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

• Published in: Biochemistry (Easton) 2017-05, Vol.56 (20), p.2627-2636
• Main Authors: Singh, Surinder M, Bandi, Swati, Mallela, Krishna M. G
• Format: Article
• Language: English
• Subjects: Actins - chemistry; Actins - metabolism; Calcium-Binding Proteins - chemistry; Calponins; Microfilament Proteins - chemistry; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Thermodynamics; Utrophin - chemistry; Utrophin - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.6b01117

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.