Acta Cryst. (2010). F66, 1354–1364
Acta Crystallographica Section F
Structure of the c-D-glutamyl-L-diamino acid
endopeptidase YkfC from Bacillus cereus in
complex with L-Ala-c-D-Glu: insights into substrate
recognition by NlpC/P60 cysteine peptidases
Qingping Xu,a,bPolat Abdubek,b,c
Tamara Astakhova,b,dHerbert L.
Xiaohui Cai,b,dDennis Carlton,b,f
Connie Chen,b,cHsiu-Ju Chiu,a,b
Michelle Chiu,b,cThomas Clayton,b,f
Debanu Das,a,bMarc C. Deller,b,fLian
Duan,b,dKyle Ellrott,b,dCarol L. Farr,b,f
Julie Feuerhelm,b,cJoanna C. Grant,b,c
Anna Grzechnik,b,fGye Won Han,b,f
Lukasz Jaroszewski,b,d,eKevin K. Jin,a,b
Heath E. Klock,b,cMark W. Knuth,b,c
Piotr Kozbial,b,eS. Sri Krishna,b,d,e
Abhinav Kumar,a,bWinnie W. Lam,a,b
David Marciano,b,fMitchell D.
Miller,a,bAndrew T. Morse,b,dEdward
Linda Okach,b,cChristina Puckett,b,c
Ron Reyes,a,bHenry J. Tien,b,f
Christine B. Trame,a,bHenry van den
Wooten,b,cAndrew Yeh,a,bKeith O.
Marc-Andre ´ Elsliger,b,fAshley M.
Deacon,a,bAdam Godzik,b,d,eScott A.
Lesleyb,c,fand Ian A. Wilsonb,f*
aStanford Synchrotron Radiation Lightsource,
SLAC National Accelerator Laboratory, Menlo
Park, CA, USA,bJoint Center for Structural
Genomics, http://www.jcsg.org, USA,cProtein
Sciences Department, Genomics Institute of the
Novartis Research Foundation, San Diego, CA,
USA,dCenter for Research in Biological Systems,
University of California, San Diego, La Jolla, CA,
USA,eProgram on Bioinformatics and Systems
Biology, Sanford–Burnham Medical Research
Institute, La Jolla, CA, USA,fDepartment of
Molecular Biology, The Scripps Research
Institute, La Jolla, CA, USA, andgPhoton
Science, SLAC National Accelerator Laboratory,
Menlo Park, CA, USA
Correspondence e-mail: firstname.lastname@example.org
Received 13 April 2010
Accepted 3 June 2010
PDB Reference: YkfC–L-Ala-?-D-Glu complex,
Dipeptidyl-peptidase VI from Bacillus sphaericus and YkfC from Bacillus
subtilis have both previously been characterized as highly specific ?-d-glutamyl-
l-diamino acid endopeptidases. The crystal structure of a YkfC ortholog from
Bacillus cereus (BcYkfC) at 1.8 A˚resolution revealed that it contains two
N-terminal bacterial SH3 (SH3b) domains in addition to the C-terminal catalytic
NlpC/P60 domain that is ubiquitous in the very large family of cell-wall-related
cysteine peptidases. A bound reaction product (l-Ala-?-d-Glu) enabled the
identification of conserved sequence and structural signatures for recognition of
l-Ala and ?-d-Glu and, therefore, provides a clear framework for understanding
the substrate specificity observed in dipeptidyl-peptidase VI, YkfC and other
NlpC/P60 domains in general. The first SH3b domain plays an important role in
defining substrate specificity by contributing to the formation of the active site,
such that only murein peptides with a free N-terminal alanine are allowed. A
conserved tyrosine in the SH3b domain of the YkfC subfamily is correlated with
the presence of a conserved acidic residue in the NlpC/P60 domain and both
residues interact with the free amine group of the alanine. This structural feature
allows the definition of a subfamily of NlpC/P60 enzymes with the same
N-terminal substrate requirements, including a previously characterized
cyanobacterial l-alanine-?-d-glutamate endopeptidase that contains the two
key components (an NlpC/P60 domain attached to an SH3b domain) for
assembly of a YkfC-like active site.
Cell-wall turnover, an enzymatic process that results in the loss of
peptidoglycan (PG) components, has been reported in many bacteria,
including Escherichia coli and Bacillus subtilis (Doyle et al., 1988).
The products of the turnover are generally re-utilized through a
process known as PG recycling (Park & Uehara, 2008). The mole-
cular processes involved in cell-wall turnover and recycling are not
currently well understood in comparison to cell-wall synthesis,
particularly in bacteria other than E. coli (Park & Uehara, 2008;
Uehara & Park, 2003, 2004, 2007; Uehara et al., 2005). In E. coli, the
cell wall is degraded by lytic transglycosylases that release anhy-
where DAP is meso-diaminopimelic acid), which are imported into
the cytoplasm, primarily by AmpG permease, and subsequently
processed by N-acetyl-anhydromuramyl-l-alanine amidase (which
cleaves between GlcNAc-anhMurNAc and l-Ala) and ld-carboxy-
peptidase LdcA (which cleaves between DAP and d-Ala). Two fates
are possible for the generated murein tripeptide l-Ala-?-d-Glu-DAP.
Under normal growth conditions, these tripeptides are recycled by
Mpl ligase and returned to the peptidoglycan-biosynthetic pathway.
During nutrient-limiting conditions, an additional pathway (Fig. 1)
is likely to be involved in the murein tripeptide metabolism, as
proposed in E. coli (Uehara & Park, 2003). MpaA endopeptidase, a
metallocarboxypeptidase, specifically cleaves l-Ala-?-d-Glu-DAP to
produce l-Ala-?-d-Glu and DAP. l-Ala-?-d-Glu is then converted to
l-Ala-l-Glu and subsequently to l-Ala and l-Glu by YcjG epimerase
and PepD peptidase, respectively.
Some of the enzymes in the cell-wall recycling of E. coli, such as
AmpG, AmpD, Mpl and MpaA, have no orthologs in B. subtilis (Park
& Uehara, 2008), suggesting that the mechanism of cell-wall recycling
may differ between the two bacteria. In B. subtilis, PG was proposed
to be cleaved by a muramidase and an amidase to produce GlcNAc-
MurNAc and stem peptides (Park & Uehara, 2008). The free peptides
are imported into the cytoplasm by an unidentified permease and
subsequently processed by YkfABC enzymes. YkfA, an ld-carboxy-
peptidase, removes the terminal d-Ala. The generated tripeptide is
further metabolized by a pathway that is functionally equivalent to
that of E. coli, with YkfC as the ?-d-Glu-DAP endopeptidase and
YkfB as the l-Ala-d-Glu epimerase (Fig. 1) (Schmidt et al., 2001).
Interestingly, while YkfB is homologous to YcjG of E. coli, YkfC is
unrelated to MpaA in sequence and structure despite having an
YkfC contains a C-terminal NlpC/P60 cysteine peptidase domain.
NlpC/P60 is a large family of cell-wall related cysteine peptidases that
are broadly distributed in bacteria, viruses, archaea and eukaryotes
(Anantharaman & Aravind, 2003; Bateman & Rawlings, 2003;
Rigden et al., 2003). Characterized NlpC/P60 enzymes are almost all
?-d-Glu-DAP (or ?-d-Glu-Lys) endopeptidases. While their bio-
chemical function seems to be conserved, the physiological roles of
NlpC/P60 proteins are diverse, including involvement in cell
separation, expansion, differentiation, cell-wall turnover, cell lysis,
protein secretion and virus infection (Smith et al., 2000). Secreted
NlpC/P60 proteins also have other roles in pathogenesis. The auto-
lysin P60 of Listeria monocytogenes is involved in host-cell invasion
(Kuhn & Goebel, 1989), enterotoxin FM of B. cereus in food
poisoning (Asano et al., 1997), and SagA of Enterococcus faecium is a
secreted antigen that binds to extracellular matrix proteins (Teng et
NlpC/P60 proteins can be lethal to bacteria owing to their ability to
compromise cell-wall integrity or cell-wall biosynthesis. Therefore,
their activities are tightly controlled through multiple mechanisms
(Smith et al., 2000); their expression is regulated at the transcription
level and their cellular localization is dependent on their physio-
logical roles. Furthermore, their atomic structures are highly
optimized to precisely define their substrate specificity (Xu et al.,
2009). NlpC/P60 proteins are often fused to auxiliary domains, many
of which are known cell-wall binding modules (e.g. LysM and the
choline-binding domain). Thus, it is generally assumed that these
auxiliary domains function as targeting domains which localize their
proteins to the cell wall. The functional synergy between the NlpC/
P60 domains and their auxiliary domains is currently not fully
understood. We have previously determined the crystal structure of a
?-d-Glu-DAP endopeptidase from cyanobacteria (AvPCP/NpPCP;
Anabaena variabilis/Nostoc punctiforme PG cysteine peptidase; Xu et
al., 2009) and showed that it contained an N-terminal bacterial SH3
(SH3b) domain and a C-terminal NlpC/P60 domain. We proposed
that the SH3b domain of this enzyme is important in defining the
substrate specificity of the peptidase domain. However, the
mechanism of substrate recognition by NlpC/P60 and SH3b was not
firmly established. Here, we report the crystal structure of YkfC from
B. cereus (BcYkfC) in complex with l-Ala-?-d-Glu. BcYkfC shares
40% sequence identity with YkfC from B. subtilis, which has
previously been biochemically characterized (Schmidt et al., 2001).
Thus, we now have the first detailed view of substrate recognition by
an NlpC/P60 protein.
2. Material and methods
2.1. Sequence analysis
Homologs of BcYkfC were identified using PSI-BLAST (Altschul
et al., 1997; three iterations) against the nonredundant (nr) protein
sequence database at the National Center for Biotechnology Infor-
mation (NCBI) using the sequence of the catalytic domain of BcYkfC
as the probe (residues 210–333). An alignment length of ?70 and an
E value of ?0.02 were used to extract a subset of hits. These proteins
(2599 sequences) were aligned using HMMALIGN (Eddy, 1998)
against the default global alignment profile of NlpC/P60 domains
(PF00877) from the PFAM database v.23 (Bateman et al., 2004).
YkfC-subfamily candidates were extracted from the above aligned
subset based on the presence of an aspartate corresponding to
position 256 of BcYkfC. The full-length sequences were then aligned
and clustered using PIPEALIGN (Plewniak et al., 2003). Only
sequences that also contained a conserved tyrosine corresponding to
position 118 of BcYkfC were classified into the YkfC subfamily. Plots
of sequence conservation in the active site were prepared using
WEBLOGO (Crooks et al., 2004).
2.2. Protein expression and purification
Clones were generated using the Polymerase Incomplete Primer
Extension (PIPE) cloning method (Klock et al., 2008). The gene
encoding BcYkfC (GenBank NP_979181; Swiss-Prot Q736M3) was
amplified by polymerase chain reaction (PCR) from B. cereus
NRS248 ATCC 10987 genomic DNA using PfuTurbo DNA poly-
merase (Stratagene) and I-PIPE (Insert) primers (forward primer,
verse primer, 50-aattaagtcgcgttaAGGTAAGTAACGACGCGCAC-
CAGCG-30; target sequence in upper case) that included sequences
for the predicted 50and 30ends. The expression vector pSpeedET,
which encodes an amino-terminal tobacco etch virus (TEV) protease-
cleavable expression and purification tag (MGSDKIHHHHHHEN-
LYFQ/G), was PCR-amplified with V-PIPE (Vector) primers
(forward primer, 50-taacgcgacttaattaactcgtttaaacggtctccagc-30; reverse
primer, 50-gccctggaagtacaggttttcgtgatgatgatgatgatg-30). V-PIPE and
I-PIPE PCR products were mixed to anneal the amplified DNA
fragments together. E. coli GeneHogs (Invitrogen) competent cells
were transformed with the I-PIPE/V-PIPE mixture and dispensed
onto selective LB–agar plates. The cloning junctions were confirmed
by DNA sequencing. Using the PIPE method, the gene segment
encoding residues Met1–Ala23 was omitted as these residues were
predicted to form a signal peptide. Expression was performed in a
selenomethionine-containing medium with suppression of normal
methionine synthesis. At the end of fermentation, lysozyme was
added to the culture to a final concentration of 250 mg ml?1and the
cells were harvested and frozen. After one freeze–thaw cycle, the
Acta Cryst. (2010). F66, 1354–1364 Xu et al.
Proposed metabolic pathways for murein peptides in E. coli and B. subtilis.
cells were sonicated in lysis buffer [50 mM HEPES pH 8.0, 50 mM
NaCl, 10 mM imidazole, 1 mM tris(2-carboxyethyl)phosphine–HCl
(TCEP)] and the lysate was clarified by centrifugation at 32 500g for
30 min. The soluble fraction was passed over nickel-chelating resin
(GE Healthcare) pre-equilibrated with lysis buffer, the resin was
washed with wash buffer [50 mM HEPES pH 8.0, 300 mM NaCl,
40 mM imidazole, 10%(v/v) glycerol, 1 mM TCEP] and the protein
was eluted with elution buffer [20 mM HEPES pH 8.0, 300 mM
imidazole, 10%(v/v) glycerol, 1 mM TCEP]. The eluate was buffer-
exchanged with TEV buffer (20 mM HEPES pH 8.0, 200 mM NaCl,
40 mM imidazole, 1 mM TCEP) using a PD-10 column (GE
Healthcare) and incubated with 1 mg TEV protease per 15 mg of
eluted protein. The protease-treated eluate was run over nickel-
chelating resin (GE Healthcare) pre-equilibrated with HEPES crys-
tallization buffer (20 mM HEPES pH 8.0, 200 mM NaCl, 40 mM
imidazole, 1 mM TCEP) and the resin was washed with the same
buffer. The flowthrough and wash fractions were combined and
concentrated to 18.8 mg ml?1as determined using the Coomassie
Plus Protein Assay Reagent (Pierce) by centrifugal ultrafiltration
(Millipore) for crystallization trials. The oligomeric state of BcYkfC
was determined using a 0.8 ? 30 cm Shodex Protein KW-803 column
(Thomson Instruments) pre-calibrated with gel-filtration standards
BcYkfC was crystallized by mixing 200 nl protein solution with
200 nl crystallization solution and equilibrating against a 50 ml
reservoir solution using the nanodroplet vapor-diffusion method
(Santarsiero et al., 2002) with standard Joint Center for Structural
Genomics (JCSG; http://www.jcsg.org) crystallization protocols
(Lesley et al., 2002). The crystallization solution was composed of
0.2 M sodium chloride, 50%(v/v) PEG 200 and 0.1 M phosphate–
citrate pH 4.2. A needle-shaped crystal of approximate dimensions
100 ? 15 ? 15 mm was harvested after 29 d at 277 K. No additional
cryoprotectant was added to the crystal. Initial screening for
diffraction was carried out using the Stanford Automated Mounting
system (SAM; Cohen et al., 2002) at the Stanford Synchrotron
Radiation Lightsource (SSRL, Menlo Park, California, USA).
2.4. Data collection, structure solution and refinement
Multi-wavelength anomalous diffraction (MAD) data were
collected at wavelengths corresponding to the peak, high-energy
remote and inflection wavelengths of a selenium MAD experiment at
100 K using a MAR CCD 325 detector (Rayonix) on SSRL beamline
11-1. Processing of the diffraction data and initial structure solution
were carried out using the automatic structure-solution script
autoXDSp developed at the JCSG (unpublished work). This script
shepherds the structure-determination process, as summarized below,
using preset rules through a decision-tree that mimics that used by an
experienced crystallographer. The calculations are parallelized on a
computer cluster such that initial maps and models can usually be
obtained within 1 h of the completion of data collection. In summary,
the MAD data were integrated and reduced using XDS and then
scaled with the program XSCALE (Kabsch, 1993, 2010). Selenium
sites were located with SHELXD (Sheldrick, 2008). Phase refinement
and automatic model building were performed using autoSHARP
(Bricogne et al., 2003) and ARP/wARP (Cohen et al., 2004). This
automated process produced an initial model that was 92% complete.
Further model completion and refinement were performed manually
with Coot (Emsley &Cowtan, 2004) and REFMAC (Murshudov et al.,
1997) from the CCP4 suite (Collaborative Computational Project,
Number 4, 1994). Data and refinement statistics are summarized in
Table 1. Analysis of the stereochemical quality of the model was
accomplished using MolProbity (Chen et al., 2010). All molecular
graphics were prepared with PyMOL (DeLano Scientific) unless
specifically stated otherwise. Atomic coordinates and experimental
structure factors for BcYkfC at 1.8 A˚resolution have been deposited
in the PDB with accession code 3h41.
2.5. Molecular modeling
Molecular docking was performed using the same protocol as
described previously (Xu et al., 2009) using Glide v.5.0 (Schro ¨dinger
LLC). The positions of the bound ligand were used as restraints such
that the l-Ala-?-d-Glu portion of the docked substrate adopted a
similar conformation as seen in the crystal structure. In order to
perform the docking studies, the tri-oxidized cysteine (OCS238) in
the crystal structure was replaced with the reduced form, which is
needed for reaction in the papain family of cysteine peptidases.
Furthermore, only the side-chain conformer with higher occupancy in
the crystal structure was considered in the docking experiments when
multiple conformations were observed for an active-site residue.
Xu et al.
Acta Cryst. (2010). F66, 1354–1364
Data-collection, phasing and refinement statistics (PDB code 3h41).
Values in parentheses are for the highest resolution shell. The high-resolution cutoff was
chosen such that the mean I/?(I) in the highest resolution shell was around 2.
Unit-cell parameters (A˚,?)
Resolution range (A˚)
a = 95.2, b = 59.4, c = 61.3, ? = 103.3
No. of observations
No. of unique reflections
Rmergeon I† (%)
No. of Se sites
Mean figure of merit
Model and refinement statistics
Resolution range (A˚)
No. of reflections (total)
No. of reflections (test)
Data set used in refinement
Restraints (r.m.s.d. observed)
Bond lengths (A˚)
Bond angles (?)
Average isotropic B value (A˚2)
ESU} based on Rfree(A˚)
All-atom clash score
Ramachandran favored (%)
No. of Ramachandran outliers
No. of rotamer outliers
|F| > 0
† Rmerge =
chosen at random and omitted from refinement.
ijIiðhklÞ ? hIðhklÞij=P
§ Rfreeis the same as Rcrystbut for 5.0% of the total reflections
} Estimated standard uncertainty in
iIiðhklÞ.‡ Rcryst =
hkljFobsj, where Fcalcand Fobsare the calculated and observed structure-factor
3. Results and discussion
3.1. Genomic context
Full-length BcYkfC (strain B. cereus ATCC 10987) contains 333
residues (molecular weight 37.3 kDa), the first 23 of which are pre-
dicted to be a signal peptide by the Phobius web server (Kall et al.,
2007). Despite being homologous to BcYkfC, B. subtilis YkfC (296
residues) does not contain a signal peptide, suggesting that the two
proteins function at different cellular locations.
As in B. subtilis, the ykfC gene (BCE_2878) is adjacent to the ykfB
l-Ala-d-Glu epimerase gene (BCE_2879) in the B. cereus genome.
The YkfB epimerases (sequence identity 50%) from both bacteria
do not contain predicted signal peptides. The genome association of
ykfB and ykfC is also observed in other bacteria (e.g. Listeria
innocua, Acidobacterium sp.,
thetaiotaomicron and Gramella forsetii). However, the genome
context of ykfBC is different in B. subtilis compared with B. cereus
(Fig. 2). The YkfA–D genes of B. subtilis are located next to the
dppB–E dipeptide ABC transporter operon, whereas the YkfBC
genes of B. cereus are located downstream of divIC, which encodes a
putative cell-division protein. Downstream of ykfC is an oppA gene
that is homologous to dppE (34% sequence identity). Both genes are
predicted to encode periplasmic dipeptide-binding proteins.
Based on the presence/absence of the signal peptide on YkfC and
YkfB, as well as their genomic contexts, we suggest that the strategy
for metabolizing murein peptides is likely to differ between B. subtilis
and B. cereus. In B. cereus, the murein peptides are likely to be
broken down outside the cell, with the resulting dipeptide l-Ala-?-d-
Glu being imported into cytoplasm for further processing by YkfB,
while the reactions catalyzed by both YkfC and YkfB are likely to
occur in the cytoplasm in B. subtilis.
3.2. Structure determination and quality of the model
The crystal structure of BcYkfC was determined using the high-
throughput structural genomics pipeline implemented at the JCSG
(Lesley et al., 2002). The selenomethionine derivative of BcYkfC was
expressed in E. coli with an N-terminal TEV-cleavable His tag and
purified by metal-affinity chromatography. In order to improve the
chance of obtaining crystals, the predicted N-terminal signal peptide
(residues 1–23) was not included in the cloned construct. The data
were indexed in space group C2 and the structure was determined at
1.79 A˚resolution with one molecule per asymmetric unit using the
MAD method (Rcryst= 16.3%, Rfree= 19.7%). The electron density
for the main chain was well defined throughout the entire molecule.
The mean residual error of the coordinates was estimated to be
0.11 A˚by the diffraction-component precision index (DPI) method
(Cruickshank, 1999). The model of BcYkfC displays good geometry,
with an all-atom clash score of 5.07, and the Ramachandran plot
produced by MolProbity (Chen et al., 2010) shows that all residues,
but one, are in allowed regions, with 97.7% in favored regions. Only
one residue is flagged as a rotamer outlier. The Ramachandran
(Pro305) and rotamer (His303) outliers are supported by well defined
electron density. Since these residues are either close to or part of the
active site, these structural deviations from ideality are likely to be of
functional relevance. Additionally, two cis-peptides (Asn57–Pro58
and Asn154–Pro155) are also supported by clear electron density.
The final model of BcYkfC contains residues 29–333, one dipeptide l-
Ala-?-d-Glu, one phosphate, six polyethylene glycol (PEG) frag-
ments from the crystallization solution and 265 waters. The residual
residue (Gly0) from the cleaved N-terminal purification tag, residues
24–28 and the side chains of Glu201 and Arg270 were disordered and
were not included in the final model. Data-collection, refinement and
model statistics are summarized in Table 1.
3.3. Overall structure
BcYkfC is likely to be a monomer in solution, as supported by
crystal-packing analysis and analytical size-exclusion chromato-
graphy. The structure of BcYkfC consists of three domains: two SH3b
domains (SH3b1, residues 29–129; SH3b2, residues 130–207) and a C-
terminal NlpC/P60 cysteine peptidase domain (residues 208–333)
(Figs. 3a and 3b). The two SH3b domains are similar to each other,
with an r.m.s.d. of 2.2 A˚for 57 aligned C?atoms (sequence identity of
13%). A structural similarity search using DALI (Holm & Sander,
1995) did not find any other structures with the same three-domain
architecture. However, a number of significantly similar substructures
were identified and are summarized in Table 2. The SH3b domains of
BcYkfC are similar to other SH3b domains (Holm & Sander, 1995),
Acta Cryst. (2010). F66, 1354–1364Xu et al.
Genomic context of the ykfB and ykfC genes in B. subtilis and B. cereus.
Structural comparisons of BcYkfC and other bacterial proteins that share at least one common domain.
The alignment was performed by the DALI structural comparison server using full-length BcYkfC and individual domains (SH3b1 and NlpC/P60) as search probes. For proteins with
multiple SH3-like domains (PDB codes 1m9s and 1xov) or multiple chains, only the best match is shown.
SH3b + NlpC/P602fg0
Xu et al. (2009)
Xu et al. (2009)
Marino et al. (2002)
Lu et al. (2006)
Korndorfer et al. (2006)
Srisailam et al. (2006)
Nasertorabi et al. (2006)
Aramini et al. (2008)
Pai et al. (2006)
TD‡ of internalin B
TD‡ of PG hydrolase ALE-1
TD‡ of endolysin PlyPSA
E. coli lipoprotein Spr
CHAP domain of GspS}
† Number of residues present in the model used for comparison.
} Glutathionylspermidine synthetase/amidase.
‡ Targeting domain. § New York SGX Research Center for Structural Genomics (unpublished
including the N-terminal domain of cyanobacterial ?-d-Glu-DAP
endopeptidases (Xu et al., 2009), the GW domains of internalin B
(Marino et al., 2002), the cell-wall-targeting domain of glycylglycine
endopeptidase ALE-1 (Lu et al., 2006), PhnA-like protein (Srisailam
et al., 2006) and endolysin PlyPSA (Korndorfer et al., 2006), as well as
many eukaryotic SH3 domains. A ?-hairpin within the so-called ‘RT
loop’ (i.e. the loop between ?A and ?B) region appears to be a
common and unique structural feature of SH3b domains compared
with their eukaryotic counterparts (Xu et al., 2009). The BcYkfC
SH3b domains both contain this conserved structural motif (?A1–
?A2). However, SH3b1 contains a novel helical insertion (?1–?3)
that is not seen in previous SH3b structures or in SH3b2 (Fig. 3b).
Prokaryotic SH3-like domains have also been implicated in poly-
peptide binding (Wylie et al., 2005) and metal binding (Pohl et al.,
1999). Although these SH3-like domains contain a similar five-
stranded (?A–?E) core, they display much larger structural differ-
ences (and are not among significant DALI hits with Z > 2.0) and lack
the ?-hairpin in the RT-loop region when compared with SH3b
Xu et al.
Acta Cryst. (2010). F66, 1354–1364
Crystal structure of YkfC from B. cereus in complex with l-Ala-?-d-Glu. (a) Ribbon representation of BcYkfC, highlighting its domain organization. SH3b1 is depicted in
blue, SH3b2 in green and NlpC/P60 in red. The bound l-Ala-?-d-Glu is shown as a stick model. (b) Ribbon representations of individual domains, showing the secondary-
structure elements. (c) Molecular surface of YkfC colored by sequence conservation. The surface color gradient indicates the level of sequence conservation from the most
conserved residues (deep red) to nonconserved residues (white).
domains. The C-terminal NlpC/P60 catalytic domain of BcYkfC is
remotely related to the papain family of cysteine peptidases
(Anantharaman & Aravind, 2003), with highest similarity to cyano-
bacterial ?-d-Glu-DAP endopeptidases (Xu et al., 2009), E. coli
lipoprotein Spr (Aramini et al., 2008) and two uncharacterized
proteins (PDB codes 2p1g and 2im9; NYSGXRC, unpublished work)
The three domains of BcYkfC are arranged in a triangle such that
each domain interacts with the two other domains. The interface
(?570 A˚2per domain) between the two SH3b domains is mostly
hydrophobic and is centered on interactions between the ?A–?A1
and ?A2–?B loops of SH3b1 and the ?A2 and ?B strands and ?D–
?E loop of SH3b2. The SH3b1–NlpC/P60 domain interface (903 A˚2
buried surface per domain) is mediated through ?2–?3–?A1 and the
?C–?D loop of SH3b1, and ?1–?2–?3 and the ?6–?7 loop of NlpC/
P60. The active site is located at this SH3b1–NlpC/P60 interface,
whereas the SH3b2 domain is distal to the active site. A multiple
sequence alignment of full-length homologs of YkfC (37 sequences;
average sequence identity of 54%) indicates that most of the highly
conserved residues are either buried inside the protein or clustered
around the active site (Fig. 3c).
3.4. Active site
The catalytic triad of the peptidase domain consists of Cys238,
His291 and His303. The catalytic Cys238 is oxidized (OCS) in the
crystal based on the electron density (Fig. 4a). Since an oxidized
cysteine can no longer function as a nucleophile in the reaction
(Storer & Me ´nard, 1994), the enzyme in the crystal is inactive. The
conformation of the cysteine side chain is not significantly affected by
the oxidation as its side chain is in a similar location and conforma-
tion as in other NlpC/P60 structures (Xu et al., 2009).
The dipeptide l-Ala-?-d-Glu was identified from well defined
electron density in the active site of BcYkfC (Fig. 4a). As the
dipeptide was not added during protein purification or crystallization,
it was most likely to have been obtained during protein expression in
E. coli. Since this dipeptide is one of the reaction products of BcYkfC
(Fig. 2), it unequivocally identifies the S2–S1 binding-site cavity,
which is formed by residues from both the SH3b1 (Glu83, Thr84 and
Tyr118) and NlpC/P60 domains (Tyr226, Trp228, Ala229, Asp237,
Arg255, Asp256, Ser257, His290 and His291). Two charged residues,
Asp237 and Arg255, which are highly conserved in NlpC/P60
domains, are involved in a hydrogen-bond network that connects
many residues in the active site (Fig. 4b).
Two active-site residues, Ser257 and His291, display two discrete
side-chain conformations (Fig. 4b). The first conformer of His291
(occupancy modeled as 0.7), which facilitates hydrogen bonding to
His303, is identical to the corresponding histidine of the catalytic
dyad in papain and other NlpC/P60 enzymes. The second rotamer
points His291 towards the solvent. The side-chain isomerism in the
active site is likely to be a consequence of the observed oxidized state
Acta Cryst. (2010). F66, 1354–1364Xu et al.
Active site and recognition of l-Ala-?-d-Glu by BcYkfC. (a) Stereoview of a 2Fo? FcOMIT map, where l-Ala-?-d-Glu and OCS238 were omitted from phasing/refinement,
contoured at 1.5?. (b) The extensive hydrogen-bond network in the active site of BcYkfC. Hydrogen bonds and distances are shown as dashed lines. (c) l-Ala-?-d-Glu (stick
representation; yellow C atoms) is located in the active site at the interface of the SH3b1 domain (blue) and the NlpC/P60 domain (red). (d) The interaction between l-Ala-
?-d-Glu and the active site of YkfC. This figure was generated using the program MOE 2008.10 (Chemical Computing Group Inc.).
of the catalytic Cys238, since the oxidized Cys238 makes multiple
hydrogen-bond interactions with nearby side chains, including
Tyr226, Ser257 and His291 (Fig. 4b).
3.5. Recognition of L-Ala-c-D-Glu
The active-site residues form a pocket that is highly complemen-
tary in shape and chemical properties to the ligand (Figs. 4c and 4d).
The average B value for the bound ligand is 26 A˚2(the overall B
value of the protein is 18 A˚2), indicating that it is well ordered with
almost full occupancy in the active site. The interface between the
dipeptide and the protein buries a total surface area of 490 A˚2. The
dipeptide is stabilized by multiple hydrogen bonds. The free amine of
l-Ala in the S2 pocket makes hydrogen bonds to Glu83 O and the
side chains of Tyr118 (OH) and Asp256 (O?1), which are highly
conserved in the YkfC subfamily of NlpC/P60 enzymes (see below).
The ?-NH group of ?-d-Glu in the S1 site forms a weak hydrogen
bond to Asp237 O?2. The ?-carboxyl of d-Glu is stabilized by
hydrogen-bonding interactions with Ser239 and Ser257 (Fig. 4d). On
the solvent-exposed side of the substrate, waters are also involved in
the hydrogen-bond network with the substrate and the enzyme (not
shown). The l-Ala methyl side chain points towards a hydrophobic
pocket defined by the side chains of Trp228 and Ala229. Additionally,
the aliphatic C atoms of d-Glu are involved in hydrophobic contacts
Xu et al.
Acta Cryst. (2010). F66, 1354–1364
Sequence alignment of YkfC from B. subtilis and B. cereus, dipeptidyl-peptidase VI (DPP VI) from B. sphaericus and a ?-d-glutamyl-l-diamino acid endopeptidase from
A. variabilis (AvPCP). The sequence numbering and secondary-structure elements of YkfC from B. cereus and AvPCP are indicated at the top and bottom, respectively. The
alignment was generated by merging and manually editing the structure-based sequence alignment of BcYkfC and AvPCP with the sequence alignment of the top three
sequences. The active-site residues are marked with colored dots at the bottom (blue, S2; orange, S1; red, catalytic triad; green, potential S0sites).
with Trp228. As a result, Trp228 contributes to both the S2 and S1
3.6. YkfC is specific for murein peptides with free N-terminal L-Ala
The active site of B. subtilis YkfC is highly conserved compared
with that of BcYkfC (Fig. 5). Dipeptidyl peptidase VI (DPP VI) from
B. sphaericus is a ?-d-Glu-DAP(Lys) dipeptidase that is found in the
cytoplasm during sporulation (Vacheron et al., 1979). DPP VI has
strict specificity for murein peptides with an N-terminal l-Ala. The
crystal structure of BcYkfC provides a structural basis for this
specificity. DPP VI and BcYkfC have only 21% sequence identity, but
the same sets of key residues are conserved, as expected given their
similar folds and function (Fig. 5). Thus, DPP VI is clearly a homolog
Acta Cryst. (2010). F66, 1354–1364Xu et al.
Models of substrate recognition by BcYkfC. (a) l-Ala-?-d-Glu-DAP was docked into the active site of BcYkfC. The protein surface is colored according to a gradient in
electrostatic potential from negative (red) to positive (blue) (MOE 2008.10; Chemical Computing Group Inc.). (b) Stereoview of the specific interactions (four polar, one
nonpolar) of ?-d-Glu (cyan) in the context of the tripeptide by five residues of YkfC (Tyr226, Trp228, Asp237, Ser239 and Ser257). The protein residues are colored
according to subsite (S1, orange; catalytic triad, magenta; S10, green). (c) Sequence conservation of the active sites in NlpC/P60 domains based on 2277 NlpC/P60 domains
with an intact catalytic dyad (Cys238 and His291) and a conserved Tyr226 (blue, S2; orange, S1; red, catalytic triad; green, S0sites). (d) Sequence conservation of the active
sites in the YkfC subfamily of NlpC/P60 enzymes based on 282 sequences selected based on the presence of a conserved aspartate residue at position 256 of BcYkfC. The
conservation of this residue is highly correlated with a conserved tyrosine in the ?D region of the SH3b domain (Tyr118 of BcYkfC); both of these residues interact with the
free amine of l-Ala of the substrate.
of BcYkfC, except that its SH3b1 domain has no large helical inser-
tion (?1–?3) between ?A1 and ?A2 and it has a shorter ?C–?D loop
(Fig. 5). Furthermore, the S2 and S1 binding sites for l-Ala-?-d-Glu
are highly conserved between DPP VI and BcYkfC, including the
conserved tyrosine (Tyr118 of YkfC), which is the only residue from
SH3b1 whose side chain interacts with l-Ala. The residues from the
catalytic domain that contribute to the S2 and S1 sites are identical in
BcYkfC and DPP VI (except for an Ala/Ser mutation at position 257
of BcYkfC). Therefore, we conclude that both the B. subtilis and the
B. cereus YkfCs are likely to have very similar substrate specificity for
murein peptides that contain an N-terminal l-Ala. Enzymes with this
specificity cannot cleave PG-biosynthesis precursors such as UDP-
MurNAc-pentapeptide and thus are not likely to interfere with the
3.7. S0 0 0sites of YkfC and docking studies
B. subtilis YkfC has previously been shown to process the tri-
peptide l-Ala-?-d-Glu-l-Lys, the tetrapeptide l-Ala-?-d-Glu-l-Lys-
(Schmidt et al., 2001). DPP VI also displayed no particular specificity
towards the additional residues attached after DAP (or Lys;
Vacheron et al., 1979). Residues in BcYkfC that potentially form the
S0sites include His290, His291, Arg306, Glu308, Arg309, Tyr321 and
Glu324. These residues are generally less conserved among close
BcYkfC homologs (Fig. 5), suggesting that the specificity of YkfC is
determined primarily at the S2 and S1 sites as described above. The
catalytic mechanism of NlpC/P60 is currently unknown, but it is likely
to be similar to that of papain based on the similar arrangement of
their catalytic residues (Anantharaman & Aravind, 2003; Aramini et
al., 2008; Xu et al., 2009), except for two interesting variations of
important catalytic residues. The third polar residue of the catalytic
triad in NlpC/P60 is more often a histidine rather than the asparagine
in papain (Bateman et al., 2004). A tyrosine (Tyr226 of BcYkfC) is
located at the position equivalent to Gln119 in papain that is thought
to be important for catalysis by stabilizing the transition state (Xu et
al., 2009; Aramini et al., 2008). Therefore, we docked the tripeptide
l-Ala-?-d-Glu-l-DAP into the active site of BcYkfC in order to
deduce a likely mode of interaction and to assess the effects of the
differences (Fig. 6a). The C?carboxyl group of ?-d-Glu is displaced
owing to the oxidation of Cys238 in the crystal structure. In the
modeled structure, Tyr226 OH interacts with the carboxyl group of
?-d-Glu (Fig. 6b). This docked structure places the C?atom close to
Cys238 SH (distance ?3.6 A˚) and could represent the chemically
productive conformation. The electrostatic surface of the binding site
is highly complementary to that of the substrate, indicating that the
polar and charged interactions are likely to be important for substrate
recognition. The basic His290 and Arg306 residues could interact
with carboxyl groups. It has previously been reported that the cata-
lytic efficiency of B. subtilis YkfC for l-Ala-?-d-Glu-Lys is signifi-
cantly less than that for tetrapeptides or pentapeptides (Schmidt et
al., 2001). This observation could be explained by the unfavorable
basic environment in the S0site. Two residues (Arg306/Glu308) at the
BcYkfC S10site are substituted by Lys/Gly in B. subtilis YkfC (Fig. 5),
resulting in a subsite with a positive charge but no negative charge
that would be less likely to accommodate the positively charged Lys
of l-Ala-?-d-Glu-Lys (Fig. 6a).
3.8. Recognition of c-D-Glu by NlpC/P60 cysteine peptidases
NlpC/P60 cysteine peptidases are ubiquitous in bacteria, with
?5000 NlpC/P60 domains in the current Pfam database when meta-
genomics data are included (Bateman et al., 2004). However, only a
few of these domains have been biochemically characterized. In the
light of the much clearer picture that we now have of substrate
binding in BcYkfC, we examined the sequence conservation of NlpC/
P60-family proteins around the active site in order to obtain further
insights into substrate specificity across the entire family.
The nonredundant (nr) database of protein sequences was sear-
ched using the PSI-BLAST program (Altschul et al., 1997) with the
NlpC/P60 domain of BcYkfC as a search probe. We extracted a total
of 2599 protein sequences using the criteria of an E value of>0.02 and
an alignment length of >70 residues from 3985 total hits, representing
a significant subset of NlpC/P60 proteins. Among these proteins, only
?1% do not possess the conserved Cys/His dyad and an additional
9% (with catalytic dyad intact) do not contain the conserved tyrosine
that is believed to be essential for catalysis. These proteins are likely
to have the NlpC/P60 fold, but either have lost cysteine peptidase
activity or are remote homologs that hydrolyze different substrates
(e.g. the CHAP family; Anantharaman & Aravind, 2003; Bateman &
Rawlings, 2003; Rigden et al., 2003). The sequence-conservation
pattern of active-site residues of the remaining 2277 proteins
(average sequence identity of 19%) is shown in Fig. 6(c). The S sites
(S2 and S1) are significantly more conserved than the S0sites in these
proteins. The most conserved site is S1, which is tailored to recognize
?-d-Glu (Fig. 6b). The three most highly conserved residues (Asp237,
Ser239 and Tyr226), aside from the catalytic dyad, are involved in
hydrogen bonding with ?-d-Glu (Fig. 6b). At position 257, residues
with small side chains (Ala/Ser/Thr) are observed, creating a cavity
for the carboxyl group of ?-d-Glu. Trp228 contributes to both the S1
and S2 sites and a conserved hydrophobic residue is usually found at
this position, which may facilitate interaction with the substrate
Ala C?. Therefore, the conservation of active-site residues indicates
that a significant percentage of the NlpC/P60 family is likely to be
specific for ?-d-Glu. More than 1300 proteins which contain the
strictly conserved Trp228, Asp237, Ser239 and Tyr226 residues (and
catalytic triads) are most likely to bind X-l-Ala-?-d-Glu moieties
(where X is H or other moieties).
3.9. Structural comparison to the cyanobacterial NlpC/P60
We have previously determined the crystal structures of two
closely related NlpC/P60 l-Ala-?-d-Glu endopeptidases from the
cyanobacteria A. variabilis (AvPCP) and N. punctiforme (NpPCP).
These enzymes contain an N-terminal SH3b domain and a C-terminal
NlpC/P60 catalytic domain (Xu et al., 2009). Surprisingly, the struc-
tures of the cyanobacterial enzymes are essentially a substructure of
YkfC: the two domains of the cyanobacterial enzymes are structurally
equivalent to the first and third domains of BcYkfC (Figs. 5, 7a and
7b). The full-length AvPCP can be superimposed onto BcYkfC with
an r.m.s.d. of 2.3 A˚and 29% sequence identity for 193 aligned C?
atoms (Table 2). Thus, the individual SH3b and NlpC/P60 domains, as
well as their relative arrangements, are highly similarly in AvPCP and
BcYkfC. Furthermore, the residues in the S2 and S1 pockets are
nearly identical (except for S257A; Fig. 7c). The striking similarity
between the cyanobacterial enzymes and YkfC was not previously
detected by sequence analysis owing to the presence of two inser-
tions: the entire SH3b2 domain and the ?1–?3 region of SH3b1.
The ?C–?D loop and the ?A1–?A2 loop of SH3b1 are longer
compared with SH3b of AvPCP. The ?C–?D loop of YkfC is located
upstream of the S2 site. The ?A1–?A2 loop of YkfC contains three
helices (?1–?3) and is located on the S0side of the binding groove in
the catalytic domain. This insertion packs against the surface of the
NlpC/P60 domain and contributes to stabilizing the interface between
Xu et al.
Acta Cryst. (2010). F66, 1354–1364
the SH3b1 and NlpC/P60 domains. Furthermore, it is also defines the
shape of the S0binding site, which appears to be more restricted for
BcYkfC than for AvPCP (Fig. 7d). Without these insertions to
stabilize this domain interface, AvPCP uses a different strategy to
achieve a similar purpose: a longer C-terminal loop in NlpC/P60
extends to interact with the SH3b domain.
We previously proposed that the cyanobacterial enzymes have the
same substrate requirement as DPP VI based on modeling studies
(Xu et al., 2009). The crystal structure of YkfC reported here further
supports DPP VI, YkfC and the cyanobacterial enzymes belonging to
a subset of NlpC/P60 ?-d-Glu-DAP endopeptidases whose substrates
possess a free l-Ala at theN-terminus. The SH3b domain helps define
the S2 binding site (Tyr118 in BcYkfC and Tyr64 in AvPCP). Addi-
tionally, it sterically hinders the docking of a large moiety beyond the
S2 site and directly contributes to specificity. This role constitutes a
new function for SH3b domains.
3.10. Identification and distribution of YkfC homologs
BcYkfC and cyanobacterial ?-d-Glu-DAP endopeptidases have
the same specificity. Other NlpC/P60 enzymes with similar properties
could potentially be detected by analyzing sequence similarities in
both the SH3b and NlpC/P60 domains. However, owing to the pre-
sence of long insertions/deletions and highly divergent sequences in
the SH3b domains, it is often difficult to detect similar enzymes using
sequence searches and the NlpC/P60 region tends to dominate the
hits. We examined an alternate method by examining specific residues
in the NlpC/P60 domain only. An acidic residue (Asp256) is essential
for YkfC specificity by neutralizing the positive charge on the free
amine of l-Ala. Among the 2599 sequences obtained above, 282
proteins were identified that contained an aspartate equivalent to
Asp256 and a conserved catalytic dyad. In 239 of the 282 proteins
(85%), the conserved tyrosine was maintained in their SH3b domains
(Tyr118 of BcYkfC), while this tyrosine was not conserved in
enzymes that lacked Asp256. Thus, the presence of an aspartate
residue at position 256 and a conserved tyrosine in SH3b is highly
correlated (Fig. 6d). Interestingly, NlpC/P60 domains with the con-
served aspartate are also found in a few single-domain or multiple-
domain proteins that do not contain any detectable SH3b domain.
The biochemical properties of these proteins are currently unknown,
but they could represent novel variants of a YkfC-type enzyme.
Nevertheless, the 239 proteins above with the conserved aspartate
(Asp256) and tyrosine (Tyr118) are most likely to define a YkfC-like
subfamily of NlpC/P60 proteins. These NlpC/P60 enzymes are
predominately distributed in four phyla of bacteria: bacteroidetes,
cyanobacteria, firmicutes and proteobacteria (?-proteobacteria).
They include all currently known enzymes with the same requirement
for a free N-terminal l-Ala, i.e. DPP VI, YkfC and AvPCP/NpPCP.
The cyanobacterial enzymes contain one SH3b domain, while the
homologs in other bacteria contain two SH3b domains. As expected,
the SH3b domains are highly divergent. The ?D strand, in which the
conserved tyrosine is located, is more conserved (Fig. 6d). This strand
Acta Cryst. (2010). F66, 1354–1364 Xu et al.
Structural comparisons between BcYkfC and the cyanobacterial NlpC/P60 endopeptidase AvPCP. (a) BcYkfC and AvPCP (PDB code 2hbw; Xu et al., 2009) are shown with
the same orientation of their common SH3b1 and NlpC/P60 domains. The structurally equivalent residues in each are shown in red. (b) Stereoview of the C?traces of the two
proteins shown in (a) [the coloring is the same as in (a)]. (c) The S binding sites of the two proteins are nearly identical. The corresponding residues of AvPCP are labeled in
parentheses. (d) Comparison of the active-site cavities and their environments. The catalytic cysteine is shown in white. A stick model of a docked murein tripeptide is shown
in the active site of BcYkfC.
is also responsible for the interface with the catalytic domain. Download full-text
BcYkfC is a member of a cluster of highly conserved homologs that
are present in closely related species such as B. anthracis, B. thur-
ingiensis and B. weihenstephanensis (sequence identity of ?95%). All
members of this group contain signal peptides. Signal peptides are
also present in some members of the bacteroidetes group, but not in
proteobacterial and cyanobacterial homologs.
It is thus likely that the YkfC subfamily of highly specialized
enzymes has evolved from a common ancestor, which was likely to
have been a P60-like general-purpose enzyme with two or moreSH3b
domains connected to a C-terminal NlpC/P60 domain. SH3b domains
may have lost the function of targeting domains and over time the
nonessential SH3b domain would have been lost, as seen in the
The structure of BcYkfC in complex with l-Ala-?-d-Glu is the first
structural representative ofan NlpC/P60 enzyme with a bound ligand.
This structure allowed us to identify the determinants of substrate
specificity, which further led to the classification of a subfamily of
highly specialized NlpC/P60 enzymes. The studies here also provide
structural and functional insights into the NlpC/P60 family of
enzymes. Additional information about BcYkfC is available from
TOPSAN(Krishnaet al., 2010)
This work was supported by the NIH, National Institute of General
Medical Sciences, Protein Structure Initiative grant U54 GM074898.
Portions ofthis research were carried out at theStanford Synchrotron
Radiation Lightsource (SSRL). The SSRL is a national user facility
operated by Stanford University on behalf of the US Department of
Energy, Office of Basic Energy Sciences. The SSRL Structural
Molecular Biology Program is supported by the Department of
Energy, Office of Biological and Environmental Research and by the
National Institutes of Health (National Center for Research
Resources, Biomedical Technology Program and the National Insti-
tute of General Medical Sciences). Genomic DNA from B. cereus
NRS248 (ATCC No. 10987D) was obtained from the American Type
Culture Collection (ATCC). The content is solely the responsibility of
the authors and does not necessarily represent the official views of
the National Institute of General Medical Sciences or the National
Institutes of Health.
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