Structure of the γ-D-glutamyl-L-diamino acid endopeptidase YkfC from Bacillus cereus in complex with L-Ala-γ-d-Glu: Insights into substrate recognition by NlpC/P60 cysteine peptidases

Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA, USA.
Acta Crystallographica Section F Structural Biology and Crystallization Communications (Impact Factor: 0.53). 10/2010; 66(Pt 10):1354-64. DOI: 10.1107/S1744309110021214
Source: PubMed


Dipeptidyl-peptidase VI from Bacillus sphaericus and YkfC from Bacillus subtilis have both previously been characterized as highly specific γ-D-glutamyl-L-diamino acid endopeptidases. The crystal structure of a YkfC ortholog from Bacillus cereus (BcYkfC) at 1.8 Å 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-γ-D-Glu) enabled the identification of conserved sequence and structural signatures for recognition of L-Ala and γ-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-γ-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.

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Available from: Hsiu-Ju Chiu, Oct 07, 2015
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    • "A structural similarity search revealed that the most similar structure is the CHAP domain of the streptococcal phage endolysin PlyC (PDB entry 4F88) [15], with a root mean square difference (r.m.s.d.) of 2.5 Å when the backbone atoms of 124 residues are superposed onto CHAPK (Z-score 11.4). The next most similar structure is the C-terminal endopeptidase domain of the NlpC/P60 family cell-wall remodelling protein Bacillus cereus PDB code 3H41; Ref. [16], with an r.m.s.d. of 2.8 Å when the backbone atoms of 114 residues are superposed (Z-score 10.2). When the PDB database is searched for sequence-similar structures, the first hit is the CHAP domain from Staphylococcus saprophyticus CHAP domain protein (PDB entry 2K3A) [17], with a sequence identity of 28% over a stretch of 94 residues. "
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    ABSTRACT: Background Bacteriophages encode endolysins to lyse their host cell and allow escape of their progeny. Endolysins are also active against Gram-positive bacteria when applied from the outside and are thus attractive anti-bacterial agents. LysK, an endolysin from staphylococcal phage K, contains an N-terminal cysteine-histidine dependent amido-hydrolase/peptidase domain (CHAPK), a central amidase domain and a C-terminal SH3b cell wall-binding domain. CHAPK cleaves bacterial peptidoglycan between the tetra-peptide stem and the penta-glycine bridge. Methods The CHAPK domain of LysK was crystallized and high-resolution diffraction data was collected both from a native protein crystal and a methylmercury chloride derivatized crystal. The anomalous signal contained in the derivative data allowed the location of heavy atom sites and phase determination. The resulting structures were completed, refined and analyzed. The presence of calcium and zinc ions in the structure was confirmed by X-ray fluorescence emission spectroscopy. Zymogram analysis was performed on the enzyme and selected site-directed mutants. Results The structure of CHAPK revealed a papain-like topology with a hydrophobic cleft, where the catalytic triad is located. Ordered buffer molecules present in this groove may mimic the peptidoglycan substrate. When compared to previously solved CHAP domains, CHAPK contains an additional lobe in its N-terminal domain, with a structural calcium ion, coordinated by residues Asp45, Asp47, Tyr49, His51 and Asp56. The presence of a zinc ion in the active site was also apparent, coordinated by the catalytic residue Cys54 and a possible substrate analogue. Site-directed mutagenesis was used to demonstrate that residues involved in calcium binding and of the proposed active site were important for enzyme activity. Conclusions The high-resolution structure of the CHAPK domain of LysK was determined, suggesting the location of the active site, the substrate-binding groove and revealing the presence of a structurally important calcium ion. A zinc ion was found more loosely bound. Based on the structure, we propose a possible reaction mechanism. Future studies will be aimed at co-crystallizing CHAPK with substrate analogues and elucidating its role in the complete LysK protein. This, in turn, may lead to the design of site-directed mutants with altered activity or substrate specificity.
    Virology Journal 07/2014; 11(1):133. DOI:10.1186/1743-422X-11-133 · 2.18 Impact Factor
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    • "Regulatory structural elements and catalytic cysteine residues are shown in red and yellow, respectively. YkfC has an additional domain adjacent to its active site and RipB contains an N-terminal extension that occludes its substrate binding cleft (Both et al., 2011; Xu et al., 2010). In Spr, residues proximal to the active site limit accessibility to catalytic residues (Aramini et al., 2008). "
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    ABSTRACT: The target range of a bacterial secretion system can be defined by effector substrate specificity or by the efficacy of effector delivery. Here, we report the crystal structure of Tse1, a type VI secretion (T6S) bacteriolytic amidase effector from Pseudomonas aeruginosa. Consistent with its role as a toxin, Tse1 has a more accessible active site than related housekeeping enzymes. The activity of Tse1 against isolated peptidoglycan shows its capacity to act broadly against Gram-negative bacteria and even certain Gram-positive species. Studies with intact cells indicate that Gram-positive bacteria can remain vulnerable to Tse1 despite cell wall modifications. However, interbacterial competition studies demonstrate that Tse1-dependent lysis is restricted to Gram-negative targets. We propose that the previously observed specificity for T6S against Gram-negative bacteria is a consequence of high local effector concentration achieved by T6S-dependent targeting to its site of action rather than inherent effector substrate specificity.
    Cell Reports 06/2012; 1(6):656-64. DOI:10.1016/j.celrep.2012.05.016 · 8.36 Impact Factor
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    • "Through its capacity to bind, with moderate affinity and selectivity, to proline-rich ligands, these domains are critical for a wide range of biological functions in eukaryotes. Their molecular functions in bacteria are not well understood, but they are widely found in proteins involved in cell wall biosynthesis, supporting the hypothesis that the YARHG domain might be binding to some component of the bacterial cell wall such as peptidoglycan [30]. "
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    ABSTRACT: We have identified a new bacterial protein domain that we hypothesise binds to peptidoglycan. This domain is called the YARHG domain after the most highly conserved sequence-segment. The domain is found in the extracellular space and is likely to be composed of four alpha-helices. The domain is found associated with protein kinase domains, suggesting it is associated with signalling in some bacteria. The domain is also found associated with three different families of peptidases. The large number of different domains that are found associated with YARHG suggests that it is a useful functional module that nature has recombined multiple times.
    PLoS ONE 05/2012; 7(5):e35575. DOI:10.1371/journal.pone.0035575 · 3.23 Impact Factor
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