifugation.
The sedimentation equilibrium data profile produced by ana-
lytical ultracentrifugation fit well to a model comprising a
single species of molecular mass of 46,178 Da (not shown),
consistent with the monomeric mass determined by SDS-PAGE
and ESI-MS, supporting the observation that Tp47 displays
monomeric characters in free solution. Consequently, Tp47
dimer formation observed within the crystal structure could be
a result of crystallization, with the high salt concentration
driving a nonspecific association of the hydrophobic surfaces,
as has been noted for other proteins undergoing crystal packing
(35, 36). In fact, when domain D (which is not required for PBP
activity) is removed from the buried surface area calculation,
only ⬃850 Å
2
surface area is buried at the monomer-monomer
interface. This is a value found at the upper limit of buried
surface area for nonspecific crystal contacts (37).
Domain Interfaces—The first three domains in cTp47 inter-
act with each other through intimate domain-domain inter-
faces. Domain A contacts domain B through its N-terminal
segment that contains

-strand A

1 and the helix-loop-helix
motif, establishing interactions with the loop regions before the
first

-strand (B

1) and after the last

-strand (B

10) in do-
main B. The first linking region between these domains (resi-
dues 34 and 44) is disordered in the crystal structure. Domain
A also interacts tightly with domain C, involving mainly side
chains in helix A
␣
2 and the loop region between

-strands A

3
and A

4 in domain A and

-strands C

3

and C

6 as well as
the loop region between strands C

6 and C

7.
Domain B interacts with domain C via a surface that has a
slightly concave, goblet-like shape. The long loops proximal to
strand B

1 and between strands B

5 and B

6 form the sides,
and helix B
␣
1 forms the bottom of the goblet. Residues in these
FIG.1.Binding of Dig-Amp to wild-type (Wt) and mutant vari-
ants of Tp47. Recombinant versions of Tp47 incubated with Dig-Amp
were separated by SDS-PAGE, electrotransferred to nylon membrane,
and developed by chemiluminescence (13, 20). Dig-Amp binding was
assessed in the presence of ZnCl
2
except where noted (⫺ZnCl
2
). ⫺Dig
-
Amp, wild-type Tp47 without Dig-Amp treatment.
Structure of the 47-kDa PBP of T. pallidum41860
regions establish a number of polar and hydrophobic interac-
tions with residues at the surface of domain C, which includes
strands C

3, C

3

, and C

4, the loop region between strands
C

5 and C

6, and helix C
␣
1. Adjacent to this interaction sur-
face is a deep cleft located between the

-hairpin B

3/B

4 and
the rest of domain B. The tip of this hairpin, as well as the
portion of this surface that is not involved in interactions with
domain C, are highly positively charged containing five argin-
ines, two histidines, and two lysines.
In contrast to domains A, B, and C, domain D is rather
isolated. It interacts only with domain C via an ionic interac-
tion between Arg-330 and Glu-404 in the linker region. Conse-
quently, the relative disposition of domain D is expected to
vary. Evidence for a larger degree of domain motion can be
found in the higher average displacement factors for the atoms
of domain D relative to the first three domains (38.8 versus 57.8
Å
2
for monomer A, 38.0 versus 67.4 Å
2
for monomer B).
Comparison of Tp47 with Other PBP Structures—The three-
dimensional structure of a conventional PBP typically is com-
prised of two structural domains, one of which is predomi-
nantly
␣
and another that is
␣
/

(38) (Fig. 5). The active site is
positioned between these two major domains, at the edge of the
central

-sheet of the
␣
/

domain. The three signature se-
quence motifs of classical PBPs that putatively were present in
Tp47 do not come together in three-dimensional space to form
a typical active site. Given that Tp47 had no similarity to other
known PBPs, it was hypothesized that it might represent a new
family of PBPs. Consistent with this possibility, DALI did not
identify Tp47 as a PBP, but rather had the highest structural
homology (Z-score ⫽ 6.1) to non-PBPs.
Acylation and Deacylation of Tp47—The interaction between
PBPs and

-lactams generally is described by the equation: E ⫹
I u E.I 3 E-I 3 E ⫹ P, where E is the PBP enzyme, I is the

-lactam, E.I is the Michaelis intermediate, E-I is the covalent
acyl-enzyme complex, and P is the reaction product (i.e.
cleaved, inactive

-lactam) (38). The formation of the enzymat-
ically inactive (covalent) acyl-enzyme complex (E-I) is known as
the acylation step. The covalent E-I complex results from the
nucleophilic attack of the carbonyl carbon atom of the

-lactam
ring by the hydroxyl group of the active site serine. The bacte-
ricidal efficiency of any

-lactam ultimately depends on the
stability of the E-I complex. However, hydrolysis of the acyl-
enzyme complex and release of the inactive

-lactam (P) occurs
by a process known as deacylation; in the case of

-lactamases,
deacylation is rapid. In former studies, Tp47 bound radiola-
beled penicillin (7), and its binding to Dig-Amp subsequently
was found to be stimulated by zinc ions (13). In the current
study, upon incubation of purified Tp47 for 2 min with penicil-
lin in the presence of zinc, two major peaks of 47,703 Da (free
Tp47) and 48,036 Da (penicillin-bound Tp47) were detected by
ESI-MS (not shown). The difference of 333 Da between the two
molecular masses corresponded with the mass of penicillin (335
Da), indicating the formation of a covalent acyl-Tp47 complex
bound predominantly in a 1:1 stoichiometry. Analogous results
were obtained using ampicillin, carbenicillin, cefuroxime, and
cephalosporin (not shown), indicating that recombinant Tp47
bound a number of

-lactams. In the absence of zinc, after 2
min of incubation, 5% of Tp47 became acylated, whereas, in the
presence of zinc, 33% of Tp47 was acylated over the same
interval (Table II), corroborating previous findings that the
PBP activity of Tp47 appears to be stimulated by zinc (13). In
the presence of zinc, acylation by penicillin was time-depend-
ent, with maximal binding observed at 6 min (Table II). How-
ever, after 6 min, marked deacylation was evident, implying
that Tp47 exhibits some intrinsic

-lactamase activity.
As shown in a previous study (13) and herein, zinc enhances
FIG.2. Stereoview of the cTp47
monomer. Domains A–D are drawn in
different colors to highlight the domain
boundaries. The residues of the hypothet-
ical PBP sequence motifs are labeled I
(
100
SVTK), II (
183
TEN), and III (
287
KTG)
(gray sticks). Figs. 2, 4, and 5 were pre-
pared with the programs BobScript (51),
POV-Ray (www.povray.org), and GLR
(L. Esser, personal communication).
FIG.3.Topology diagram of domain A (green) and domain B
(red) of cTp47. Strands are depicted as arrows, and helices are shown
as rectangular boxes. Strands and helices are numbered sequentially for
each domain. The N and C termini of domain A are shown.
Structure of the 47-kDa PBP of T. pallidum 41861
the binding of

-lactams to Tp47. This led to the initial idea
that Tp47 was a zinc-dependent PBP (13). Two lines of evidence
now challenge this view. First, we now show that rather than
promoting acylation, zinc actually inhibits the deacylation of
Tp47 (see below). Second, an in vitro carboxypeptidase assay
using the synthetic depsipeptide substrate Sle (an analog of
D-Ala-D-Ala) initially suggested that Sle was hydrolyzed by
Tp47 in the presence of zinc, as indicated by an apparent
increase in UV absorption at 254 nm (13). However, subse-
quent experiments have revealed that this apparent absorption
increase is due, at least in part, to scattering caused by Tp47
aggregates that form in the presence of zinc (not shown).
Hence, the initial contention that Tp47 might be a zinc-depend-
ent carboxypeptidase (13) remains tenuous at this time.
Mass spectrometry has been employed for the identification
of the penicillin-binding site in Staphylococcus aureus PBP 2a
(21). Using a similar strategy, liganded Tp47 was digested with
trypsin, and peptide fragments were assessed by MALDI-TOF
MS. Attempts to identify a particular peptide fragment to
which penicillin was bound were unsuccessful, suggesting that
the acylated product was unstable during the procedure. One
potential explanation for this was the intrinsic

-lactamase
activity inferred in Table II.
FIG.4.Stereoview of the cTp47 dimer. The xenon atoms used in phasing the structure are represented as cyan spheres. The N termini of both
monomers, which occur on the same face of the dimer, are oriented (as predicted) toward the cytoplasmic membrane surface.
FIG.5. Comparison of the cTp47
structure to representative

-lacta-
mases and PBPs. Representative struc-
tures from the major classes of

-lactama-
ses plus a
D-Ala-D-Ala-peptidase/PBP are
shown with domains A–C of Tp47 (do-
main D is not required for PBP/

-lacta-
mase activity). Black arrows highlight the
known active sites of the representative
structures. The green sphere in the Class
B structure represents a Zn
2⫹
ion. The
Class A structure is the TEM1

-lacta-
mase from E. coli (Protein Data Bank
code 1btl), the Class B structure is the
zinc metallolactamase from Bacillus
cereus (Protein Data Bank code 1bmc),
the Class C structure is the cephalospori-
nase from Enterobacter cloacae (Protein
Data Bank code 2blt), the Class D struc-
ture is the Oxa-10

-lactamase from
Pseudomonas aeruginosa (Protein Data
Bank code 1e3u), and the PBP structure
is the
D-Ala-D-Ala-peptidase/PBP from P.
aeruginosa (Protein Data Bank code
1ceg).
T
ABLE II
Percent of Tp47 remaining acylated with penicillin at various times
Acylation reactions were carried out in either the presence (⫹)or
absence (⫺)of2m
M ZnCl
2
.
Incubation time
% Acylation
⫹Zn
2⫹
⫺Zn
2⫹
min
2335
440—
a
650—
10 35 5
20 28 —
30 27 ND
b
a
—, undetermined.
b
ND, not detectable.
Structure of the 47-kDa PBP of T. pallidum41862
Kinetic Parameters for Tp47

-Lactamase Activities—Cer-
tain PBPs have intrinsic

-lactamase activity (30, 39). Kinetic
analysis of

-lactam hydrolysis was used to assess whether the
deacylation of Tp47 (Table II) was because of a similar intrinsic
ability to hydrolyze

-lactams. The kinetic parameters of hy-
drolytic activities of Tp47 were determined for three

-lactams
and are summarized in Table III. Tp47 exhibited an unexpect-
edly high level of

-lactam hydrolytic activity. Although the
turnover rates (k
cat
) for

-lactam hydrolysis by Tp47 were
10–20-fold lower than for typical

-lactamases (40), they are
substantially higher than the

-lactamase activity of E. coli
PBP5, which has an unusually high

-lactamase activity (k
cat
⫽ 0.07 s
⫺1
) (39). On this basis, it could be conjectured that Tp47
is a

-lactamase. However, from a biological perspective, this
notion is strongly inconsistent with the exquisite sensitivity of
T. pallidum to

-lactams, particularly when the extraordinary
abundance of Tp47 in T. pallidum is taken into account (41).
Thus, the biological relevance of the putative Tp47 in vitro

-lactamase activity remains suspect, as it may be of little or no
consequence to the biology of T. pallidum in vivo (i.e. during
human infection). Interestingly, a higher level of penicillin
binding to Tp47 was observed in the presence of zinc (Fig. 1 and
Table II). As noted earlier, zinc also induces the aggregation of
Tp47 (not shown), which appears as a suppression of in vitro

-lactamase activity. Taken together, it is tempting to specu-
late that the enhanced PBP activity of Tp47 has been observ-
able, at least in part, by virtue of the inhibitory action of zinc on
the intrinsic

-lactamase activity of Tp47. Finally, the in vitro
hydrolytic activity of wild-type Tp47 was inhibited by tazobac-
tam, an inhibitor of class A

-lactamases (42, 43), suggesting
that competitive inhibition is active site directed. The apparent
K
i
value for hydrolysis of penicillin by wild-type Tp47 was
26.95 ⫾ 0.35 n
M.
Potential Active Site—Catalytic centers of PBPs have a con-
served topology wherein three conserved motifs comprise the
catalytic center (10, 11). The sequence of Tp47 has three such
hypothetical signature motifs (13). However, mutations in the
Ser of the putative SVTK motif and Lys of the KTG motif did
not abrogate the PBP activity of Tp47 (Fig. 1). Furthermore, all
three motifs of classical PBPs initially thought to be present in
Tp47 are found in three different domains separated by dis-
tances greater than 30 Å (Fig. 2), supporting the contention
that the three hypothetical motifs do not comprise the active
site for

-lactam binding in Tp47. We thus conclude that Tp47
exhibits a unique mechanism for

-lactam binding. Further
inspection of the structure therefore was undertaken to iden-
tify the active site. Emphasis was placed on searching for
another reasonable PBP active site cleft, which might contain a
Ser nucleophile spatially near another residue suitable for
abstraction of a proton from the hydroxyl group of Ser (e.g. a
positively charged amino acid such as Lys). Such efforts were
not successful.
The predominance of hydrophobic residues and the immuno-
globulin fold of domain D suggested that it might be utilized for
protein-protein interaction(s) when in its native membrane
setting within T. pallidum. In addition, the location, flexibility,
and relative disposition of domain D suggests that it might not
be involved in PBP and

-lactamase activities. In this regard, a
domain D deletion mutant of Tp47 (Tp47⌬D) retained wild-
type levels of both activities (not shown). Thus, it is reasonable
to conclude that domain D has no catalytic role in the PBP
activity of Tp47.
An analysis of the charge distribution on the surface of
domains A–C of the Tp47 monomer is shown in Fig. 6. A
positively charged cleft is found at the intersection of domains
B and C, close to the domain B

-hairpin formed by strands
B

3 and B

4. This cleft might function as a binding site for the
carboxylate of
D-Ala-D-Ala, and hence

-lactams. In the crystal
structure, this cleft is found near the noncrystallographic 2-fold
axis of the dimer. A dextran sulfate polysaccharide with an
␣
136 linkage was modeled into the positive difference density
found in this cleft. Approximately one-half of the electron den-
sity assigned to the polysaccharide is associated with each
protein monomer, and the hydrogen-bonding pattern between
the protein and each sulfated dextrose monosaccharide is sim-
ilar. An attempt to model the polysaccharide backbone of nat-
urally occurring peptidoglycan (repeating N-acetylmuramic
acid (NAM)

134 linked to N-acetylglucosamine) into this
density was not successful. An NAM monomer could be mod-
eled into the density, but the

134 linkage of NAM-N-acetyl-
glucosamine was inconsistent with the local 2-fold symmetry of
the cleft. If Tp47 utilizes this cleft for the interaction of

134-
linked peptidoglycan subunits, it appears that steric con-
straints dictate that the protein be in the monomeric state, as
supported by our sedimentation equilibrium experiments.
Whereas small crystals of cTp47 normally can be grown in the
absence of dextran sulfate, crystallization with NAM or N-
acetylglucosamine monosaccharides in place of dextran sulfate
did not yield cTp47 crystals.
Further attempts to identify the active site of Tp47 by co-
crystallization and/or soaking of crystals with

-lactams were
unsuccessful, probably because of the deacylation activity
noted earlier. A 3.8-Å data set was obtained from a co-crystal-
lization and soak of cTp47 with the

-lactamase inhibitor ta-
zobactam. The electron density map revealed changes in the
positively charged cleft that may be because of a partial dis-
placement of the dextran sulfate polysaccharide by the tazobac-
tam, but an unambiguous fit of the inhibitor into this low
resolution map was not possible.
Biological Significance and Implications—Tp47 was first
noted in early molecular studies of T. pallidum, due largely to
its abundance and profound immunogenicity (41). It thus ini-
tially was targeted for study as a potential syphilis serodiag-
nostic reagent (41, 44), and many newer generation serological
tests for syphilis now include Tp47 as a principal, if not sole,
antigenic component (45, 46). Tp47 initially also was thought to
be an outer membrane protein (41). However, a more extensive
TABLE III
Kinetic parameters for wild-type Tp47 hydrolytic activities using
ampicillin, penicillin G, or nitrocefin at pH 7.4 (37 °C)
Substrate k
cat
K
m
k
cat
/K
m
s
⫺1
M s
⫺1
M
⫺1
Ampicillin 187 ⫾ 22 75 ⫾ 7 2.49 ⫾ 0.06
Penicillin G 271 ⫾ 682⫾ 3 3.31 ⫾ 0.20
Nitrocefin 131 ⫾ 28 94 ⫾ 1 1.38 ⫾ 0.28
FIG.6.The cTp47 monomer has a positively charged cleft. A
surface representation of the electrostatic charge distribution for the
Tp47 monomer (domains A–C) is shown at the left of the figure and is
in the same orientation as in Fig. 2. The central figure was obtained via
a rotation of 90° about the horizontal axis of the monomer. For com-
parison, the charge distribution for the active site cleft in the
D-Ala-D-
Ala-peptidase/PBP (Protein Data Bank code 1ceg) is shown at the right
of the figure. The displayed surface potential varies approximately from
⫺10 to 10 kT with acidic surfaces in red and basic in blue. The electro-
static surface potential was calculated and rendered in the program
GRASP (52).
Structure of the 47-kDa PBP of T. pallidum 41863
body of work, which has taken into account the previously
unrecognized fragility of the unusual T. pallidum outer mem-
brane (1, 47), later supported that Tp47 likely is a cytoplasmic
membrane lipoprotein that, according to convention, would
protrude into the periplasmic space (47, 48). This finding was
more consistent with earlier studies that implicated it as a PBP
(7, 13), inasmuch as PBPs reside at the cytoplasmic membrane
(2). However, the precise role of Tp47 in the biosynthesis of T.
pallidum peptidoglycan remains unclear. Although corrobora-
tive data are lacking, it is possible, implicated largely by its
molecular mass, that Tp47 is a DD-carboxypeptidase. If so, the
marked abundance of Tp47 would imply that it serves to limit
the degree of cross-linking in the peptidoglycan of T. pallidum,
thereby promoting the rather remarkable, highly flexuous mo-
tility pattern of the spirochete (49). Consistent with this view,
other preliminary data have suggested that the expression of
full-length, lipidated Tp47 in E. coli (13) reduces the degree of
cross-linking in E. coli peptidoglycan.
2
Despite both mutagenesis and x-ray crystallography data
presented herein, identification of the putative active site of
Tp47 for

-lactam binding remains unresolved. The three-di-
mensional structure of Tp47 has revealed a positively charged
cleft that may bind monosaccharides and/or possibly tazobac-
tam, and that cleft might function as an interaction site for the
relevant carboxylate group of
D-Ala-D-Ala (and

-lactams), but
more conclusive evidence awaits further mutagenesis studies.
Regardless, the combined data provide compelling evidence
that Tp47 represents a new class of PBP. It also is not known
to what extent this novel type of PBP might be found in other
bacterial pathogens, but it is anticipated that the burgeoning
genomics field will eventually shed additional light on this.
Finally, it is noteworthy that although not sharing homology
with Tp47, a completely
␣
-helical cysteine-rich protein B of
Helicobacter pylori recently was described as representing an-
other new class of PBP (50). Although molecular modeling
inferred that a site within the
␣
-helical cysteine-rich protein B
might bind to NAM, the crystal structure also did not defini-
tively reveal the active site. Tp47 and the
␣
-helical cysteine-
rich protein B thus now seem to represent two examples of
PBPs that do not satisfy classical PBP paradigms, the ramifi-
cations of which remain to be more fully explored.
Acknowledgments—We thank Taissia Popova and Martin Goldberg
for assistance with mutant constructions, Sandra Hill for excellent
technical assistance with crystal preparation and handling, Bikash
Pramanik for mass spectrometry, John Buynak for supplying tazobac-
tam, Joseph Albanesi for guidance with analytical ultracentrifugation,
Zbyszek Otwinowski for assistance in processing the laboratory source
xenon-derivatized data set, and Andrzej Joachimiak and the staff of the
Structural Biology 19-ID beamline for expert aid in data collection.
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2
M. V. Norgard and M. S. Goldberg, unpublished data.
Structure of the 47-kDa PBP of T. pallidum41864
