M16B Metallopeptidase
Created by Xin Zhao
Bacillus halodurans M16B metallo peptidase (PDB ID: 3HDI)(
pic.1) is one prokaryotic member of M16 peptidase family. The M16 family of zinc peptidases comprises a pair of homologous domains that form two halves of a clam-shell surrounding the active site(
pic.2)(
pic.3)(
pic.4), which has been observed in closed, open, and locked-open states. For example, M16A and M16C subfamilies form one class (peptidasomes): they degrade 30-70 residue peptides, and adopt both open and closed conformations. The eukaryotic M16B subfamily forms a second class (processing proteases): they adopt a single partly-open conformation that enables them to cleave signal sequences from larger proteins. For Bacillus halodurans M16B metallo peptidase, which is a prokaryotic M16B peptidase, it has features of both classes: thus, it forms stable open homodimers in solution that resemble the processing proteases; but the clam-shell closes upon binding substrate, a feature of the M16A/C peptidasomes. Moreover, clam-shell closure is required for proteolytic activity.
physiological form, secondary structure and Subunit structure
BHP, the crystal structure of the BH2405 gene product of Bacillus halodurans C-125 strain, forms a monodimer in solution at physiological salt concentrations, with a Kd % > 20 nM, and that an inactive mutant preincubated with insulin-B had a similar Kd. In the crystals, each active site is occupied by a single metal ion. Each monomer of BHP has 14 a helixes and 11 b sheets(
pic.5()
pic.6) and consists of two structurally similar subdomains (residues 1-207 and 223-415) connected by a long linker and related by an approximate 2-fold axis of symmetry(
pic.7). Each subdomain has a core with a 3-dimensional structure comprising a 5-stranded antiparallel b sheet and a helical bundle that packs against the convex face of the sheet. BHP forms a homodimer in the crystals with a closed clamshell architecture similar to those of the M16A/C peptidasomes, IDE and PreP. The two halves of the BHP dimer are very similar, with a root-mean-square deviation (rmsd) of 0.38 Å for 408 superposed Ca atoms. When compared with the other known structures of M16 proteases, the BHP monomer overlays with a typical rmsd of ~1.5 Å for ~70% of their Ca atoms. The interface between the BHP monomers involves multiple interactions involving ~73 residues (out of 414) from each monomer, burying a large surface area (~2700 Å ), which is comparable in size with the closed clamshells of IDE and PreP. The interface creates a closed water-filled chamber with a volume of ~10,000 Å, similar to PreP, but smaller than in IDE (Malito et al., 2008). Because there are no channels through which a peptide could enter, the clam-shell must open at least transiently in order to bind a substrate.
important residues
Because BHP is a homodimer, it has two active sites. Both sites contain a continuous stretch of electron density consistent with a bound peptide ligand.(
pic.9
pic.10) The relatively weak electron density suggests an occupancy of 50%. However, it is also possible that each BHP dimer binds a single peptide at full occupancy, but that given a stochastic distribution of peptides between the two crystallographically distinct sites, only an averaged image with partial occupancy is observed. The electron density was not of sufficient clarity to resolve the peptide sequence(s), but N and C termini of the peptides interact with opposing halves of the clam-shell, stabilizing the closed conformation. The two monomers contributing to the active site are in green and blue; the substrate is built as a polyalanine chain in orange/red/blue (
pic.11). The cobalt ion is labeled (Co).
A previous analysis of the cleavage specificity of the close ortholog, BH4, revealed a preference for aromatic residues at P1 (upstream of the cleavage site), and a broader preference at P10 (downstream of the cleavage site), including hydrophobic/ aromatic residues and arginine (Dabonne et al., 2005). The structure of BHP is consistent with these preferences. Thus, the S1 site is flat, and its preference for aromatic residues may arise from favorable stacking interactions with F78 from the b4 strand, which is conserved in M16 peptidasomes. The S10 site is a deep pocket (
pic.12) with hydrophobic walls and acidic residues at the base. The hydrophobic lining presumably favors hydrophobic residues, whereas the side chain of arginine is long enough to extend to the base of the pocket and interact with the acidic groups.
Clam-Shell Closure Enhances Catalytic Activity
The tight binding between BHP monomers (Kd > 20 nM) indicates that it is predominantly a homodimer both in the presence and absence of substrate. An opening mechanism that maintains an extensive interface is critical for BHP, because it comprises two polypeptide chains that must remain paired throughout the catalytic cycle. By contrast, the M16A/C domains are connected covalently by a linker that wraps around the hinge point, which seems to allow for a much smaller interface in the open conformation.
More detailed comparison between BHP and the eukaryotic M16B enzymes reveals an exception to the rigid body rotation; helix a12 packs against the second monomer, making very similar interactions in both the open and closed conformations. In particular, L-344 (L-386 in MPP) seems to act as a linchpin by packing into a pocket at one corner of the N-terminal b sheet of the second monomer. That is, helix a12 remains affixed to the second monomer, and the rotational hinge points leading to clam-shell opening actually occur at either end of this helix. One important prediction of this model is that the environment of a12 changes within its own monomer as a result of clam-shell opening. Of particular note, helix a14 rolls or ratchets around helix a12, burying an exposed hydrophobic surface on a12 and forms new contacts with the top edge of the N-terminal b sheet. The burial of hydrophobic surfaces in this local region may compensate, at least in part, for the loss of the more extensive interface in the closed conformation of BHP.(
pic.13).
Summary and Conclusions
The summaries of this study are as follows: (i) BHP functions as a homodimer. (ii) The active site of BHP is similar to thoseof the M16A/C peptidasomes, consistent with its peptidasome function, and distinct from M16B eukaryotic processing peptidases. (iii) BHP exists in an open dimeric conformation in the absence of substrate. (iv)The open structure likely resembles the eukaryotic M16B proteins, and closes by means of hinge points at either ends of two fixed helices (a12). (v) Closure of the clam-shell is required for catalytic activity.