Structural basis for high-affinity actin binding revealed by a \u03b2-III-spectrin SCA5 missense mutation

Adam W Avery,Albina Orlova,Thomas S Hays,Michael E Fealey,Edward H Egelman,David D Thomas,Andrew R Thompson,Fengbin Wang

doi:10.1038/s41467-017-01367-w

Adam W Avery, Albina Orlova + Show 6 more

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https://doi.org/10.1038/s41467-017-01367-w

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Abstract

Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disease caused by mutations in the cytoskeletal protein β-III-spectrin. Previously, a SCA5 mutation resulting in a leucine-to-proline substitution (L253P) in the actin-binding domain (ABD) was shown to cause a 1000-fold increase in actin-binding affinity. However, the structural basis for this increase is unknown. Here, we report a 6.9 Å cryo-EM structure of F-actin complexed with the L253P ABD. This structure, along with co-sedimentation and pulsed-EPR measurements, demonstrates that high-affinity binding caused by the CH2-localized mutation is due to opening of the two CH domains. This enables CH1 to bind actin aided by an unstructured N-terminal region that becomes α-helical upon binding. This helix is required for association with actin as truncation eliminates binding. Collectively, these results shed light on the mechanism by which β-III-spectrin, and likely similar actin-binding proteins, interact with actin, and how this mechanism can be perturbed to cause disease.

Highlights

Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disease caused by mutations in the cytoskeletal protein β-III-spectrin
SCA5 pathogenesis results from a functional deficit in Purkinje cells, in which the expression of β-III-spectrin is required for normal cerebellar control of motor coordination3. β-III-spectrin is thought to form a heterotetrameric complex with α-II-spectrin, and to cross-link actin filaments to form a cytoskeleton localizing to the shafts and spines of Purkinje cell dendrites. β-III-spectrin is required for normal dendrite structure[4] and synaptic transmission[5, 6]
Consistent with this, many mutations in the CH2 domains of both α-actinin and filamin impart modest gains in actin-binding domain (ABD) affinity for actin[17, 18]. These studies suggest that the L253P mutation of β-III-spectrin, which is localized to CH2, causes high-affinity actin binding by disrupting a regulatory mechanism that shifts the ABD structural equilibrium from a closed to more open binding-competent state

Summary

Introduction

Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disease caused by mutations in the cytoskeletal protein β-III-spectrin. We report a 6.9 Å cryo-EM structure of F-actin complexed with the L253P ABD This structure, along with co-sedimentation and pulsed-EPR measurements, demonstrates that high-affinity binding caused by the CH2-localized mutation is due to opening of the two CH domains. Our group reported that a SCA5 missense mutation, L253P, localized to the β-III-spectrin N-terminal actin-binding domain (ABD), causes a ~1000-fold increase in actin-binding affinity[7]. Consistent with this, many mutations in the CH2 domains of both α-actinin and filamin impart modest gains in ABD affinity for actin[17, 18] These studies suggest that the L253P mutation of β-III-spectrin, which is localized to CH2, causes high-affinity actin binding by disrupting a regulatory mechanism that shifts the ABD structural equilibrium from a closed to more open binding-competent state. We report cryo-EM, co-sedimentation, and pulsed electron paramagnetic resonance (EPR) data consistent with such a mechanism

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