Increased actin binding is a shared molecular consequence of numerous spinocerebellar ataxia mutations in β-III-spectrin

Abstract

Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disease caused by mutations in the SPTBN2 gene encoding the cytoskeletal protein β-III-spectrin. Previously, we demonstrated that a L253P missense mutation, localizing to the β-III-spectrin actin-binding domain (ABD), causes increased actin-binding affinity. Here we investigate the molecular consequences of nine additional ABD-localized, SCA5 missense mutations: V58M, K61E, T62I, K65E, F160C, D255G, T271I, Y272H, and H278R. We show that all of the mutations, similar to L253P, are positioned at or near the interface of the two calponin homology subdomains (CH1 and CH2) comprising the ABD. Using biochemical and biophysical approaches, we demonstrate that the mutant ABD proteins can attain a well-folded state. However, thermal denaturation studies show that all nine mutations are destabilizing, suggesting a structural disruption at the CH1-CH2 interface. Importantly, all nine mutations cause increased actin binding. The mutant actin-binding affinities vary greatly, and none of the nine mutations increase actin-binding affinity as much as L253P. ABD mutations causing high-affinity actin binding, with the notable exception of L253P, appear to be associated with early age of symptom onset. Altogether, the data indicate increased actin-binding affinity is a shared molecular consequence of numerous SCA5 mutations, which has important therapeutic implications.

Cardiomyopathy-associated variants alter the structure and function of the α-actinin-2 actin-binding domain

“…The previously characterized A119T ABD also lacked the N-terminal 18 residues; we previously showed that N-terminal residues preceding CH1 impact the stability of the related ABD of β-III-spectrin [27]. Interestingly, loss of cooperative unfolding was not observed for any of ten different missense variants in the related ABD of β-III-spectrin [35]. Most of the β-III-spectrin variants localized to the CH1/CH2 interface, including L253P and T271I, which are at the equivalent residue positions as α-actinin-2 M228T and T247M, respectively.…”