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A conformational switch in a partially unwound helix selectively determines the pathway for substrate release from the carnitine/?-butyrobetaine antiporter CaiT.

ABSTRACT: CaiT is a homotrimeric antiporter that exchanges l-carnitine (CRN) with ?-butyrobetaine (GBB) across the bacterial membrane. Three structures have been resolved to date for CaiT, all in the inward-facing state: CRN-bound (with four CRNs per subunit), GBB-bound (two GBBs per subunit), and apo. One of the reported binding sites is the counterpart of the primary site observed in structurally similar transporters. However, the mechanism and pathway(s) of CRN/GBB unbinding and translocation, or even the ability of the substrates to dislodge from the reported binding sites, are yet to be determined. To shed light on these issues, we performed a total of 1.3 ?s of molecular dynamics simulations and examined the dynamics of substrate-bound CaiT structures under different conditions. We find that both CRN and GBB are able to dissociate completely from their primary site into the cytoplasm. Substrate molecules initially located at the secondary sites dissociate even faster (within tens of nanoseconds) into the extra- or intracellular regions. Interestingly, the unbinding pathway from the primary site appears to be dictated by the geometry of the unwound part of the transmembrane (TM) helix 3, mostly around Thr(100) therein. Arg(262) on TM7, which apparently mimics the role of Na(+) in CaiT structural homologues, plays a key role in triggering the dissociation of the substrate away from the primary site and guiding its release to the cytoplasm provided that the unwound part of TM3 switches from a shielding to a yielding pose.

SUBMITTER: Zomot E

PROVIDER: S-EPMC3442516 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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A conformational switch in a partially unwound helix selectively determines the pathway for substrate release from the carnitine/γ-butyrobetaine antiporter CaiT.

Zomot Elia E Bahar Ivet I

The Journal of biological chemistry 20120729 38

CaiT is a homotrimeric antiporter that exchanges l-carnitine (CRN) with γ-butyrobetaine (GBB) across the bacterial membrane. Three structures have been resolved to date for CaiT, all in the inward-facing state: CRN-bound (with four CRNs per subunit), GBB-bound (two GBBs per subunit), and apo. One of the reported binding sites is the counterpart of the primary site observed in structurally similar transporters. However, the mechanism and pathway(s) of CRN/GBB unbinding and translocation, or even ...[more]

PMID: 22843728

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Project description:We report a conformational switch between two distinct intrinsically disordered subensembles within the active site of a transcription factor. This switch highlights an evolutionary benefit conferred by the high plasticity of intrinsically disordered domains, namely, their potential to dynamically sample a heterogeneous conformational space housing multiple states with tailored properties. We focus on proto-oncogenic basic-helix-loop-helix (bHLH)-type transcription factors, as these play key roles in cell regulation and function. Despite intense research efforts, the understanding of structure-function relations of these transcription factors remains incomplete as they feature intrinsically disordered DNA-interaction domains that are difficult to characterize, theoretically as well as experimentally. Here we characterize the structural dynamics of the intrinsically disordered region DNA-binding site of the vital MYC-associated transcription factor X (MAX). Integrating nuclear magnetic resonance (NMR) measurements, molecular dynamics (MD) simulations, and electron paramagnetic resonance (EPR) measurements, we show that, in the absence of DNA, the binding site of the free MAX2 homodimer samples two intrinsically disordered conformational subensembles. These feature distinct structural properties: one subensemble consists of a set of highly flexible and spatially extended conformers, while the second features a set of "hinged" conformations. In this latter ensemble, the disordered N-terminal tails of MAX2 fold back along the dimer, forming transient long-range contacts with the HLH-region and thereby exposing the DNA binding site to the solvent. The features of these divergent substates suggest two mechanisms by which protein conformational dynamics in MAX2 might modulate DNA-complex formation: by enhanced initial recruitment of free DNA ligands, as a result of the wider conformational space sampled by the extended ensemble, and by direct exposure of the binding site and the corresponding strong electrostatic attractions presented while in the hinged conformations.

Dataset Information

A conformational switch in a partially unwound helix selectively determines the pathway for substrate release from the carnitine/?-butyrobetaine antiporter CaiT.

Publications

A conformational switch in a partially unwound helix selectively determines the pathway for substrate release from the carnitine/γ-butyrobetaine antiporter CaiT.

Similar Datasets

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