A standard orientation for metallopeptidases.

Visualization of three-dimensional structures is essential to the transmission of information to the general reader and the comparison of related structures. Therefore, it would be useful to provide a common framework. Based on the work of Schechter and Berger, and the finding that most peptidases bind their substrates in extended conformation, we suggest a "standard orientation" for the overall description of metallopeptidases (MPs) as done before for peptidases of other classes. This entails a frontal view of the horizontally-aligned active-site cleft. A substrate is bound N- to C-terminally from left (on the non-primed side of the cleft) to right (on the primed side), and the catalytic metal ion resides at the cleft bottom at roughly half width. This view enables us to see that most metalloendopeptidases are bifurcated into an upper and a lower sub-domain by the cleft, whose back is framed by a nearly horizontal "active-site helix." The latter comprises a short zinc-binding consensus sequence, either HEXXH or HXXEH, which provides two histidines to bind the single catalytic metal and the general-base/acid glutamate required for catalysis. In addition, an oblique "backing helix" is observed behind the active-site helix, and a mixed ß-sheet of at least three strands is positioned in the upper sub-domain paralleling the cleft. The lowermost "upper-rim" strand of the sheet runs antiparallel to the substrate bound in the cleft and therefore contributes both to delimitating the cleft top and to binding of the substrate main-chain on its non-primed side through ß-ribbon-like interactions. In contrast, in metalloexopeptidases, which chop off N- or C-terminal residues only, extensive binding on both sides of the cleft is not required and a different overall scaffold is generally observed. This consists of an αßα-sandwich, which is reminiscent of, but clearly distinct from, the archetypal α/ß-hydrolase fold. Metalloexopeptidases have their active sites at the C-terminal end of a central, eight-stranded twisted ß-sheet, and can contain one or two catalytic metal ions. As the zinc-binding site and the residues engaged in substrate binding and catalysis are mainly provided by loops connecting the ß-sheet strands and the helices on either side, the respective standard orientations vary with respect to the position of the sheets. The standard orientation of eight prototypic MP structures is presented and discussed. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.

Structural and functional analyses reveal that Staphylococcus aureus antibiotic resistance factor HmrA is a zinc-dependent endopeptidase.

HmrA is an antibiotic resistance factor of methicillin-resistant Staphylococcus aureus. Molecular analysis of this protein revealed that it is not a muramidase or ß-lactamase but a nonspecific double-zinc endopeptidase consisting of a catalytic domain and an inserted oligomerization domain, which probably undergo a relative interdomain hinge rotation upon substrate binding. The active-site cleft is located at the domain interface. Four HmrA protomers assemble to a large ∼170-kDa homotetrameric complex of 125 Å. All four active sites are fully accessible and ∼50-70 Å apart, far enough apart to act on a large meshwork substrate independently but simultaneously. In vivo studies with four S. aureus strains of variable resistance levels revealed that the extracellular addition of HmrA protects against loss of viability in the presence of oxacillin and that this protection depends on proteolytic activity. All of these results indicate that HmrA is a peptidase that participates in resistance mechanisms in vivo in the presence of ß-lactams. Furthermore, our results have implications for most S. aureus strains of known genomic sequences and several other cocci and bacilli, which harbor close orthologs. This suggests that HmrA may be a new widespread antibiotic resistance factor in bacteria.