Dataset Information

Two Deafness-Causing Actin Mutations (DFNA20/26) Have Allosteric Effects on the Actin Structure.

ABSTRACT: Point mutations in ?-cytoplasmic actin have been shown to result in autosomal-dominant, nonsyndromic, early-onset deafness. Two mutations at the same site, K118M and K118N, provide a unique opportunity to compare the effects of two dissimilar amino acid substitutions that produce a similar phenotype in humans. K118 resides in a helix that runs from K113 to T126, and mutations that alter the position, dynamics, and/or biochemistry of this helix can result in a wide range of pathologies. Using a combination of computational and experimental studies, both employing yeast actin, we find that these mutations at K118 result in changes in the structure and dynamics of the DNase-I loop, alterations in the structure of the H73 loop as well as the side-chain orientations of W79 and W86, changes in nucleotide exchange rates, and significant shifts in the twist of the actin monomer. Interestingly, in the case of K118N, the twist of the monomer is nearly identical to that of the F-actin protomer, and in vitro polymerization assays show that this mutation results in faster polymerization. Taken together, these results indicate that mutations at this site give rise to a series of small changes that can be tolerated in vivo but result in misregulation of actin assembly and dynamics.

SUBMITTER: Jepsen L

PROVIDER: S-EPMC4968419 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Two Deafness-Causing Actin Mutations (DFNA20/26) Have Allosteric Effects on the Actin Structure.

Jepsen Lauren L Kruth Karina A KA Rubenstein Peter A PA Sept David D

Biophysical journal 20160701 2

Point mutations in γ-cytoplasmic actin have been shown to result in autosomal-dominant, nonsyndromic, early-onset deafness. Two mutations at the same site, K118M and K118N, provide a unique opportunity to compare the effects of two dissimilar amino acid substitutions that produce a similar phenotype in humans. K118 resides in a helix that runs from K113 to T126, and mutations that alter the position, dynamics, and/or biochemistry of this helix can result in a wide range of pathologies. Using a c ...[more]

PMID: 27463135

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Project description:Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased tendency to aggregate. Thus, mutations in TM4 cause a range of phenotypes of dysfunctional gap junction channels that are discussed within the context of the X-ray crystallographic structure.

Dataset Information

Two Deafness-Causing Actin Mutations (DFNA20/26) Have Allosteric Effects on the Actin Structure.

Publications

Two Deafness-Causing Actin Mutations (DFNA20/26) Have Allosteric Effects on the Actin Structure.

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