PT - JOURNAL ARTICLE AU - Andrew R Harris AU - Brian Belardi AU - Pamela Jreij AU - Kathy Wei AU - Andreas Bausch AU - Daniel A Fletcher TI - Steric Regulation of Tandem Calponin Homology Domain Actin-Binding Affinity AID - 10.1101/598359 DP - 2019 Jan 01 TA - bioRxiv PG - 598359 4099 - http://biorxiv.org/content/early/2019/05/27/598359.short 4100 - http://biorxiv.org/content/early/2019/05/27/598359.full AB - Tandem calponin homology domains are common actin-binding motifs found in proteins such as α-actinin, filamin, utrophin, and dystrophin that organize actin filaments into functional structures. Despite a conserved structural fold and regions of high sequence similarity, tandem calponin homology (CH1-CH2) domains can have remarkably different actin-binding properties, with disease-associated point mutants known to cause increased as well as decreased affinity for f-actin. How small variations in sequence can lead to significant differences in binding affinity and resulting actin organization remains unclear. Here, we show that the affinity of CH1-CH2 domains for f-actin can be both increased and decreased by diverse modifications that change the effective ‘openness’ of CH1 and CH2 that sterically regulates binding to f-actin. We find that mutations to the interdomain linker, as well as changes to the inter-CH domain interface distinct from a well characterized cation-π interaction, ‘open’ the domain and increases binding affinity, while a modification that adds molecular size to the CH2 ‘closes’ the domain and decreases binding affinity. Interestingly, we observe non-uniform sub-cellular localization of CH1-CH2 domains to f-actin that depend on the N-terminal flanking region of CH1 but not on the overall affinity to f-actin. This dependence of CH1-CH2 binding properties on sequence and ‘openness’ contributes to a mechanistic understanding of disease-associated mutations and has implications for engineering actin-binding domains with specific properties.