Dataset Information

Structural Basis for Differences in Dynamics Induced by Leu Versus Ile Residues in the CD Loop of Kir Channels.

ABSTRACT: The effect of the conserved Leu/Ile site in the CD loop on the gating dynamics of Kir channels and corresponding micro-structural mechanism remains unclear. Molecular dynamics simulations were performed to investigate the structural mechanism of chicken Kir2.2. Compared to WT, the I223L mutant channel bound to PIP2 more strongly, was activated more rapidly, and maintained the activation state more stably after PIP2 dissociation. Cellular electrophysiology assays of mouse Kir2.1 and human Kir2.2 indicated that, consistent with simulations, the Leu residue increased the channel responses to PIP2 through increased binding affinity and faster activation kinetics, and the deactivation kinetics decreased upon PIP2 inhibition. The Ile residue induced the opposite responses. This difference was attributed to the distinct hydrophobic side chain symmetries of Leu and Ile; switching between these residues caused the interaction network to redistribute and offered effective conformation transduction in the Leu systems, which had more rigid and independent subunits.

SUBMITTER: Lu S

PROVIDER: S-EPMC5085999 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Structural Basis for Differences in Dynamics Induced by Leu Versus Ile Residues in the CD Loop of Kir Channels.

Lü Shouqin S An Hailong H Zhang Hailin H Long Mian M

Molecular neurobiology 20151031 9

The effect of the conserved Leu/Ile site in the CD loop on the gating dynamics of Kir channels and corresponding micro-structural mechanism remains unclear. Molecular dynamics simulations were performed to investigate the structural mechanism of chicken Kir2.2. Compared to WT, the I223L mutant channel bound to PIP<sub>2</sub> more strongly, was activated more rapidly, and maintained the activation state more stably after PIP<sub>2</sub> dissociation. Cellular electrophysiology assays of mouse Ki ...[more]

PMID: 26520451

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Project description:Agnoprotein is a small multifunctional regulatory protein required for sustaining the productive replication of JC virus (JCV). It is a mostly cytoplasmic protein localizing in the perinuclear area and forms highly stable dimers/oligomers through a Leu/Ile/Phe-rich domain. There have been no three-dimensional structural data available for agnoprotein due to difficulties associated with the dynamic conversion from monomers to oligomers. Here, we report the first nuclear magnetic resonance (NMR) structure of a synthetic agnoprotein peptide spanning amino acids Thr17 to Glu55 where Lys23 to Phe39 encompassing the Leu/Ile/Phe-rich domain forms an amphipathic ?-helix. On the basis of these structural data, a number of Ala substitution mutations were made to investigate the role of the ?-helix in the structure and function of agnoprotein. Single L29A and L36A mutations exhibited a significant negative effect on both protein stability and viral replication, whereas the L32A mutation did not. In addition, the L29A mutant displayed a highly nuclear localization pattern, in contrast to the pattern for the wild type (WT). Interestingly, a triple mutant, the L29A+L32A+L36A mutant, yielded no detectable agnoprotein expression, and the replication of this JCV mutant was significantly reduced, suggesting that Leu29 and Leu36 are located at the dimer interface, contributing to the structure and stability of agnoprotein. Two other single mutations, L33A and E34A, did not perturb agnoprotein stability as drastically as that observed with the L29A and L36A mutations, but they negatively affected viral replication, suggesting that the role of these residues is functional rather than structural. Thus, the agnoprotein dimerization domain can be targeted for the development of novel drugs active against JCV infection.Agnoprotein is a small regulatory protein of JC virus (JCV) and is required for the successful completion of the viral replication cycle. It forms highly stable dimers and oligomers through its hydrophobic (Leu/Ile/Phe-rich) domain, which has been shown to play essential roles in the stability and function of the protein. In this work, the Leu/Ile/Phe-rich domain has been further characterized by NMR studies using an agnoprotein peptide spanning amino acids T17 to Q54. Those studies revealed that the dimerization domain of the protein forms an amphipathic ?-helix. Subsequent NMR structure-based mutational analysis of the region highlighted the critical importance of certain amino acids within the ?-helix for the stability and function of agnoprotein. In conclusion, this study provides a solid foundation for developing effective therapeutic approaches against the dimerization domain of the protein to inhibit its critical roles in JCV infection.

Dataset Information

Structural Basis for Differences in Dynamics Induced by Leu Versus Ile Residues in the CD Loop of Kir Channels.

Publications

Structural Basis for Differences in Dynamics Induced by Leu Versus Ile Residues in the CD Loop of Kir Channels.

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