Molecular insight into the regulation of vimentin by cysteine modifications and zinc binding

Abstract

23 p.-7 fig. ; The intermediate filament protein vimentin is involved in essential cellular processes, including cell division and stress responses, as well as in the pathophysiology of cancer, pathogen infection, and autoimmunity. The vimentin network undergoes marked reorganizations in response to oxidative stress, in which modifications of vimentin single cysteine residue, Cys328, play an important role, and is modulated by zinc availability. However, the molecular basis for this regulation is not fully understood. Here, we show that Cys328 displays a low pKa, supporting its reactivity, and is readily alkylated and oxidized in vitro. Moreover, combined oxidation and crosslinking assays and molecular dynamics simulations support that zinc ions interact with Cys328 in its thiolate form, whereas Glu329 and Asp331 stabilize zinc coordination. Vimentin oxidation can induce disulfide crosslinking, implying the close proximity of Cys328 from neighboring dimers in certain vimentin conformations, supported by our computational models. Notably, micromolar zinc concentrations prevent Cys328 alkylation, lipoxidation, and disulfide formation. Moreover, zinc selectively protects vimentin from crosslinking using short-spacer cysteine-reactive but not amine-reactive agents. These effects are not mimicked by magnesium, consistent with a lower number of magnesium ions hosted at the cysteine region, according to molecular dynamics simulations. Importantly, the region surrounding Cys328 is involved in interaction with several drugs targeting vimentin and is conserved in type III intermediate filaments, which include glial fibrillary acidic protein and desmin. Altogether, our results identify this region as a hot spot for zinc binding, which modulates Cys328 reactivity. Moreover, they provide a molecular standpoint for vimentin regulation through the interplay between cysteine modifications and zinc availability. ; This work was supported by the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie Grant Agreement No. 675132 "Masstrplan", Grants SAF2015-68590-R and RTI2018-097624-B-I00 from Agencia Estatal de Investigación, MICINN/FEDER, Spain, and Instituto de Salud Carlos III/FEDER, RETIC ARADyAL RD16/0006/0021 to DPS; Grant CTQ2017-88353-R from MICINN to SMS; Grant BES-2015-071588 from MICINN to JGC. ; Peer reviewed