Interdomain Linker Determines Primarily the Structural Stability of Dystrophin and Utrophin Tandem Calponin-Homology Domains Rather than Their Actin-Binding Affinity.
Tandem calponin-homology (CH) domains are the most common actin-binding domains in proteins. However, structural principles underlying their function are poorly understood. These tandem domains exist in multiple conformations with varying degrees of inter-CH-domain interactions. Dystrophin and utrophin tandem CH domains share high sequence similarity (∼82%), yet differ in their structural stability and actin-binding affinity. We examined whether the conformational differences between the two tandem CH domains can explain differences in their stability and actin binding. Dystrophin tandem CH domain is more stable by ∼4 kcal/mol than that of utrophin. Individual CH domains of dystrophin and utrophin have identical structures but differ in their relative orientation around the interdomain linker. We swapped the linkers between dystrophin and utrophin tandem CH domains. Dystrophin tandem CH domain with utrophin linker (DUL) has similar stability as that of utrophin tandem CH domain. Utrophin tandem CH domain with dystrophin linker (UDL) has similar stability as that of dystrophin tandem CH domain. Dystrophin tandem CH domain binds to F-actin ∼30 times weaker than that of utrophin. After linker swapping, DUL has twice the binding affinity as that of dystrophin tandem CH domain. Similarly, UDL has half the binding affinity as that of utrophin tandem CH domain. However, changes in binding free energies due to linker swapping are much lower by an order of magnitude compared to the corresponding changes in unfolding free energies. These results indicate that the linker region determines primarily the structural stability of tandem CH domains rather than their actin-binding affinity.
Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, and ‡Program in Structural Biology and Biochemistry, University of Colorado Anschutz Medical Campus , Aurora, Colorado 80045, United States.
Swati
S
Bandi
Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, and ‡Program in Structural Biology and Biochemistry, University of Colorado Anschutz Medical Campus , Aurora, Colorado 80045, United States.
Surinder M
SM
Singh
Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, and ‡Program in Structural Biology and Biochemistry, University of Colorado Anschutz Medical Campus , Aurora, Colorado 80045, United States.
Krishna M G
KM
Mallela
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D016189:Dystrophin / Q000737:chemistry / Q000378:metabolism*; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D055550:Protein Stability; D017433:Protein Structure, Secondary; D049411:Utrophin / Q000737:chemistry / Q000378:metabolism*; D000096985:Calponins
D000199:Actins; D002135:Calcium-Binding Proteins; D016189:Dystrophin; D008840:Microfilament Proteins; D049411:Utrophin; D000096985:Calponins