Structure of the γ-D-glutamyl-L-diamino acid endopeptidase YkfC fromBacillus cereusin complex withL-Ala-γ-D-Glu: insights into substrate recognition by NlpC/P60 cysteine peptidases

Abstract

PDB Reference: YkfC-L-Ala--D-Glu complex, 3h41.Dipeptidyl-peptidase VI from Bacillus sphaericus and YkfC from Bacillus subtilis have both previously been characterized as highly specific -d-glutamyll-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--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.

Structural Insights into the Pseudomonas aeruginosa Type VI Virulence Effector Tse1 Bacteriolysis and Self-protection Mechanisms

“…Structural homology searches by DALI (23) reveal that Tse1 shares a notable structural similarity with characterized NlpC/P60 domains of other DL-endopeptidases. The significant hits retrieved by DALI include DL-endopeptidase YkfC from Bacillus cereus (Z ϭ 10.0, PDB code 3H41) (24), membrane-anchored cell-wall hydrolase Spr from E. coli (Z ϭ 8.8, PDB code 2K1G) (25), DL-endopeptidases NpPCP and AvPCP from cyanobacteria Nostoc punctiforme and Anabaena variabilis (Z ϭ 8.6 and 8.5, PDB codes 2EVR and 2HBW) (26), and the peptidoglycan endopeptidases RipA and RipB from Mycobacterium tuberculosis (Z ϭ 7.1 and 7.2, PDB code 3NE0 and 3PBI) (Fig. 1C) (27,28).…”

“…Several of the endopeptidases act on the simple murein tetrapeptide comprising the specific ␥-D-glutamyl-DAP bond. The fused auxiliary domains confer substrate specificity on these peptidoglycan hydrolases (24,26). However, as an exogenous peptidoglycan hydrolase delivered by T6SS, Tse1 directly faces the intact cell wall peptidoglycan and targets its specific ␥-D-glutamyl-DAP linkage occurring in the crosslinked murein peptide.…”

Structure of the Type VI Effector-Immunity Complex (Tae4-Tai4) Provides Novel Insights into the Inhibition Mechanism of the Effector by Its Immunity Protein*

“…The Dali server was used to find the structural neighbors to Tae4 (29). There are two CHAP (cysteine, histidine-dependent amidohydrolases/peptidase) family functional proteins with Z-scores greater than 5, including endopeptidase YkfC from Bacillus cereus (PDB ID code 3H41) (30) and bacteriolytic effector Tae1 (Tse1) from P. aeruginosa (PDB ID code 4F0V) (10). Despite the low sequence similarity, superpositions of Tae4 with the two proteins give r.m.s.d.…”

Functional metagenomic discovery of bacterial effectors in the human microbiome and isolation of commendamide, a GPCR G2A/132 agonist

“…A number of predicted catabolic enzymes resemble peptidoglycan hydrolyzing enzymes with different arrangements of NlpC/p60-like cell wall peptidase domains and lysozyme-like glycosyl hydrolase domains. For example, Cbeg7 has both an NlpC/p60 and a lysozyme-like domain, whereas Cbeg8 and -9 have either an NlpC/p60 (Cbeg9) or lysozyme-like (Cbeg8) domain in addition to either a M23 zinc metallopeptidase (Cbeg9) or LysM carbohydrate binding domain (Cbeg8) (44)(45)(46). The domain architecture seen in Cbeg8 is typical of bacterial autolysins that are associated with both reorganizing cell membranes for cell division and host invasion by pathogens (47).…”