Mutational screen identifies critical amino acid residues of beta-actin mediating interaction between its folding intermediates and eukaryotic cytosolic chaperonin CCT

The three-dimensional reconstruction of apo-CCT-alpha -actin by cryoelectro n microscopy shows that actin binds either the CCT beta -CCT delta or the C CT epsilon -CCT delta subunit pairs of the chaperonin in an open and appare ntly quasi-native conformation. The CCT-binding sites are seen located at t he tips of the two arms of actin and these same regions of actin have been implicated in CCT binding through beta -actin peptide-array screening. Thre e main CCT binding regions exist: actin Sites I, II, and III, which are com posed of loops that are surface-exposed in native actin. Sixty-eight amino acid residues on beta3-actin have been screened by mutagenesis for effects on CCT interaction in quantitative in vitro translation assays in rabbit re ticulocyte lysate. Actin seems to be folding cooperatively on chaperonin, s ince certain mutants discriminate CCT binding from processing. Actin Site I I, located at the tip of actin subdomain 4, is the major determinant for CC T binding. Site II is composed of two anti-parallel extended beta -strands, with F200-T203 and D244 contributing substantially to the binding site. Th e substrate recognition chemistry of CCT thus seems different from that of Group I chaperonins and probably reflects the fact that it needs to be high ly specific to enable capture and folding of the actins and tubulins. (C) 2 001 Academic Press.