A standard orientation for metallopeptidases

Abstract

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 β-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 β-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 αβα-sandwich, which is reminiscent of, but clearly distinct from, the archetypal α/β-hydrolase fold. Metalloexopeptidases have their active sites at the C-terminal end of a central, eight-stranded twisted β-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 β-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.

A Novel Family of Soluble Minimal Scaffolds Provides Structural Insight into the Catalytic Domains of Integral Membrane Metallopeptidases

“…41, 42 and also see Table 2 in Ref. 44). Superposition of both proabylysin and projannalysin onto thermolysin reveals that the polypeptide chains are very similar along almost the entire structure of the former (M 1 -K 99 ) and V71-P195 of the latter (Fig.…”

“…2 in Ref. 44), and is only very distantly related to proabylysin (PDB 3B4R; DALI Z score 1.2; r.m.s.d. 3.5; 51 aligned residues; 10% sequence identity) and its structural relatives.…”

“…The insertion of the C-terminal tail also provides information on the distinct subsites on the primed side of the cleft. M 104 nestles into what is most likely the S 1 subsite of the cleft (cleft and substrate subsites in boldface type), which is usually the major determinant for specificity in endolytic MPs (44). The methionine side chain is present in double conformation, and the surrounding residues give rise to a site that is wide and hydrophobic, thus suggesting that mature abylysin could easily accommodate bulky hydrophobic residues in P 1 , as observed in the otherwise unrelated matrix metalloproteinases, snapalysins, and ADAMs/adamalysins, which are metzincins (77-79).…”

“…Preceding the latter strand is the "bulge-edge segment" (L 26 -I 30 ), which contributes to shaping the top of the cleft on its primed side (for cleft and substrate subsite nomenclature, see Refs. 44,76). After ␤3, a short 3 10 -helix (1; the "linking helix") at the right top of the molecule links the sheet with the "active-site helix" ␣2, which contains the short zinc-binding consensus sequence 60 HEXXH 64 (see below).…”

“…2 in Ref. 44) between transmembrane helices (TMHs) provides a useful starting point for the identification of such CDs, which are mostly cytoplasmic (Fig. 1).…”