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

Abstract

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 β-sandwich N-terminal domain followed by the classical carboxypeptidase α/β-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.

Functional segregation and emerging role of cilia‐related cytosolic carboxypeptidases (CCPs)

“…Notably, the enzymes acting as deglycylases in holozoans remain unknown, but recently two enzymes belonging to the M20 family of peptidases, which display deglycylase activity on the multifunctional proteins 14‐3‐3 in Giardia duodenalis , have been identified (49). Interestingly, our recently resolved structure of the Pseudomona aeruginosa CCP (18) is related to M20 peptidases. Finally, human CCP2 found in the basal bodies of ciliated HeLa cells colocalized with γ‐tubulin across all stages of the cell cycle (Fig.…”

“…A total of 147 CCP sequences (Supplemental Table S3) were aligned using T‐Coffee (17). The tridimensional structure of the CCP recently released (18) led us to define the conserved core of CCPs and consequently to refine the sequence‐structural alignment of CCPs used in the phylogenetic analyses. The alignment was manually corrected, taking into account the structural alignment information described previously (5).…”

Cytosolic Carboxypeptidase 5 Removes α- and γ-Linked Glutamates from Tubulin

“…Recently, purified CCP1 was reported to release Glu from small peptides containing 2-3 Glu residues and a blocked N terminus (34). The structure of a bacterial CCP, as determined by x-ray crystallography, shows classical metallocarboxypeptidase-like motifs, including the presence of an active site and substrate-binding pockets generally similar to those of enzymatically active metallocarboxypeptidases, such as mammalian carboxypeptidases A and B (35). The purified CCP used for the crystallization studies was inactive toward all substrates that were tested, despite the presence of a functional active site pocket in the structure, possibly due to the presence of an N-terminal domain that occludes access of the substrate to the active site (35).…”

Zebrafish Cytosolic Carboxypeptidases 1 and 5 Are Essential for Embryonic Development

“…In addition to the CP domain, which has homology to the CP domain of A/B and N/E subfamily enzymes, all CCPs in humans and zebrafish contain a ␤-sheet-rich domain to the N-terminal side of the CP domain. Although there is no sequence similarity between this domain in the CCPs and the prodomain in the CPA/B subfamily of enzymes, the size of the domain and the high degree of ␤-sheet secondary structure are similar among the A/B and CCP subfamilies (31). The ␤-sheet domain in the CCPs is likely to be functional based on the high degree of sequence identity between human and zebrafish orthologs ( Fig.…”