Actin interacts with CCT via discrete binding sites: a binding transition-release model for CCT-mediated actin folding.
The chaperones prefoldin and the cytosolic chaperonin CCT-containing TCP-1 (CCT) guide the cytoskeletal protein actin to its native conformation. Performing an alanine scan of actin, we identified discrete recognition determinants for CCT interaction. Interestingly, one of these is similar and functional in the non-homologous protein Cdc20, suggesting that some of the binding information in the CCT target proteins is shared. The information in actin for recognition by CCT and for folding is different, as all but one of the mutants in the recognition determinants are folding-competent. In addition, some other actin mutants remain CCT-arrested and are not released in a native conformation, whereas others do fold but remain bound to CAP. Kinetic experiments provide evidence that CCT-mediated folding of non-native actin occurs in at least two steps, in which initially the recognition determinant 245-249 contacts CCT and the other determinants interact at later stages. Actin mutants that are CCT-arrested demonstrate that some regions neighbouring the recognition determinants are involved in modulating the correct folding transitions of actin on CCT, or its release from this chaperonin. Further, we found that the ATP binding of actin is not a prerequisite for its release, and we suggest that CAP may be involved in charging the nucleotide. Based on the kinetics of CCT binding and folding of actin and actin mutants, we propose a multi-step recognition-transition-release model. This also implies that the currently accepted notion of CCT-mediated actin folding is probably more complex.
Neirynck
Katrien
K
Flanders Interuniversity Institute for Biotechnology (VIB 09) and Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University. A. Baertsoenkaai 3, 9000 Gent, Belgium.
Waterschoot
Davy
D
Vandekerckhove
Joël
J
Ampe
Christophe
C
Rommelaere
Heidi
H
D000199:Actins / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D001665:Binding Sites; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D020125:Mutation, Missense; D011485:Protein Binding; D017510:Protein Folding
D000199:Actins; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins