Plasticity in the Oxidative Folding Pathway of the High Affinity Nerita Versicolor Carboxypeptidase Inhibitor (NvCI)

Nerita Versicolor carboxypeptidase inhibitor (NvCI) is the strongest inhibitor reported so far for the M14A subfamily of carboxypeptidases. It comprises 53 residues and a protein fold composed of a two-stranded antiparallel β sheet connected by three loops and stabilized by three disulfide bridges. Here we report the oxidative folding and reductive unfolding pathways of NvCI. Much debate has gone on whether protein conformational folding guides disulfide bond formation or instead they are disulfide bonds that favour the arrangement of local or global structural elements. We show here that for NvCI both possibilities apply. Under physiological conditions, this protein folds trough a funnelled pathway involving a network of kinetically connected native-like intermediates, all sharing the disulfide bond connecting the two β-strands. In contrast, under denaturing conditions, the folding of NvCI is under thermodynamic control and follows a "trial and error" mechanism, in which an initial quasi-stochastic population of intermediates rearrange their disulfide bonds to attain the stable native topology. Despite their striking mechanistic differences, the efficiency of both folding routes is similar. The present study illustrates thus a surprising plasticity in the folding of this extremely stable small disulfide-rich inhibitor and provides the basis for its redesign for biomedical applications

Plasticity in the Oxidative Folding Pathway of the High Affinity Nerita Versicolor Carboxypeptidase Inhibitor (NvCI)

Nerita Versicolor carboxypeptidase inhibitor (NvCI) is the strongest inhibitor reported so far for the M14A subfamily of carboxypeptidases. It comprises 53 residues and a protein fold composed of a two-stranded antiparallel β sheet connected by three loops and stabilized by three disulfide bridges. Here we report the oxidative folding and reductive unfolding pathways of NvCI. Much debate has gone on whether protein conformational folding guides disulfide bond formation or instead they are disulfide bonds that favour the arrangement of local or global structural elements. We show here that for NvCI both possibilities apply. Under physiological conditions, this protein folds trough a funnelled pathway involving a network of kinetically connected native-like intermediates, all sharing the disulfide bond connecting the two β-strands. In contrast, under denaturing conditions, the folding of NvCI is under thermodynamic control and follows a "trial and error" mechanism, in which an initial quasi-stochastic population of intermediates rearrange their disulfide bonds to attain the stable native topology. Despite their striking mechanistic differences, the efficiency of both folding routes is similar. The present study illustrates thus a surprising plasticity in the folding of this extremely stable small disulfide-rich inhibitor and provides the basis for its redesign for biomedical applications.Instituto Multidisciplinario de Biología Celula

Fine modulation of the respiratory syncytial virus M2-1 protein quaternary structure by reversible zinc removal from its Cys(3)-His(1) motif

Human respiratory syncytial virus (hRSV) is a worldwide distributed pathogen that causes respiratory disease mostly in infants and the elderly. The M2-1 protein of hRSV functions as a transcription antiterminator and partakes in virus particle budding. It is present only in Pneumovirinae, namely, Pneumovirus (RSV) and Metapneumovirus, making it an interesting target for specific antivirals. hRSV M2-1 is a tight tetramer bearing a Cys3-His1 zinc-binding motif, present in Ebola VP30 protein and some eukaryotic proteins, whose integrity was shown to be essential for protein function but without a biochemical mechanistic basis. We showed that removal of the zinc atom causes dissociation to a monomeric apo-M2-1 species. Surprisingly, the secondary structure and stability of the apo-monomer is indistinguishable from that of the M2-1 tetramer. Dissociation reported by a highly sensitive tryptophan residue is much increased at pH 5.0 compared to pH 7.0, suggesting a histidine protonation cooperating in zinc removal. The monomeric apo form binds RNA at least as well as the tetramer, and this interaction is outcompeted by the phosphoprotein P, the RNA polymerase cofactor. The role of zinc goes beyond stabilization of local structure, finely tuning dissociation to a fully folded and binding competent monomer. Removal of zinc is equivalent to the disruption of the motif by mutation, only that the former is potentially reversible in the cellular context. Thus, this process could be triggered by a natural chelator such as glutathione or thioneins, where reversibility strongly suggests a modulatory role in the participation of M2-1 in the assembly of the polymerase complex or in virion budding.Fil: Esperante, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Noval, María Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Altieri, Tamara A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: de Oliveira, Guilherme A. P.. Universidade Federal Do Rio de Janeiro. Instituto de Biologia; BrasilFil: Silva, Jerson L.. Universidade Federal Do Rio de Janeiro. Instituto de Biologia; BrasilFil: de Prat Gay, Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin