Secondary binding site of the potato carboxypeptidase inhibitor. Contribution to its structure, folding, and biological properties.
The contribution of each residue of the potato carboxypeptidase inhibitor (PCI) secondary binding site to the overall properties of this protein has been examined using alanine-scanning mutagenesis. Structural and enzymatic studies, performed on a series of PCI mutants, demonstrate that the proper positioning of the primary site for efficient binding and inhibition of carboxypeptidase A is significantly dependent on such a secondary contact region. The aromatic residues in this region play a key role in the stabilization of the PCI-enzyme complex, whereas polar residues contribute little to this task. A comparative study of the oxidative folding of these PCI mutants has been carried out using the disulfide quenching approach. The data, together with the structural characterization of some of these mutants, clearly indicate that noncovalent forces drive the refolding of this small disulfide-rich protein at the reshuffling stage, the rate-limiting step of the process. Moreover, it reveals that by introducing new noncovalent intramolecular contacts in PCI, we may create more stable variants, which also show improved folding efficiency. Taken together, the collected results clarify the folding determinants of the primary and secondary binding sites of PCI and their contribution to the inhibition of the carboxypeptidase, providing clues about PCI evolution and knowledge for its biotechnological redesign.
Arolas
Joan L
JL
Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain.
Lorenzo
Julia
J
Rovira
Ana
A
Vendrell
Josep
J
Aviles
Francesc X
FX
Ventura
Salvador
S
D001665:Binding Sites; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011480:Protease Inhibitors; D011487:Protein Conformation; D017510:Protein Folding; D011198:Solanum tuberosum / Q000201:enzymology*; D013816:Thermodynamics
D010940:Plant Proteins; D011480:Protease Inhibitors