title: The actin binding affinity of the utrophin tandem calponin-homology domain is primarily determined by its N-terminal domain.
abstract: The structural determinants of the actin binding function of tandem calponin-homology (CH) domains are poorly understood, particularly the role of individual domains. We determined the actin binding affinity of isolated CH domains from human utrophin and compared them with the affinity of the full-length tandem CH domain. Traditional cosedimentation assays indicate that the C-terminal CH2 domain binds to F-actin much weaker than the full-length tandem CH domain. The N-terminal CH1 domain is less stable and undergoes severe protein aggregation; therefore, traditional actin cosedimentation assays could not be used. To address this, we have developed a folding-upon-binding method. We refolded the CH1 domain from its unfolded state in the presence of F-actin. This results in a competition between actin binding and aggregation. A differential centrifugation technique was used to distinguish actin binding from aggregation. Low-speed centrifugation pelleted CH1 aggregates, but not F-actin or its bound protein. Subsequent high-speed centrifugation resulted in the cosedimentation of bound CH1 along with F-actin. The CH1 domain binds to F-actin with an affinity similar to that of the full-length tandem CH domain, unlike the CH2 domain. The actin binding cooperativity between the two domains was quantitatively calculated from the association constants of the full-length tandem CH domain and its CH domains, and found to be much smaller than the association constant of the CH1 domain alone. These results indicate that the actin binding affinity of the utrophin tandem CH domain is primarily determined by its CH1 domain, when compared to that of its CH2 domain or the cooperativity between the two CH domains.
authors.affiliation: Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus , Aurora, Colorado 80045, United States.
authors.forename: Surinder M
authors.initials: SM
authors.lastname: Singh
authors.forename: Swati
authors.initials: S
authors.lastname: Bandi
authors.forename: Steve J
authors.initials: SJ
authors.lastname: Winder
authors.forename: Krishna M G
authors.initials: KM
authors.lastname: Mallela
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D002417:Cattle; D018360:Crystallography, X-Ray; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D049411:Utrophin / Q000737:chemistry* / Q000378:metabolism*; D000096985:Calponins
chemical_list: D000199:Actins; D002135:Calcium-Binding Proteins; D008840:Microfilament Proteins; D049411:Utrophin; D000096985:Calponins; C483599:UTRN protein, human