Crystal Structure of the Bacillus subtilis Penicillin-binding Protein 4a, and its Complex with a Peptidoglycan Mimetic Peptide

Abstract

The genome of Bacillus subtilis encodes 16 penicillin-binding proteins (PBPs) involved in the synthesis and/or remodelling of the peptidoglycan during the complex life cycle of this sporulating Gram-positive rod-shaped bacterium. PBP4a (encoded by the dacC gene) is a low-molecular mass PBP clearly exhibiting in vitro DD-carboxypeptidase activity. We have solved the crystal structure of this protein alone and in complex with a peptide (D-α-aminopymelyl-ε-D-alanyl-D-alanine) that mimics the C-terminal end of the Bacillus peptidoglycan stem peptide. PBP4a is composed of three domains: the penicillin-binding domain with a fold similar to the class A β-lactamase structure and two domains inserted between the conserved motifs 1 and 2 characteristic of the penicillin-recognizing enzymes. The soaking of PBP4a in a solution of D-α-aminopymelyl-ε-D-alanyl-D-alanine resulted in an adduct between PBP4a and a D-α-aminopimelyl-ε-D-alanine dipeptide and an unbound D-alanine, i.e. the products of acylation of PBP4a by D-α-aminopymelyl-ε-D-alanyl-D-alanine with the release of a D-alanine. The adduct also reveals a binding pocket specific to the diaminopimelic acid, the third residue of the peptidoglycan stem pentapeptide of B. subtilis. This pocket is specific for this class of PBPs.

Crystal Structures of Complexes of Bacterial dd-Peptidases with Peptidoglycan-Mimetic Ligands: The Substrate Specificity Puzzle

“…10 An analogous site is likely present on B. subtilis PBP4a. 18 In what appears to be sharp contrast, essentially no firm electron density for the side chain, and none particularly for the polar terminus, was observed in the complexes of E. coli PBP5 with β-lactams 5 and 6 ( Fig. 1c and d).…”

“…It should be noted, however, that other low-molecular-mass class C enzymes that have structures very similar to those of the R39 enzyme (e.g., E. coli PBP4 and B. subtilis PBP4a) 14,17,18 do appear to have real, if nonessential (in the same sense as E. coli PBP5 perhaps; see below), roles in bacterial cell wall construction and maintenance. 34,35 The latter of these enzymes has also been shown to have high hydrolytic activity against the peptide 7.…”

“…The aminopimelyl ammonium-terminus hydrogen bonds to the Asp142 side chain. It is striking that, as with B. subtilis PBP4a, 18 the elements of the side-chain binding site are derived from two separate domains of the protein: 16 the β-lactam-binding domain per se and domain II, with the latter supplying Trp139, Asp142, and Tyr147 (Fig. 1a).…”

“…18 The positions of the active-site residues of both structures are nearly identical, and the side chain of peptide 7 occupies exactly the same position, making identical interactions, particularly with Asp142 and Ser415 (R39 numbering); the one notable difference is that Arg351 is replaced by a histidine in PBP4a. In the primary structure of most PBPs homologous to R39 (e.g., E. coli PBP4 and Neisseria gonorrhoeae PBP3), this residue is a histidine or an arginine, and the other residues forming the meso-diaminopimelate-binding pocket are well conserved.…”

“…14,15 Crystal structures have confirmed the structural resemblance. [16][17][18] The R39 DD-peptidase is a water-soluble enzyme that is loosely associated with the bacterial cell membrane. Although the precise role of the enzyme in vivo is not known, under in vitro conditions, it has been shown to catalyze the hydrolysis and aminolysis of small D-alanyl peptides and esters.…”