J. Mol. Biol. (1995) 249, 138–152 The Origins and Consequences of Asymmetry in the Chaperonin Reaction Cycle Steven G. Burston*, Neil A. Ranson and Anthony R. Clarke The binding of nucleotides and chaperonin-10 (cpn10) to the Escherichia coli Molecular Recognition Centre and Department of chaperonin-60 (cpn60) and their effect upon the molecular symmetry has Biochemistry, University of been examined both kinetically and at equilibrium. ATP binds tightly and is hydrolysed on only one heptameric ring of the cpn60 tetradecamer at a Bristol, School of Medical Sciences, University Walk time, thus inducing asymmetry in the cpn60 oligomer even in the absence of cpn10. In the absence of cpn10 these seven ATP molecules hydrolyse to Bristol BS8 1TD, UK form a cpn60:ADP 7 complex in which ADP is tightly bound (K d = 2–7 M); further ADP binding to form a cpn60:ADP 14 complex is weak (K 1/2 = 2.3 mM). We conclude that symmetrical nucleotide complexes (with 14 ATP or 14 ADPs) are unstable, demonstrating negative co-operativity between the rings. When cpn60 is mixed with cpn10 and ATP the resultant cpn60:ATP 7 :cpn10 complex is formed rapidly (the rate constant for cpn10 association is >4 x 10 7 M -1 s -1 ) and before ATP is hydrolysed (k = 0.12 s -1 per active subunit) to produce an extremely stable cpn60:ADP 7 :cpn10 complex. This allows ATP association on the unoccupied ring and nucleotide asymmetry in the double toroid is preserved. In ‘‘trapping’’ experiments, where the cpn60:ADP 7 :cpn10 is challenged with ATP, cpn10 was observed to dissociate at a rate identical to that of steady-state ATP hydrolysis in the presence of cpn10 (k = 0.042 s -1 per active subunit). The spontaneous decay of cpn60:ADP 7 :cpn10 and any of the major steady-state complexes, under conditions where free nucleotides had been removed, occurred at a rate tenfold lower than ATP hydrolysis. Since the binding of the non-hydrolysable analogue AMP-PNP was unable to induce dissociation of the co-chaperonin it was concluded that a transient state following ATP hydrolysis is necessary for the rapid dissociation of cpn10, which occurs once in every cycle. Trapping experiments using sub-stoichiometric concentrations of cpn10, relative to cpn60, show an unchanged rate of cpn10 exchange upon ATP hydrolysis, indicating that the formation of a symmetric, ‘‘football’’-shaped complex in which two molecules of the co-chaperonin are bound to cpn60, is not an obligatory intermediate in the exchange process. Keywords: molecular chaperone; chaperonin; ATPase; protein folding *Corresponding author Introduction Molecular chaperones are described as a class of proteins whose physiological role is to assist in the correct transport, folding and assembly of other polypeptide chains whilst not themselves being part of the final structure (Ellis & van der Vies, 1991). The chaperonins form a subset of the molecular chaper- ones of which the homologues from Escherichia coli are the most widely studied. They consist of two oligomeric proteins; the first being cpn60, a tetradec- amer composed of 57 kDa subunits (Hemmingsen et al ., 1988), and its co-protein cpn10, a hep- tamer of 10.4 kDa subunits (Chandrasekhar et al ., 1986). These proteins are essential for cell viability at all temperatures (Fayet et al ., 1989). cpn60 appears under the electron microscope as a double toroid in which each ring is composed of seven subunits whilst cpn10 is a single ring of seven subunits which binds to the apical domains of one ring of cpn60 in the presence of Mg-ATP and Mg-ADP (Saibil et al ., 1991; Chen et al ., 1994) or, under certain non-physiological conditions to both rings of cpn60 (Llorca et al ., 1994; Schmidt et al ., 1994a). The X-ray determined structure Abbreviations used: cpn, chaperonin; mMDH, mitochondrial L-malate dehydrogenase; AMP-PNP, 5'-adenylyl imidodiphosphate; TEA, triethanolamine hydrochloride; PMSF, phenylmethylsulphonyl fluoride; GuHCl, guanidinium hydrochloride. 0022–2836/95/210138–15 $08.00/0  1995 Academic Press Limited