The Crystal Structure of the Inhibitor-complexed Carboxypeptidase D Domain II and the Modeling of Regulatory Carboxypeptidases

Abstract

The three-dimensional crystal structure of duck carboxypeptidase D domain II has been solved in a complex with the peptidomimetic inhibitor, guanidinoethylmercaptosuccinic acid, occupying the specificity pocket. This structure allows a clear definition of the substrate binding sites and the substrate funnel-like access. The structure of domain II is the only one available from the regulatory carboxypeptidase family and can be used as a general template for its members. Here, it has been used to model the structures of domains I and III from the former protein and of human carboxypeptidase E. The models obtained show that the overall topology is similar in all cases, the main differences being local and because of insertions in non-regular loops. In both carboxypeptidase D domain I and carboxypeptidase E slightly different shapes of the access to the active site are predicted, implying some kind of structural selection of protein or peptide substrates. Furthermore, emplacement of the inhibitor structure in the active site of the constructed models showed that the inhibitor fits very well in all of them and that the relevant interactions observed with domain II are conserved in domain I and carboxypeptidase E but not in the non-active domain III because of the absence of catalytically indispensable residues in the latter protein. However, in domain III some of the residues potentially involved in substrate binding are well preserved, together with others of unknown roles, which also are highly conserved among all carboxypeptidases. These observations, taken together with others, suggest that domain III might play a role in the binding and presentation of proteins or peptide substrates, such as the pre-S domain of the large envelope protein of duck hepatitis B virus. Carboxypeptidases (CPs)1 are enzymes that catalyze the cleavage of C-terminal peptide bonds in proteins and peptides. From a mechanistic point of view, CPs can be classified in two groups, metalloCPs and serine CPs. The metalloCPs possess a Zn 2ϩ cofactor in the active site. In mammals, this family currently contains 13 members subdivided into two subfamilies, the digestive enzymes and the regulatory enzymes (1-5). Whereas the biological function of the digestive CPs is to contribute to protein degradation, the regulatory ones are generally involved in physiological processes that require a higher specificity. Within each group, the members have 25-63% amino acid sequence identity, but it decreases to only 15-25% when comparison is performed between subfamilies. This low overall homology between subfamilies implies that they diverged early in time.The digestive CPs are soluble, non-glycosylated proteins that are synthesized as inactive precursors containing a 90 -95-amino acid N-terminal pro-segment (5, 6). The regulatory CPs have been purified and characterized from biological fluids and tissues, where they are found in soluble or in membrane-attached forms, in minor quantities. This subfamily includes CPD, CPE, CPM, CPN, CPZ, and novel proteins with ...

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