The novel structure of a cytosolic M14 metallocarboxypeptidase (CCP) from Pseudomonas aeruginosa: a model for mammalian CCPs.

PaCCP is a metallocarboxypeptidase (MCP) of the M14 family from Pseudomonas aeruginosa, which belongs to a bacterial clade of carboxypeptidases that are homologous to the recently discovered M14D subfamily of human nonsecretory cytosolic carboxypeptidases (CCPs). CCPs are intracellular peptidases involved, among other roles, in the post-translational modifications of tubulin. Here we report the crystal structure of PaCCP at high resolution (1.6 Å). Its 375 residues are folded in a novel ß-sandwich N-terminal domain followed by the classical carboxypeptidase α/ß-hydrolase domain, this one in a shorter and more compact form. The former is unique in the whole family and does not have sequential or structural homology with other domains that are usually flanking the latter, like the prodomain of the M14A subfamily or the C-terminal transthyretin/prealbumin-like domains of the M14B subfamily. PaCCP does not display activity against small carboxypeptidase substrates, so in this form it might constitute an inactive precursor of the protease. Structural results derived from cocrystallization with well-known inhibitors of MCPs indicate that the enzyme might only possess C-terminal hydrolase activity against cellular substrates of particular specificity and/or when undergoes structural rearrangements. The derived PaCCP structure allows a first structural insight into the more complex and largely unknown mammalian CCP subfamily.

Tri-domain bifunctional inhibitor of metallocarboxypeptidases A and serine proteases isolated from marine annelid Sabellastarte magnifica.

This study describes a novel bifunctional metallocarboxypeptidase and serine protease inhibitor (SmCI) isolated from the tentacle crown of the annelid Sabellastarte magnifica. SmCI is a 165-residue glycoprotein with a molecular mass of 19.69 kDa (mass spectrometry) and 18 cysteine residues forming nine disulfide bonds. Its cDNA was cloned and sequenced by RT-PCR and nested PCR using degenerated oligonucleotides. Employing this information along with data derived from automatic Edman degradation of peptide fragments, the SmCI sequence was fully characterized, indicating the presence of three bovine pancreatic trypsin inhibitor/Kunitz domains and its high homology with other Kunitz serine protease inhibitors. Enzyme kinetics and structural analyses revealed SmCI to be an inhibitor of human and bovine pancreatic metallocarboxypeptidases of the A-type (but not B-type), with nanomolar K(i) values. SmCI is also capable of inhibiting bovine pancreatic trypsin, chymotrypsin, and porcine pancreatic elastase in varying measures. When the inhibitor and its nonglycosylated form (SmCI N23A mutant) were overproduced recombinantly in a Pichia pastoris system, they displayed the dual inhibitory properties of the natural form. Similarly, two bi-domain forms of the inhibitor (recombinant rSmCI D1-D2 and rSmCI D2-D3) as well as its C-terminal domain (rSmCI-D3) were also overproduced. Of these fragments, only the rSmCI D1-D2 bi-domain retained inhibition of metallocarboxypeptidase A but only partially, indicating that the whole tri-domain structure is required for such capability in full. SmCI is the first proteinaceous inhibitor of metallocarboxypeptidases able to act as well on another mechanistic class of proteases (serine-type) and is the first of this kind identified in nature.

A novel metallocarboxypeptidase-like enzyme from the marine annelid Sabellastarte magnifica--a step into the invertebrate world of proteases.

After screening 25 marine invertebrates, a novel metallocarboxypeptidase (SmCP) has been identified by activity and MS analytical approaches, and isolated from the marine annelid Sabellastarte magnifica. The enzyme, which is a minor component of the molecularly complex animal body, as shown by 2D gel electrophoresis, has been purified from crude extracts to homogeneity by affinity chromatography on potato carboxypeptidase inhibitor and by ion exchange chromatography. SmCP is a protease of 33792 Da, displaying N-terminal and internal sequence homologies with M14 metallocarboxypeptidase-like enzymes, as determined by MS and automated Edman degradation. The enzyme contains one atom of Zn per molecule, is activated by Ca2+ and is drastically inhibited by the metal chelator 1,10-phenanthroline, as well as by excess Zn2+ or Cu2+, but moderately so by EDTA. SmCP is also strongly inhibited by specific inhibitors of metallocarboxypeptidases, such as benzylsuccinic acid and the protein inhibitors found in potato and leech (i.e. recombinant forms, both at nanomolar levels). The enzyme displays high peptidase efficiency towards pancreatic carboxypeptidase-A synthetic substrates, such as those with hydrophobic residues at the C-terminus but, remarkably, also towards the acidic ones. This property, previously described as for carboxypeptidase O-like activity, has been shown on long peptide substrates by MS. The results obtained in the present study indicate that SmCP is a novel member of the M14 metallocarboxypeptidases family (assignable to the M14A or pancreatic-like subfamily) with a wider specificity that has not been described previously.

Nna1-like proteins are active metallocarboxypeptidases of a new and diverse M14 subfamily.

Nna1 has some sequence similarity to metallocarboxypeptidases, but the biochemical characterization of Nna1 has not previously been reported. In this work we performed a detailed genomic scan and found >100 Nna1 homologues in bacteria, Protista, and Animalia, including several paralogs in most eukaryotic species. Phylogenetic analysis of the Nna1-like sequences demonstrates a major divergence between Nna1-like peptidases and the previously known metallocarboxypeptidases subfamilies: M14A, M14B, and M14C. Conformational modeling of representative Nna1-like proteins from a variety of species indicates an unusually open active site, a property that might facilitate its action on a wide variety of peptide and protein substrates. To test this, we expressed a recombinant form of one of the Nna1-like peptidases from Caenorhabditis elegans and demonstrated that this protein is a fully functional metallocarboxypeptidase that cleaves a range of C-terminal amino acids from synthetic peptides. The enzymatic activity is activated by ATP/ADP and salt-inactivated, and is preferentially inhibited by Z-Glu-Tyr dipeptide, which is without precedent in metallocarboxypeptidases and resembles tubulin carboxypeptidase functioning; this hypothesis is strongly reinforced by the results depicted in Kalinina et al. published as accompanying paper in this journal. Our findings demonstrate that the M14 family of metallocarboxypeptidases is more complex and diverse than expected, and that Nna1-like peptidases are functional variants of such enzymes, representing a novel subfamily (we propose the name M14D) that contributes substantially to such diversity.

Structural basis of the resistance of an insect carboxypeptidase to plant protease inhibitors.

Corn earworm (Helicoverpa zea), also called tomato fruitworm, is a common pest of many Solanaceous plants. This insect is known to adapt to the ingestion of plant serine protease inhibitors by using digestive proteases that are insensitive to inhibition. We have now identified a B-type carboxypeptidase of H. zea (CPBHz) insensitive to potato carboxypeptidase inhibitor (PCI) in corn earworm. To elucidate the structural features leading to the adaptation of the insect enzyme, the crystal structure of the recombinant CPBHz protein was determined by x-ray diffraction. CPBHz is a member of the A/B subfamily of metallocarboxypeptidases, which displays the characteristic metallocarboxypeptidase alpha/beta-hydrolase fold, and does not differ essentially from the previously described Helicoverpa armigera CPA, which is very sensitive to PCI. The data provide structural insight into several functional properties of CPBHz. The high selectivity shown by CPBHz for C-terminal lysine residues is due to residue changes in the S1' substrate specificity pocket that render it unable to accommodate the side chain of an arginine. The insensitivity of CPBHz to plant inhibitors is explained by the exceptional positioning of two of the main regions that stabilize other carboxypeptidase-PCI complexes, the beta8-alpha9 loop, and alpha7 together with the alpha7-alpha8 loop. The rearrangement of these two regions leads to a displacement of the active-site entrance that impairs the proper interaction with PCI. This report explains a crystal structure of an insect protease and its adaptation to defensive plant protease inhibitors.