Defining the eukaryotic cytosolic chaperonin-binding sites in human tubulins

The actins and tubulins are the obligate substrates in vivo of the chaperon in-containing TCP-1 (CCT). The precise elements of recognition between the chaperonin and its substrates remain largely unknown. We have used a solid phase peptide binding assay to screen the human alpha, beta and gamma -tubu lin sequences for CCT recognition. Multiple regions seem to be implicated i n interactions between tubulins and CCT. These potential CCT-binding sites are highly dispersed throughout the primary sequences of the human tubulins . Ln addition, using site-directed mutagenesis we assessed the contribution of the selected residues in the C-terminal domain of beta -tubulin to CCT binding. Various hot spots have been identified even though, in each case, their replacement by alanine does not reduce dramatically the total affinit y of beta -tubulin for CCT. The CCT-binding information in the tubulins is probably confined to multiple specific regions each having weak or moderate affinity for CCT apical domains. The main binding region seems to be locat ed between residues 263 and 384, but there are no single amino acid residue s in this region, which make large contributions to the binding energy, alt hough we have detected a minor contribution by F377. These biochemical resu lts are understandable in the context of our recent structural analysis of CCT-tubulin complexes by cryo-electron microscopy and image reconstruction, which shows that, in one stage of an in vitro binding reaction between apo -CCT and tubulin diluted from guanidinium chloride, ten major, stable conta cts between tubulin and CCT are involved. Therefore, specificity is achieve d through the co-operation of many specific, albeit weak, interactions. (C) 2000 Academic Press.