Versatile substrate protein recognition mechanism of the eukaryotic chaperonin CCT.

Group II chaperonins, found in eukaryotic and archaeal organisms, recognize substrate proteins through diverse mechanisms that involve either hydrophobic- or electrostatic-dominated interactions. This action is distinct from the universal substrate recognition mechanism of group I chaperonins, which bind a wide spectrum of non-native proteins primarily through hydrophobic interactions. We use computational approaches to pinpoint the substrate protein binding sites of the gamma-subunit of the eukaryotic chaperonin CCT and to identify its interactions with the stringent substrate beta-tubulin. Protein-protein docking methods reveal intrinsic binding sites of CCT comprising a helical (HL) region, homologous to the GroEL-binding site, and the helical protrusion (HP) region. We performed molecular dynamics simulations of the solvated CCTgamma apical domain, beta-tubulin peptide-CCTgamma complexes, and isolated beta-tubulin peptides. We find that tubulin binds to CCTgamma through an extensive interface that spans both the HL region and the HP region. HL interactions involve both hydrophobic and electrostatic contacts, while binding to the HP region is stabilized almost exclusively by a salt bridge network. On the basis of additional simulations of a beta-tubulin-CCTgamma complex that involves a reduced interface, centered onto the HP region, we conclude that this salt bridge network is the minimal stabilizing interaction required. Strong conservation of the charged amino acids that participate in the salt bridge network, Arg306 and Glu271, indicates a general mechanism across the nonidentical CCT subunits and group II chaperonins.