Mutational Screen Identifies Critical Amino Acid Residues of β-Actin Mediating Interaction between Its Folding Intermediates and Eukaryotic Cytosolic Chaperonin CCT

Abstract

Thethree-dimensional reconstruction of apo-CCT-α-actin by cryoelectron microscopy shows that actin binds either the CCTβ–CCTδ or the CCTϵ–CCTδ subunit pairs of the chaperonin in an open and apparently quasi-native conformation. The CCT-binding sites are seen located at the tips of the two arms of actin and these same regions of actin have been implicated in CCT binding through β-actin peptide-array screening. Three main CCT binding regions exist: actin Sites I, II, and III, which are composed of loops that are surface-exposed in native actin. Sixty-eight amino acid residues on β-actin have been screened by mutagenesis for effects on CCT interaction in quantitative in vitro translation assays in rabbit reticulocyte lysate. Actin seems to be folding cooperatively on chaperonin, since certain mutants discriminate CCT binding from processing. Actin Site II, located at the tip of actin subdomain 4, is the major determinant for CCT binding. Site II is composed of two anti-parallel extended β-strands, with F200–T203 and D244 contributing substantially to the binding site. The substrate recognition chemistry of CCT thus seems different from that of Group I chaperonins and probably reflects the fact that it needs to be highly specific to enable capture and folding of the actins and tubulins.