Biology subjects

Kopke Salinas, R.

Publications and source records attributed to Kopke Salinas, R..

3 recordsLinked to original sources

bioRxiv2020

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

Na+/Ca2+ exchangers (NCX) are secondary active transporters that couple the translocation of Na+ 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 Na+/Ca2+ exchanger of Drosophila, CALX. NMR spectroscopy and SAXS studies showed that CALX and NCX CBD12 constructs display significant inter-domain flexibility in the Apo state, but assume rigid inter-domain 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 (1H-15N RDCs) and Small-Angle X-Ray Scattering (SAXS) data in a combined modelling strategy that generated atomistic information on the most representative conformations. This joint approach demonstrated that CALX-CBD12 preferentially samples closed conformations, while the wide-open inter-domain 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 towards extended conformers, which could be a key step in the Na+/Ca2+ exchangers allosteric regulation mechanism. The present strategy, combining MD with NMR and SAXS, provides a powerful approach to select representative structures from ensembles of conformations, which could be applied to other flexible multi-domain systems. SIGNIFICANCEThe conformational ensemble of CALX-CBD12, the main Ca2+-sensor of Drosophilas Na+/Ca2+ exchanger, was characterized by a combination of MD simulations with SAXS and NMR data using the EOM approach. This analysis showed that this two-domain construct experiences opening-closing motions, providing molecular information about CALX-CBD12 in the Apo state. Ca2+-binding shifts the conformational ensemble towards extended conformers. These findings are consistent with a model according to which Ca2+ modulation of CBD12 plasticity is a key step in the Ca2+-regulation mechanism of the full-length exchanger.

biophysics↗

bioRxiv2020

Molecular dynamics reveals complex compensatory effects of ionic strength on the SARS-CoV-2 Spike/hACE-2 interaction

The SARS-CoV-2 pandemic has already killed more than 800,000 people worldwide. To gain entry, the virus uses its spike protein to bind to host hACE-2 receptors on the host cell surface and mediate fusion between viral and cell membranes. As initial steps leading to virus entry involves significant changes in protein conformation as well as in the electrostatic environment in the vicinity of the spike-hACE-2 complex, we explored the sensitivity of the interaction to changes in ionic strength through computational simulations and surface plasmon resonance. We identified two regions in the receptor-binding domain (RBD), E1 and E2, which interact differently with hACE-2. At high salt concentration, E2-mediated interactions are weakened but are compensated by strengthening E1-mediated hydrophobic interactions. These results provide a detailed molecular understanding of spike RBD/hACE-2 complex formation and stability under a wide range of ionic strengths. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=192 SRC="FIGDIR/small/267351v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@1a7dc02org.highwire.dtl.DTLVardef@15d3c78org.highwire.dtl.DTLVardef@2d09c1org.highwire.dtl.DTLVardef@db78a9_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2019

CALX-CBD1 Ca2+-binding cooperativity studied by NMR spectroscopy and ITC with Bayesian statistics

The Na+/Ca2+ exchanger of Drosophila melanogaster, CALX, is the main Ca2+-extrusion mechanism in olfactory sensory neurons and photoreceptor cells. Na+/Ca2+ exchangers have two Ca2+ sensor domains, CBD1 and CBD2. In contrast to the mammalian homologues, CALX is inhibited by Ca2+-binding to CALX-CBD1, while CALX-CBD2 does not bind Ca2+ at physiological concentrations. CALX-CBD1 consists of a {beta}-sandwich and displays four Ca2+ binding sites at the tip of the domain. In this study, we used NMR spectroscopy and isothermal titration calorimetry (ITC) to investigate the cooperativity of Ca2+-binding to CALX-CBD1. We observed that this domain binds Ca2+ in the slow exchange regime at the NMR chemical shift time scale. Ca2+-binding restricts the dynamics in the Ca2+-binding region. Experiments of 15N CEST and 15N R2 dispersion allowed the determination of Ca2+ dissociation rates ({approx} 20 s-1). NMR titration curves of residues in the Ca2+-binding region were sigmoidal due to the contribution of chemical exchange to transverse magnetization relaxation rates, R2. Hence, a novel approach to analyze NMR titration curves was proposed. Ca2+-binding cooperativity was examined assuming two different stoichiometric binding models and using a Bayesian approach for data analysis. Fittings of NMR and ITC binding curves to the Hill model yielded nHill = 2.9 - 3.1, near maximum cooperativity (nHill = 4). By assuming a stepwise model to interpret the ITC data, we found that the probability of binding from 2 up to 4 Ca2+ is at least three orders of magnitude higher than that of binding a single Ca2+. Hence, four Ca2+ ions bind almost simultaneously to CALX-CBD1. Cooperative Ca2+-binding is key to enable this exchanger to efficiently respond to changes in the intracellular Ca2+-concentration in sensory neuronal cells.\n\nSIGNIFICANCECALX-CBD1 is the Ca2+-sensor domain of the Na+/Ca2+ exchanger of Drosophila melanogaster. It consists of a {beta}-sandwich, and contains four Ca2+ binding sites at the distal loops. In this study, we examined the cooperative binding of four Ca2+ ions to CALX-CBD1 using NMR spectroscopy and isothermal titration calorimetry (ITC) experiments. NMR and ITC data were analyzed using the framework of the binding polynomial formalism and Bayesian statistics. A novel approach to analyze NMR titration data in the slow exchange regime was proposed. These results support the view that CALX-CBD1 binds four Ca2+ with high cooperativity. The significant ligand binding cooperativity exhibited by this domain is determinant for the efficient allosteric regulation of this exchanger by intracellular Ca2+.

biophysics↗