Biology subjects

Granata, D.

Publications and source records attributed to Granata, D..

3 recordsLinked to original sources

bioRxiv2018

A consistent picture of TRPV1 activation emerges from molecular simulations and experiments

Although the structure of TRPV1 has been experimentally determined in both the closed and open states, very little is known about its activation mechanism. In particular, the conformational changes occurring in the pore domain and resulting in ionic conduction have not been identified yet. Here, we suggest a hypothetical molecular mechanism for TRPV1 activation, which involves the rotation of a conserved asparagine in S6 from the S4-S5 linker toward the pore. This rotation is correlated with the dehydration of four peripheral cavities located between S6 and the S4-S5 linker and the hydration of the pore. In light of our hypothesis, we perform bioinformatics analyses of TRP and other evolutionary related ion channels, analyze newly available structures and re-examine previously reported water accessibility and mutagenesis experiments. Overall, we provide several independent lines of evidence that corroborate our hypothesis. Finally, we show that the proposed molecular mechanism is compatible with the currently existing idea that in TRPV1 the selectivity filter acts as a secondary gate.

biophysics↗

bioRxiv2017

TRPV1 activation relies on hydration/dehydration of nonpolar cavities

TRPV1 promotes cationic currents across cellular membranes in response to multiple stimuli such as increased temperature, binding of chemicals, low pH and voltage. The molecular underpinnings of TRPV1 gating, in particular the mechanism of temperature sensitivity, are still largely unknown. Here, we used molecular simulations and electrophysiology to shed light on the closed to open transition. Specifically, we found that gating of TRPV1 relies on the motion of an evolutionarily conserved amino acid (N676) in the middle of the S6 helix. On rotation, the side chain of this asparagine faces either the central pore or the S4-S5 linker. Only in the former case is the central pore hydrated and thus conductive. Interestingly, when N676 rotates toward the linker, we observe hydration of four so far unreported small nonpolar cavities. Based on these findings, we propose a model for TRPV1 gating involving the dynamic hydration of these four cavities. Free energy calculations indicate that this gating mechanisms is markedly temperature dependent favoring the open state at high temperature. On the basis of this model, which is able to rationalize a wealth of seemingly conflicting and/or unrelated experimental observations, we predicted the behavior of two single residue mutants, M572A and F580Y, the consequences of which we confirmed experimentally.

biophysics↗

bioRxiv2017

From Sequence to Function: Coevolving Amino Acids Encode Structural and Functional Domains

Amino acids interactions within protein families are so optimized that the sole analysis of evolutionary co-mutations can identify pairs of contacting residues. It is also known that evolution conserves functional dynamics, i.e., the concerted motion or displacement of large protein regions or domains. Is it, therefore, possible to use a pure sequence-based analysis to identify these dynamical domains? To address this question, we introduce here a general co-evolutionary coupling analysis strategy and apply it to a curated sequence database of hundreds of protein families. For most families, the sequence-based method partitions amino acids into few clusters. When viewed in the context of the native structure, these clusters have the signature characteristics of viable protein domains: they are spatially separated but individually compact. They have a direct functional bearings too, as shown for various reference cases. We conclude that even large-scale structural and functionally-related properties can be recovered from inference methods applied to evolutionary-related sequences. The method introduced here is available as a software package and web server (http://spectrus.sissa.it/spectrus-evo_webserver).

bioinformatics↗