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Cholesterol-mediated allosteric regulation of the mitochondrial translocator protein structure.

ABSTRACT: Cholesterol is an important regulator of membrane protein function. However, the exact mechanisms involved in this process are still not fully understood. Here we study how the tertiary and quaternary structure of the mitochondrial translocator protein TSPO, which binds cholesterol with nanomolar affinity, is affected by this sterol. Residue-specific analysis of TSPO by solid-state NMR spectroscopy reveals a dynamic monomer-dimer equilibrium of TSPO in the membrane. Binding of cholesterol to TSPO's cholesterol-recognition motif leads to structural changes across the protein that shifts the dynamic equilibrium towards the translocator monomer. Consistent with an allosteric mechanism, a mutation within the oligomerization interface perturbs transmembrane regions located up to 35?Å away from the interface, reaching TSPO's cholesterol-binding motif. The lower structural stability of the intervening transmembrane regions provides a mechanistic basis for signal transmission. Our study thus reveals an allosteric signal pathway that connects membrane protein tertiary and quaternary structure with cholesterol binding.

SUBMITTER: Jaipuria G

PROVIDER: S-EPMC5379104 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Cholesterol-mediated allosteric regulation of the mitochondrial translocator protein structure.

Jaipuria Garima G Leonov Andrei A Giller Karin K Vasa Suresh Kumar SK Jaremko Łukasz Ł Jaremko Mariusz M Linser Rasmus R Becker Stefan S Zweckstetter Markus M

Nature communications 20170330

Cholesterol is an important regulator of membrane protein function. However, the exact mechanisms involved in this process are still not fully understood. Here we study how the tertiary and quaternary structure of the mitochondrial translocator protein TSPO, which binds cholesterol with nanomolar affinity, is affected by this sterol. Residue-specific analysis of TSPO by solid-state NMR spectroscopy reveals a dynamic monomer-dimer equilibrium of TSPO in the membrane. Binding of cholesterol to TSP ...[more]

PMID: 28358007

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Project description:Engineering novel allostery into existing proteins is a challenging endeavor to obtain novel sensors, therapeutic proteins, or modulate metabolic and cellular processes. The RG13 protein achieves such allostery by inserting a circularly permuted TEM-1 ?-lactamase gene into the maltose binding protein (MBP). RG13 is positively regulated by maltose yet is, serendipitously, inhibited by Zn(2+) at low µM concentration. To probe the structure and allostery of RG13, we crystallized RG13 in the presence of mM Zn(2+) concentration and determined its structure. The structure reveals that the MBP and TEM-1 domains are in close proximity connected via two linkers and a zinc ion bridging both domains. By bridging both TEM-1 and MBP, Zn(2+) acts to "twist tie" the linkers thereby partially dislodging a linker between the two domains from its original catalytically productive position in TEM-1. This linker 1 contains residues normally part of the TEM-1 active site including the critical ?3 and ?4 strands important for activity. Mutagenesis of residues comprising the crystallographically observed Zn(2+) site only slightly affected Zn(2+) inhibition 2- to 4-fold. Combined with previous mutagenesis results we therefore hypothesize the presence of two or more inter-domain mutually exclusive inhibitory Zn(2+) sites. Mutagenesis and molecular modeling of an intact TEM-1 domain near MBP within the RG13 framework indicated a close surface proximity of the two domains with maltose switching being critically dependent on MBP linker anchoring residues and linker length. Structural analysis indicated that the linker attachment sites on MBP are at a site that, upon maltose binding, harbors both the largest local C? distance changes and displays surface curvature changes, from concave to relatively flat becoming thus less sterically intrusive. Maltose activation and zinc inhibition of RG13 are hypothesized to have opposite effects on productive relaxation of the TEM-1 ?3 linker region via steric and/or linker juxtapositioning mechanisms.

Dataset Information

Cholesterol-mediated allosteric regulation of the mitochondrial translocator protein structure.

Publications

Cholesterol-mediated allosteric regulation of the mitochondrial translocator protein structure.

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