High-resolution crystal structures of villin headpiece and mutants with reduced F-actin binding activity.
Villin-type headpiece domains are approximately 70 amino acid modular motifs found at the C terminus of a variety of actin cytoskeleton-associated proteins. The headpiece domain of villin, a protein found in the actin bundles of the brush border epithelium, is of interest both as a compact F-actin binding domain and as a model folded protein. We have determined the high-resolution crystal structures of chicken villin headpiece (HP67) at 1.4 A resolution as well as two mutants, R37A and W64Y, at 1.45 and 1.5 A resolution, respectively. Replacement of R37 causes a 5-fold reduction in F-actin binding affinity in sedimentation assays. Replacement of W64 results in a much more drastic reduction in F-actin binding affinity without significant changes in headpiece structure or stability. The detailed comparison of these crystal structures with each other and to our previously determined NMR structures of HP67 and the 35-residue autonomously folding subdomain in villin headpiece, HP35, provides the details of the headpiece fold and further defines the F-actin binding site of villin-type headpiece domains.
Meng
Jianmin
J
Department of Physiology and Biophysics, Boston University School of Medicine, 715 Albany Street, Boston, Massachusetts 02118, USA.
Vardar
Didem
D
Wang
Yeming
Y
Guo
Hwai-Chen
HC
Head
James F
JF
McKnight
C James
CJ
D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D030161:Avian Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002645:Chickens / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D013696:Temperature
D000199:Actins; D030161:Avian Proteins; D008840:Microfilament Proteins; C026615:villin