Albany 2015:Book of Abstracts

Albany 2015
Conversation 19
June 9-13 2015
©Adenine Press (2012)

The crystal structure of a tri domain BPTI Kunitz inhibitor in complex with human carboxypeptidase A4 revealed a novel and non canonical inhibition mechanism

Protein metallo carboxypeptidase inhibitors (CPIs) are one of the less abundant in nature. Only nine CPIs have been reported and can be divided in two groups according their inhibition mechanism: substrate like recognition or steric blockage of the active site. The first group is the most representative and includes CPIs that insert their C-terminal in the enzyme active site in a substrate feature, forming a stable complex. The others inhibitors block the access to the active site by steric blockage.

SmCI is a CPI isolated from the sea annelid Sabellastarte magnifica able to inhibit metallo CPA (Carboxypeptidase A) through a novel inhibition mechanism. It is a one-chain protein of 19703 Da, N-glycosilated, and folded in three BPTI/Kunitz domains. The BPTI/Kunitz family represents typical serine protease inhibitors. However, SmCI is the only BPTI/Kunitz inhibitor able to additionally block CPA activity.

In this work, we elucidated the inhibition mechanism of CPA by SmCI using kinetic and structural techniques. For the kinetic approach, we obtained the recombinant SmCI bi-domains D1D2 and D2D3. Bi-domain D1D2 was the only one able to inhibit CPA. However, its 3-folds higher Ki value against this enzyme, compared with the natural and recombinant tri-domain forms (SmCI and SmCIr), indicates its lower inhibition strength. This kinetic data suggests that the first domain has the essential role in CPA inhibition, but also, the requirements of the whole tri-domain molecule to reach the maximal inhibition. The interaction mechanism was later detailed from the crystallographic structure of the SmCI-CPA complex. For better results during the crystallization trials, we firstly obtained and characterized the non-glycosilated mutant SmCI N23A. The SmCI N23A-humanCPA4 complex structure was obtained by X ray crystallography; and it surprisingly revealed the N-terminal tail of SmCI inserted in the enzyme active site. These results represent the first non-canonical mechanism described for metallo CPA enzymes. SmCI N-tail is able to keep the same kind and pattern of interaction in the primary site compared to inhibitors with a substrate-like mechanism. Interestingly, the P1-P2-P3 sites (the first three residues inserted in the active site) in both kinds of inhibitors share a similar backbone and side chain orientation. They are only different in the capability to chelate the Zn atom present in the active site. CPIs with a substrate-like interaction mechanism chelate Zn atom with the carboxylate terminal group and exclude catalytic water from its functional position. In contrast, SmCI only indirectly contact the Zn atom by a water molecule, which could explain its one order higher Ki value in comparison to those inhibitors.


Figure:Tri-dimensional structure of the SmCI-humanCPA4 complex. SmCI (pink) and CPA (green) were shown in ribbon representation with the Zn2+ atom as a yellow sphere. The SmCI N-tail (I-S-V, labeled in black) and the nine disulfide bridges (labeled in yellow) are shown in stick representation.

This work describes a novel non-canonical metallo CPA interaction mechanism and the molecular details of the first inhibitor belonging to BPTI/Kunitz family able to block metallo CPA enzymes and formed by three domains. Our results contribute to current basic biochemical knowledge in the metallo CPA-inhibitor structure-function relationship. We support a new potential structural motive capable to inhibit metallo CPA, which also represents a starting point in the design and development of biomedical and biotechnological applications of SmCI or new synthetic inhibitors. In addition, SmCI domain architecture suggests the possibility of a simultaneous inhibition of metallo CPA and serine proteases by different primary sites found in the inhibitor for the binding of each enzyme catalytic type. This feature could suggest a possible SmCI role as a defence mechanism against S. magnifica predators.

Mey Ling Reytor1
David Reverter2
Sebastian Trejo2
Maria de los Angeles Chavez1
Francesc X. Aviles2
Maday Alonso del Rivero1

1Centro de Estudio de Proteínas
Facultad de Biología
Universidad de la Habana
2Institut de Biotecnología i Biomedicina
Universitat Autonoma de Barcelona