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 cryoelectron microscopy shows that actin binds either the CCTbeta-CCTdelta or the CCTepsilon-CCTdelta 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 beta-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 beta-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 beta-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.
McCormack
E A
EA
Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London, SW3 6JB, United Kingdom.
Rohman
M J
MJ
Willison
K R
KR
D000199:Actins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D018797:Cell Cycle Proteins; D002474:Cell-Free System; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins / Q000737:chemistry / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D003600:Cytosol; D029721:Drosophila Proteins; D005057:Eukaryotic Cells; D006801:Humans; D007700:Kinetics; D051379:Mice; D008840:Microfilament Proteins; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
D000199:Actins; C489549:CAP1 protein, human; C533298:CCT2 protein, human; C533300:CCT4 protein, human; C533301:CCT5 protein, human; C410502:Capt protein, Drosophila; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D029721:Drosophila Proteins; D008840:Microfilament Proteins; C533315:Tcp1 protein, mouse; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins