Structural Study of the WH2 Family and Filamin: Implications for Actin Cytoskeleton Regulation

• Main Author: Aguda, Adeleke H.
• Format: Doctoral Thesis
• Language: English
• Published: Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi 2006
• Subjects: Biochemistry; Actin; Thymosin; Ciboulot; N-WASP; Filamin; Calponin homology domain; WH2; Protein Crystallography; Biokemi
• Online Access: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7188; http://nbn-resolving.de/urn:isbn:91-554-6679-6

Cellular processes like motility, chemotaxis, phagocytosis and morphogenesis are dependent on the dynamic regulation of the actin cytoskeleton. This cytoskeleton system is tightly controlled by a number of diverse actin-binding proteins (ABPs) by various mechanisms described as nucleation, polymerization, capping, severing, depolymerization and sequestration. The ABPs are grouped based on sequence identity as in the Wiskott-Aldrich Syndrome protein homology domain 2 (WH2), and the calponin homology domain (CH) containing proteins. In this work, we elucidate the crystal structures of hybrids of gelsolin domain 1 with thymosin β4, ciboulot domain 2, and the second WH2 domain of N-WASP each bound to actin. We show that the single WH2 motif containing protein thymosin β4 in part sequesters actin by binding its pointed end via a C-terminal helix. This interaction prevents the addition of bound actin protomers to the barbed end of the filament. We propose that sequence variations in some WH2 motifs conferred F-actin binding ability to multiple repeat-containing proteins. These F-actin binding domains interact with the barbed end of a filament and the adjacent WH2 motifs are then freed to add their bound actin to the growing filament end. We demonstrate the binding of ciboulot domains 2 and 3 to both G- and F-actin and that full length ciboulot is capable of binding two actin monomers simultaneously. We have also cloned, expressed, purified and crystallized rod domains 14-16 from the actin crosslinking protein a-filamin. Preliminary X-ray crystallography data gives us hope that we shall be able to solve the structure of this triple domain repeat.