Structural Basis of Substrate Selectivity of E. coli Prolidase

Abstract

Prolidases, metalloproteases that catalyze the cleavage of Xaa-Pro dipeptides, are conserved enzymes found in prokaryotes and eukaryotes. In humans, prolidase is crucial for the recycling of collagen. To further characterize the essential elements of this enzyme, we utilized the Escherichia coli prolidase, PepQ, which shares striking similarity with eukaryotic prolidases. Through structural and bioinformatic insights, we have extended previous characterizations of the prolidase active site, uncovering a key component for substrate specificity. Here we report the structure of E. coli PepQ, solved at 2.0 Å resolution. The structure shows an antiparallel, dimeric protein, with each subunit containing N-terminal and C-terminal domains. The C-terminal domain is formed by the pita-bread fold typical for this family of metalloproteases, with two Mg(II) ions coordinated by five amino-acid ligands. Comparison of the E. coli PepQ structure and sequence with homologous structures and sequences from a diversity of organisms reveals distinctions between prolidases from Gram-positive eubacteria and archaea, and those from Gram-negative eubacteria, including the presence of loop regions in the E. coli protein that are conserved in eukaryotes. One such loop contains a completely conserved arginine near the catalytic site. This conserved arginine is predicted by docking simulations to interact with the C-terminus of the substrate dipeptide. Kinetic analysis using both a charge-neutralized substrate and a charge-reversed variant of PepQ support this conclusion, and allow for the designation of a new role for this key region of the enzyme active site.

Substrate specificity and reaction mechanism of human prolidase

“…Depending on the particular protein, the preferences for the identity of the metal ions can vary . The physiologically relevant metal ions in the case of human prolidase are Mn 2+ although some residual activity has been reported in the presence of other divalent ions , among them Mg 2+ . In order to examine the metal binding in detail, apo‐prolidase crystals have been soaked with MnCl 2 solution.…”

“…Furthermore, the substrate specificity of Escherichia coli prolidase was analyzed previously based on docking experiments. This pointed to an essential role of a certain Arg residue . This hypothesis is interesting, but still needs to be confirmed by experimental evidence.…”

GroEL actively stimulates folding of the endogenous substrate protein PepQ

“…DapA, like RuBisCO, is a member of the TIM-barrel family of proteins, a canonical α/β-fold that is highly represented in the subset of E. coli proteins that depend on GroEL for folding3132. By contrast, PepQ is a member the so-called pita-bread proteins282930, a protein fold that is fundamentally distinct from the TIM-barrel fold27. To date, no pita-bread fold has been examined in detail as a GroEL-substrate protein.…”

“…PepQ catalyses the hydrolysis of dipeptides that contain C-terminal proline residues2728. It forms a homodimer, with each monomer (∼50 kDa) built from two domains: a small, mixed α/β N-terminal domain and a pita-bread fold2930 C-terminal domain that contains the active site (Fig.…”

Mutations in pepQ Confer Low-Level Resistance to Bedaquiline and Clofazimine in Mycobacterium tuberculosis

“…high level of conservation with E. coli pepP and both prokaryotic and eukaryotic prolidases [25]), not in close proximity to the active site. Thus, L44P is likely a structure-destabilizing mutation, as is the frameshift mutation in Ala14.…”