Designing out disulfide bonds of leech carboxypeptidase inhibitor: implications for its folding, stability and function.
Leech carboxypeptidase inhibitor (LCI) is a 67-residue, tight-binding metallocarboxypeptidase inhibitor composed of a compact domain with a five-stranded beta-sheet and a short alpha-helix that are strongly stabilized by four disulfide bonds. In this study, we investigated the contribution of each particular disulfide to the folding, stability and function of LCI by constructing a series of single and multiple mutants lacking one to four disulfide bonds. The results allow a better understanding of how individual disulfide bonds shape and restrict the conformational space that LCI must explore before attaining its native conformation. The work also dissected the role played by intramolecular rearrangements of disulfides during LCI folding, providing a new kinetic scheme in which the 2S ensemble suffers a non-specific oxidation into the 3S ensemble. These 3-disulfide-bonded species reshuffle to preferentially form III-A and III-B, two major native-like folding intermediates that need structural rearrangements through the formation of scrambled isomers to finally render native LCI. The designed multiple mutants of LCI are unable to fold correctly, displaying a highly unstructured conformation and a very low inhibitory capability, which indicates the importance of disulfide bonds in LCI for both correct folding and achievement of a functional structure. In contrast, the elimination of a single disulfide bond in LCI only results in a significant reduction of conformational stability, but the mutations have a rather moderate impact on carboxypeptidase inhibition, allowing the possibility to target the intrinsic stability and specific activity of LCI independently. In this way, the findings reported provide a basis for the design of novel variants of the molecule with improved therapeutic properties.
Arolas
Joan L
JL
Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Barcelona, Spain.
Castillo
Virginia
V
Bronsoms
Sílvia
S
Aviles
Francesc X
FX
Ventura
Salvador
S
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors*; D004220:Disulfides / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D007865:Leeches / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*
D004220:Disulfides; D004791:Enzyme Inhibitors; C116332:LCI protein, Hirudo medicinalis; D011506:Proteins; D002268:Carboxypeptidases