Biology subjects

Perez-Conesa, S.

Publications and source records attributed to Perez-Conesa, S..

4 recordsLinked to original sources

bioRxiv2021

Allosteric effect of nanobody binding on ligand-specific active states of the β2-Adrenergic Receptor

Nanobody binding stabilizes the active state of G-protein-coupled receptor (GPCR) and modulates its affinity for bound ligands. However, the atomic level basis for this allosteric regulation remains elusive. Here, we investigate the conformational changes induced by the binding of a nanobody (Nb80) on the active-like {beta}2 adrenergic receptor ({beta}2AR) via enhanced sampling molecular dynamics simulations. Dimensionality reduction analysis shows that Nb80 stabilizes a highly active state of the {beta}2AR with a ~14 [A] outward movement of transmembrane helix 6 and close proximity of transmembrane (TM) helices 5 and 7. This is further supported by the residues located at hotspots located on TMs 5, 6 and 7, as shown by supervised machine learning methods. Besides, ligand-specific subtle differences in the conformations assumed by intercellular loop 2 and extracellular loop 2 are captured from the trajectories of various ligand-bound receptors in the presence of Nb80. Dynamic network analysis further reveals that Nb80 binding can enhance the communication between the binding sites of Nb80 and of the ligand. We identify unique allosteric signal transmission mechanisms between the Nb80-binding site and the extracellular domains in presence of full agonist and G-protein biased partial agonist, respectively. Altogether, our results provide insights into the effect of intracellular binding partners on the GPCR activation mechanism, which could be useful for structure-based drug discovery. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=153 SRC="FIGDIR/small/451885v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@16f8ebforg.highwire.dtl.DTLVardef@104a47dorg.highwire.dtl.DTLVardef@1cb1659org.highwire.dtl.DTLVardef@284699_HPS_FORMAT_FIGEXP M_FIG Graphical Table of Contents C_FIG

biophysics↗

bioRxiv2021

Biophysical characterization of the SARS-CoV-2 E protein

SARS-CoV-2 contains four structural proteins in its genome. These proteins aid in the assembly and budding of new virions at the ER-Golgi intermediate compartment (ERGIC). Current fundamental research efforts largely focus on one of these proteins - the spike (S) protein. Since successful antiviral therapies are likely to target multiple viral components, there is considerable interest in understanding the biophysical role of its other structural proteins, in particular structural membrane proteins. Here, we have focused our efforts on the characterization of the full-length envelope (E) protein from SARS-CoV-2, combining experimental and computational approaches. Recombinant expression of the full-length E protein from SARS-CoV-2 reveals that this membrane protein is capable of independent multimerization, possibly as a tetrameric or smaller species. Fluorescence microscopy shows that the protein localizes intracellularly, and coarse-grained MD simulations indicate it causes bending of the surrounding lipid bilayer, corroborating a potential role for the E protein in viral budding. Although we did not find robust electrophysiological evidence of ion-channel activity, cells transfected with the E protein exhibited reduced intracellular Ca2+, which may further promote viral replication. However, our atomistic MD simulations revealed that previous NMR structures are relatively unstable, and result in models incapable of ion conduction. Our study highlights the importance of using high-resolution structural data obtained from a full-length protein to gain detailed molecular insights, and eventually permitting virtual drug screening.

biophysics↗

bioRxiv2020

Informing NMR experiments with molecular dynamics simulations to characterize the dominant activated state of the KcsA ion channel

As the first potassium channel with a X-ray structure determined, and given its homology to eukaryotic channels, the pH-gated prokaryotic channel KcsA has been extensively studied. Nevertheless, questions related in particular to the allosteric coupling between its gates remain open. The many currently available X-ray crystallography structures appear to correspond to various stages of activation and inactivation, offering insights into the molecular basis of these mechanisms. Since these studies have required mutations, complexation with antibodies, and substitution of detergents in place of lipids, examining the channel under more native conditions is desirable. Solid-state NMR (SSNMR) can be used to study the wild-type protein under activating conditions (low pH), at room temperature, and in bacteriomimetic liposomes. In this work, we sought to structurally assign the activated state present in SSNMR experiments. We used a combination of molecular dynamics (MD) simulations, chemical shift prediction algorithms, and Bayesian inference techniques to determine which of the most plausible X-ray structures resolved to date best represents the activated state captured in SSNMR. We first identified specific nuclei with simulated NMR chemical shifts that differed significantly when comparing partially open vs. fully open ensembles from MD simulations. The simulated NMR chemical shifts for those specific nuclei were then compared to experimental ones, revealing that the simulation of the partially open state was in good agreement with the SSNMR data. Nuclei that discriminate effectively between partially and fully open states belong to residues spread over the sequence and provide a molecular level description of the conformational change.

biophysics↗

bioRxiv2020

Network analysis reveals how lipids and other cofactors influence membrane protein allostery

Many membrane proteins are modulated by external stimuli, such as small molecule binding or change in pH, transmembrane voltage or temperature. This modulation typically occurs at sites that are structurally distant from the functional site. Revealing the communication, known as allostery, between these two sites is key to understanding the mechanistic details of these proteins. Residue interaction networks of isolated proteins are commonly used to this end. Membrane proteins, however, are embedded in a lipid bilayer which may contribute to allosteric communication. The fast diffusion of lipids hinders direct use of standard residue interaction networks. Here, we present an extension which includes cofactors such as lipids and small molecules in the network. The novel framework is applied to three membrane proteins: a voltage-gated ion channel (KCNQ1), a G-protein coupled receptor (GPCR - {beta}2 adrenergic receptor) and a pH-gated ion channel (KcsA). Through systematic analysis of the obtained networks and their components, we demonstrate the importance of lipids for membrane protein allostery. Finally, we reveal how small molecules may stabilize different protein states by allosterically coupling and decoupling the protein from the membrane.

biophysics↗