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<p>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.</p>

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

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年7月21日

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  • Maya Noureddine,
  • Halina Mikolajek,
  • Nathan Cowieson,
  • Nikos Pinotsis,
  • Paul Robinson,
  • Alexandre Slater,
  • Charles Redwood,
  • Siobhan Loughna,
  • Chris Denning,
  • Fiyaz Mohammed,
  • Katja Gehmlich

摘要

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.