The N-Terminal Flanking Region Modulates the Actin Binding Affinity of the Utrophin Tandem Calponin-Homology Domain

Abstract

Despite sharing a high degree of sequence similarity, the tandem calponin-homology (CH) domain of utrophin binds to actin 30 times stronger than that of dystrophin. We have previously shown that this difference in actin binding affinity could not be ascribed to the differences in inter-CH-domain linkers [Bandi, S., et al. (2015) Biochemistry 54, 5480-5488]. Here, we examined the role of the N-terminal flanking region. The utrophin tandem CH domain contains a 27-residue flanking region before its CH1 domain. We examined its effect by comparing the structure and function of full-length utrophin tandem CH domain Utr(1-261) and its truncated Utr(28-261) construct. Both full-length and truncated constructs are monomers in solution, with no significant differences in their secondary or tertiary structures. Truncated construct Utr(28-261) binds to actin 30 times weaker than that of the full-length Utr(1-261), similar to that of the dystrophin tandem CH domain with a much shorter flanking region. Deletion of the N-terminal flanking region stabilizes the CH1 domain. The magnitude of the change in binding free energy upon truncation is similar to that of the change in thermodynamic stability. The isolated N-terminal peptide by itself is significantly random coil and does not bind to F-actin in the affinity range of Utr(1-261) and Utr(28-261). These results indicate that the N-terminal flanking region significantly affects the actin binding affinity of tandem CH domains. This observation further stresses that protein regions other than the three actin-binding surfaces identified earlier, irrespective of whether they directly bind to actin, also contribute to the actin binding affinity of tandem CH domains.

Steric Regulation of Tandem Calponin Homology Domain Actin-Binding Affinity

“…This construct (Δ-n-term) was largely cytoplasmic and had a low bound fraction (0.25±0.03, p<0.05), indicating a low binding affinity to F-actin compared to the native utrophin CH1-CH2. This is consistent with previous results highlighting the importance of this region for actin-binding affinity (Iwamoto et al, 2018;Avery et al, 2017a;Singh et al, 2017).…”

“…These mutations are consistent with a model in which the degree to which the CH1 and CH2 domains are 'open' or 'closed' regulates affinity to f-actin. Interestingly, we also find that the N-terminal region of CH1, which was recently shown to affect f-actin binding affinity (Avery et al, 2017a;Singh et al, 2017;Iwamoto et al, 2018), is sufficient to alter the subcellular localization of the CH1-CH2 domain in live cells, independent of affinity. The ability of small sequence changes in CH1-CH2 domains to not only increase and decrease affinity but also alter sub-cellular localization provides new insight into disease-associated mutations and raises new questions about how the localization of actin-binding proteins and spatial organization of the actin cytoskeleton are regulated.…”

“…The N-terminal flanking region varies significantly between CH1-CH2 domain proteins, both in sequence and in length (Iwamoto et al, 2018;Singh et al, 2017). This region has recently been shown to be important for actin-binding affinity, as its deletion in either utrophin (Singh et al, 2017) or ß-spectrin (Avery et al, 2017a) abrogates actin binding. Interestingly, the N-terminal flanking regions from filamin B is significantly shorter than that from ß-spectrin, despite the relatively high reported binding affinity (Kd~7µM) of filamin B's ABD for f-actin ).…”

Multivariate Analysis of Two-Dimensional 1H, 13C Methyl NMR Spectra of Monoclonal Antibody Therapeutics To Facilitate Assessment of Higher Order Structure

“…However, the utility of this approach is limited when large numbers of datasets must be analyzed as well as if individual peaks are not all fully resolved, as is the case for mAbs. 11 Fortunately, practical solutions to these limitations can be found in one-dimensional (1D) 1 H NMR methods, such as in application to complex mixtures, where statistical based approaches for analysis of spectra have been well established. 12 Indeed a number of 1D 1 H NMR based methods have been recently proposed for chemometric HOS analysis of mAbs.…”

Biased localization of actin binding proteins by actin filament conformation

“…Recent cryo-Electron Microscopy studies have mapped the interacting residues between the actin binding domain from filamin A and factin (Iwamoto et al, 2018), and between the actin binding domain of utrophin and factin to 3.6Å resolution (Kumari et al, 2019). We chose to examine residues predicted to lie within actin binding surface 2 (ABS2) on utrophin CH1 (Iwamoto et al, 2018), at the CH1-CH2 domain interface (Galkin et al, 2010a;Harris et al, 2019) and within the n-terminal flanking region (Avery et al, 2017;Harris et al, 2019;Singh et al, 2017), which we had found altered actin-binding affinity in a previous study .…”