Synthetic actin-binding domains reveal compositional constraints for function.
The actin-binding domains of many proteins consist of a canonical type 1/type 2 arrangement of the structurally conserved calponin homology domain. Using the actin-binding domain of alpha-actinin-1 as a scaffold we have generated synthetic actin-binding domains by altering position and composition of the calponin homology domains. We show that the presence of two calponin homology domains alone and in the context of an actin-binding domain is not sufficient for actin-binding, and that both single and homotypic type 2 calponin homology domain tandems fail to bind to actin in vitro and in transfected cells. In contrast, single and tandem type 1 calponin homology domain arrays bind actin directly but result in defective turnover rates on actin filaments, and in aberrant actin bundling when introduced into the full-length alpha-actinin molecule. An actin-binding domain harboring the calponin homology domains in an inverted position, however, functions both in isolation and in the context of the dimeric alpha-actinin molecule. Our data demonstrate that the dynamics and specificity of actin-binding via actin-binding domains requires both the filament binding properties of the type 1, and regulation by type 2 calponin homology domains, and appear independent of their position.
Lorenzi
Maria
M
Consorzio Mario Negri Sud, Department of Cell Biology and Oncology, Via Nazionale 8a, I-66030 Santa Maria Imbaro, Italy.
Gimona
Mario
M
D000185:Actinin / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D000096985:Calponins
D000185:Actinin; D002135:Calcium-Binding Proteins; D008840:Microfilament Proteins; D000096985:Calponins