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Remodeling of the interdomain allosteric linker upon membrane binding of CCT? pulls its active site close to the membrane surface.

ABSTRACT: The rate-limiting step in the biosynthesis of the major membrane phospholipid, phosphatidylcholine, is catalyzed by CTP:phosphocholine cytidylyltransferase (CCT), which is regulated by reversible membrane binding of a long amphipathic helix (domain M). The M domain communicates with the catalytic domain via a conserved ?20-residue linker, essential for lipid activation of CCT. Previous analysis of this region (denoted as the ?EC/J) using MD simulations, cross-linking, mutagenesis, and solvent accessibility suggested that membrane binding of domain M promotes remodeling of the ?EC/J into a more compact structure that is required for enzyme activation. Here, using tryptophan fluorescence quenching, we show that the allosteric linker lies superficially on the membrane surface. Analyses with truncated CCTs show that the ?EC/J can interact with lipids independently of the M domain. We observed strong FRET between engineered tryptophans in the ?EC/J and vesicles containing dansyl-phosphatidylethanolamine that depended on the native J sequence. These data are incompatible with the extended conformation of the ?E helix observed in the previously determined crystal structure of inactive CCT but support a bent ?E helix conformation stabilized by J segment interactions. Our results suggest that the membrane-adsorbed, folded allosteric linker may partially cover the active site cleft and pull it close to the membrane surface, where cytidyl transfer can occur efficiently in a relatively anhydrous environment.

SUBMITTER: Knowles DG

PROVIDER: S-EPMC6802524 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Remodeling of the interdomain allosteric linker upon membrane binding of CCTα pulls its active site close to the membrane surface.

Knowles Daniel G DG Lee Jaeyong J Taneva Svetla G SG Cornell Rosemary B RB

The Journal of biological chemistry 20190904 42

The rate-limiting step in the biosynthesis of the major membrane phospholipid, phosphatidylcholine, is catalyzed by CTP:phosphocholine cytidylyltransferase (CCT), which is regulated by reversible membrane binding of a long amphipathic helix (domain M). The M domain communicates with the catalytic domain via a conserved ∼20-residue linker, essential for lipid activation of CCT. Previous analysis of this region (denoted as the αE<sub>C</sub>/J) using MD simulations, cross-linking, mutagenesis, and ...[more]

PMID: 31488548

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Project description:The retroviral Gag capsid (Gag-CA) interdomain linker is an unstructured peptide segment connecting structured N-terminal and C-terminal domains. Although the region is reported to play roles in virion morphogenesis and infectivity, underlying molecular mechanisms remain unexplored. To address this issue, we determined biological and molecular phenotypes of HIV-1 CA linker mutants by experimental and in silico approaches. Among the nine linker mutants tested, eight exhibited attenuation of viral particle production to various extents mostly in parallel with a reduction in viral infectivity. Sucrose density gradient, confocal microscopy, and live-cell protein interaction analyses indicated that the defect is accompanied by attenuation of Gag-Gag interactions following Gag plasma membrane targeting in the cells. In silico analyses revealed distinct distributions of interaction-prone hydrophobic patches between immature and mature CA proteins. Molecular dynamics simulations predicted that the linker mutations can allosterically alter structural fluctuations, including the interaction surfaces apart from the mutation sites in both the immature and mature CA proteins. These results suggest that the HIV-1 CA interdomain linker is a cis-modulator of the CA interaction surfaces to optimize efficiency of Gag assembly, virion production, and viral infectivity.IMPORTANCE HIV-1 particle production and infection are highly ordered processes. Viral Gag proteins play a central role in the assembly and disassembly of viral molecules. Of these, capsid protein (CA) is a major contributor to the Gag-Gag interactions. CA consists of two structured domains, i.e., N-terminal (NTD) and C-terminal (CTD) domains, connected by an unstructured domain named the interdomain linker. While multiple regions in the NTD and CTD are reported to play roles in virion morphogenesis and infectivity, the roles of the linker region in Gag assembly and virus particle formation remain elusive. In this study, we showed by biological and molecular analyses that the linker region functions as an intramolecular modulator to tune Gag assembly, virion production, and viral infectivity. Our study thus illustrates a hitherto-unrecognized mechanism, an allosteric regulation of CA structure by the disordered protein element, for HIV-1 replication.

Dataset Information

Remodeling of the interdomain allosteric linker upon membrane binding of CCT? pulls its active site close to the membrane surface.

Publications

Remodeling of the interdomain allosteric linker upon membrane binding of CCTα pulls its active site close to the membrane surface.

Similar Datasets

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