Molecular insights on CALX-CBD12 interdomain dynamics from MD simulations, RDCs, and SAXS

Autor(es)

de Souza Degenhardt, Maximilia F. ^[1] ; Vitale, Phelipe A. M. ^[2] ; Abiko, Layara A. ^[2] ; Zacharias, Martin ^[3] ; Sattler, Michael ^[4] ; Oliveira, Cristiano L. P. ^[1] ; Salinas, Roberto K. ^[2]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Inst Phys, Dept Expt Phys, Sao Paulo - Brazil

^[2] Univ Sao Paulo, Inst Chem, Dept Biochem, Sao Paulo - Brazil

^[3] Tech Univ Munich, Phys Dept, Munich - Germany

^[4] Tech Univ Munich, Chem Dept, Munich - Germany

• Tipo de documento: Artigo Científico
• Fonte: BIOPHYSICAL JOURNAL; v. 120, n. 17, p. 3664-3675, SEP 7 2021.
• Citações Web of Science: 0

Resumo

Na+/Ca2+ exchangers (NCXs) are secondary active transporters that couple the translocation of Nathorn with the transport of Ca2+ in the opposite direction. The exchanger is an essential Ca2+ extrusion mechanism in excitable cells. It consists of a transmembrane domain and a large intracellular loop that contains two Ca2+-binding domains, CBD1 and CBD2. The two CBDs are adjacent to each other and form a two-domain Ca2+ sensor called CBD12. Binding of intracellular Ca2+ to CBD12 activates the NCX but inhibits the NCX of Drosophila, CALX. NMR spectroscopy and SAXS studies showed that CALX and NCX CBD12 constructs display significant interdomain flexibility in the apo state but assume rigid interdomain arrangements in the Ca2+-bound state. However, detailed structure information on CBD12 in the apo state is missing. Structural characterization of proteins formed by two or more domains connected by flexible linkers is notoriously challenging and requires the combination of orthogonal information from multiple sources. As an attempt to characterize the conformational ensemble of CALX-CBD12 in the apo state, we applied molecular dynamics (MD) simulations, NMR (H-1-N-15 residual dipolar couplings), and small-angle x-ray scattering (SAXS) data in a combined strategy to select an ensemble of conformations in agreement with the experimental data. This joint approach demonstrated that CALX-CBD12 preferentially samples closed conformations, whereas the wide-open interdomain arrangement characteristic of the Ca2+-bound state is less frequently sampled. These results are consistent with the view that Ca2+ binding shifts the CBD12 conformational ensemble toward extended conformers, which could be a key step in the NCXs' allosteric regulation mechanism. This strategy, combining MD with NMR and SAXS, provides a powerful approach to select ensembles of conformations that could be applied to other flexible multidomain systems. (AU)

• Processo FAPESP: 18/16092-5 - Investigando sistemas coloidais por métodos de espalhamento
• Beneficiário: Cristiano Luis Pinto de Oliveira
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 16/24531-3 - Propriedades estruturais e biofísicas de lipoproteínas nativa e modificada
• Beneficiário: Antonio Martins Figueiredo Neto
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 19/19968-1 - Estudos estruturais acerca do trocador de Na+/Ca2+ (CALX) por espectroscopia de ressonância magnética nuclear em solução
• Beneficiário: Roberto Kopke Salinas
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 17/05614-8 - Trocadores de Na+/Ca2+: relação entre estrutura e função
• Beneficiário: Phelipe Augusto Mariano Vitale
• Modalidade de apoio: Bolsas no Brasil - Doutorado Direto
• Processo FAPESP: 16/07490-1 - Estudos estruturais acerca do trocador de Na+/Ca2+ e de proteínas do vírus Zika por espectroscopia de Ressonância Magnética Nuclear em solução
• Beneficiário: Roberto Kopke Salinas
• Modalidade de apoio: Auxílio à Pesquisa - Regular