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An Electrostatic Funnel in the GABA-Binding Pathway.

ABSTRACT: The ?-aminobutyric acid type A receptor (GABAA-R) is a major inhibitory neuroreceptor that is activated by the binding of GABA. The structure of the GABAA-R is well characterized, and many of the binding site residues have been identified. However, most of these residues are obscured behind the C-loop that acts as a cover to the binding site. Thus, the mechanism by which the GABA molecule recognizes the binding site, and the pathway it takes to enter the binding site are both unclear. Through the completion and detailed analysis of 100 short, unbiased, independent molecular dynamics simulations, we have investigated this phenomenon of GABA entering the binding site. In each system, GABA was placed quasi-randomly near the binding site of a GABAA-R homology model, and atomistic simulations were carried out to observe the behavior of the GABA molecules. GABA fully entered the binding site in 19 of the 100 simulations. The pathway taken by these molecules was consistent and non-random; the GABA molecules approach the binding site from below, before passing up behind the C-loop and into the binding site. This binding pathway is driven by long-range electrostatic interactions, whereby the electrostatic field acts as a 'funnel' that sweeps the GABA molecules towards the binding site, at which point more specific atomic interactions take over. These findings define a nuanced mechanism whereby the GABAA-R uses the general zwitterionic features of the GABA molecule to identify a potential ligand some 2 nm away from the binding site.

SUBMITTER: Carpenter TS

PROVIDER: S-EPMC4847780 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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An Electrostatic Funnel in the GABA-Binding Pathway.

Carpenter Timothy S TS Lightstone Felice C FC

PLoS computational biology 20160427 4

The γ-aminobutyric acid type A receptor (GABAA-R) is a major inhibitory neuroreceptor that is activated by the binding of GABA. The structure of the GABAA-R is well characterized, and many of the binding site residues have been identified. However, most of these residues are obscured behind the C-loop that acts as a cover to the binding site. Thus, the mechanism by which the GABA molecule recognizes the binding site, and the pathway it takes to enter the binding site are both unclear. Through th ...[more]

PMID: 27119953

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Project description:HasR in the outer membrane of Serratia marcescens binds secreted, heme-loaded HasA and translocates the heme to the periplasm to satisfy the cell's demand for iron. The previously published crystal structure of the wild-type complex showed HasA in a very specific binding arrangement with HasR, apt to relax the grasp on the heme and assure its directed transfer to the HasR-binding site. Here, we present a new crystal structure of the heme-loaded HasA arranged with a mutant of HasR, called double mutant (DM) in the following that seemed to mimic a precursor stage of the abovementioned final arrangement before heme transfer. To test this, we performed first molecular dynamics (MD) simulations starting at the crystal structure of the complex of HasA with the DM mutant and then targeted MD simulations of the entire binding process beginning with heme-loaded HasA in solution. When the simulation starts with the former complex, the two proteins in most simulations do not dissociate. When the mutations are reverted to the wild-type sequence, dissociation and development toward the wild-type complex occur in most simulations. This indicates that the mutations create or enhance a local energy minimum. In the targeted MD simulations, the first protein contacts depend upon the chosen starting position of HasA in solution. Subsequently, heme-loaded HasA slides on the external surface of HasR on paths that converge toward the specific arrangement apt for heme transfer. The targeted simulations end when HasR starts to relax the grasp on the heme, the subsequent events being in a time regime inaccessible to the available computing power. Interestingly, none of the ten independent simulation paths visits exactly the arrangement of HasA with HasR seen in the crystal structure of the mutant. Two factors which do not exclude each other could explain these observations: the double mutation creates a non-physiologic potential energy minimum between the two proteins and /or the target potential in the simulation pushes the system along paths deviating from the low-energy paths of the native binding processes. Our results support the former view, but do not exclude the latter possibility.

Dataset Information

An Electrostatic Funnel in the GABA-Binding Pathway.

Publications

An Electrostatic Funnel in the GABA-Binding Pathway.

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