Steric Regulation of Tandem Calponin Homology Domain Actin-Binding Affinity

Abstract

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 cationπ 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.

Biased localization of actin binding proteins by actin filament conformation

“…Secondly, CH2 acts as a negative regulator of F-actin binding affinity, by sterically clashing with the actin filament. Our previous work 37 , and the work of others 41,43,44 , has shown that mutations targeting residues involved in CH1-CH2 interactions can promote opening of the tandem domain (for example, Q33A T36A on utrnWT). This relieves the steric interactions between CH2 and F-actin and thus increases binding affinity (Fig.…”

“…Two mechanisms regulate CH1-CH2 binding to F-actin. CH1-CH2 domains are found in many actin crosslinking and regulatory proteins 37 , including α-actinins in stress fibers 38 and filamins in the actin cortex 39 . The minimal actin-binding domain of utrophin (CH1-CH2) is often used as a generic marker for Factin 40 , which raises the question of whether it uniformly labels all filaments in cells or might be biased towards a specific actin filament conformation.…”

Molecular mechanism for direct actin force-sensing by α-catenin

“…Proteins from the large Calponin Homology (CH) domain ABP superfamily have diverse functions as cytoskeletal cross-linkers and plasma membrane/organelle tethers ( Liem, 2016 ; Razinia et al, 2012 ). Their ABDs have also been reported to have sequence elements that undergo folding transitions associated with filament engagement ( Avery et al, 2017 ; Galkin et al, 2010a ; Iwamoto et al, 2018 ) which can sterically regulate their actin binding ( Harris et al, 2019 ). This suggests members of this family could plausibly employ force-activated binding mechanisms similar to α-catenin to coordinate diverse mechanotransduction pathways throughout the cell, as could other ABPs with similar properties.…”

Molecular mechanism for direct actin force-sensing by α-catenin

“…Proteins from the large Calponin Homology (CH) domain ABP superfamily have diverse functions as cytoskeletal cross-linkers and plasma membrane / organelle tethers (Liem, 2016;Razinia et al, 2012). Their ABDs have also been reported to have sequence elements that undergo folding transitions associated with filament engagement (Avery et al, 2017;Galkin et al, 2010a;Iwamoto et al, 2018) which have the capacity to sterically regulate their actin binding (Harris et al, 2019). This suggests members of this family could plausibly employ force-activated binding mechanisms similar to a-catenin to coordinate diverse mechanotransduction pathways throughout the cell, as could other ABPs with similar properties.…”