Comprehensive biophysical and structural profiling of alpha-actinin-2 variants reveals mechanistic diversity in hypertrophic cardiomyopathy

Journal article

Hypertrophic cardiomyopathy (HCM) is a genetic disease associated with sudden cardiac death. Variants in alpha-actinin-2 (ACTN2), a Z-disc protein that anchors actin thin filaments have been implicated in HCM, yet their structural consequences remain poorly defined. Here, we characterise seventeen HCM-associated ACTN2 variants spanning multiple domains using an integrated and tiered workflow combining high-throughput assays, structural modelling and biophysical approaches. All variants display reduced solubility, with actin-binding domain (ABD) substitutions showing pronounced thermal instability by differential scanning fluorimetry. Modelling of nine variants predicts diverse pathogenic mechanisms including compromised actin-binding, impaired ABD regulatory conformations, disrupted dimerisation interfaces, and perturbed domain architecture. Crystal structures of two rod-domain variants reveal intact dimerisation despite modelling predictions. Actin-binding assays for ABD variants confirm altered actin engagement suggesting that binding dynamics may drive pathogenicity. Limited proteolysis indicates reduced structural stability across variants, while size-exclusion chromatography coupled with multi-angle light scattering or small-angle X-ray scattering (SEC-MALS/SAXS) shows a strong propensity for aggregation. Batch-mode SAXS further demonstrates early aggregation onset in selected ABD variants at elevated temperatures. Collectively, these findings establish that HCM-linked ACTN2 variants compromise protein integrity through multiple mechanisms, highlight the ABD as a hotspot of vulnerability and provide a potential framework for interpreting cardiomyopathy-associated variants.

Authors: Noureddine, M; Mikolajek, H; Cowieson, N; Pinotsis, N; Robinson, P

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Nature Research
• Journal: Nature Communications
• Volume: 17
• Issue: 1
• Publication date: 2026-07-21
• Acceptance date: 2026-06-26
• DOI: 10.1038/s41467-026-75392-z
• EISSN: 2041-1723
• ISSN: 2041-1723
• Language: English
• Keywords: Protein domain; In silico; Protein structure; Small-angle X-ray scattering; Structural variation; Actin; Hypertrophic cardiomyopathy; Proteolysis; Genetic variants