Crystal Structures of the Group II Chaperonin from Thermococcus strain KS-1: Steric Hindrance by the Substituted Amino Acid, and Inter-subunit Rearrangement between Two Crystal Forms

Abstract

The crystal structures of the group II chaperonins consisting of the α subunit with amino acid substitutions of G65C and/or I125T from the hyperthermophilic archaeum Thermococcus strain KS-1 were determined. These mutants have been shown to be active in ATP hydrolysis but inactive in protein folding. The structures were shown to be double-ring hexadecamers in an extremely closed form, which was consistent with the crystal structure of native α₈β₈-chaperonin from Thermoplasma acidophilum. Comparisons of the present structures with the atomic structures of the GroEL₁₄–GroES₇–(ADP)₇ complex revealed that the deficiency in protein-folding activity with the G65C amino acid substitution is caused by the steric hindrance of the local conformational change in an equatorial domain. We concluded that this mutant chaperonin with G65C substitution is deprived of the smooth conformational change in the refolding-reaction cycle. We obtained a new form of crystal with a distinct space group at a lower concentration of sulfate ion in the presence of nucleotide. The crystal structure obtained at the lower concentration of sulfate ion tilts outward, and has much looser inter-subunit contacts compared with those in the presence of a higher concentration of sulfate ion. Such subunit rotation has never been characterized in group II chaperonins. The crystal structure obtained at the lower concentration of sulfate ion tilts outward, and has much looser inter-subunit contacts compared with those in the presence of a higher concentration of sulfate ion.

Introduction

Chaperonins, a class of molecular chaperones,¹ function as a protein-folding chamber in an ATP-dependent manner. A back-to-back stacked toroidal assembly is common among most chaperonins. Chaperonins have been classified into two subfamilies on the basis of their structural differences and sequence homology.² GroEL from Escherichia coli, whose properties have been well studied,3., 4., 5. is a member of group I chaperonins, and the chaperonins from other bacteria and eukaryotic organelles belong to group I. On the other hand, group II chaperonins exist in archaea,6., 7. and the eukaryotic cytosol.8., 9. One of the major differences between the two groups is that group I chaperonins require a single ring cofactor (termed GroES in E. coli) for enclosing their cavities, while group II chaperonins have a built-in lid in their ring, and no homologue of the cofactor has been found. Although most group I chaperonins consist of a double homo-heptameric ring, group II chaperonins consist of a double hetero (or homo)-octameric or nonameric ring.¹⁰

One common feature between the groups is that each subunit has three domains: an equatorial domain that contains an ATPase site and is involved in inter-ring and intra-ring contacts; an apical domain for the recognition of the non-native protein; and an intermediate domain, which connects the other two domains via flexible hinges. The arrangement of these domains was revealed by the determination of the crystal structures.11., 12. The function of chaperonins as a folding machine for the substrate proteins accompanies an extensive conformational rearrangement of these domains during an ATP-dependent cycle. In the case of group I chaperonins, electron microscopic13., 14. and X-ray crystallographic studies15., 16., 17. have contributed to the identification of various conformers. Especially, the crystal structure of the GroEL₁₄–GroES₇–(ADP)₇ complex demonstrated that the lining of the cavity in the polypeptide acceptor state (open state) is hydrophobic, whereas it is hydrophilic in the protein-folding state (closed state). The ATP binding to the open state triggers the downward en bloc rotation of an intermediate domain and the upward motion of an apical domain, followed by GroES binding to the apical domain, which enlarges the upward apical domain movement and caps the ring.18., 19., 20. The binding site for the substrate protein is shared with the GroES-binding site,²¹ and therefore, GroES-binding results in substrate release into the cavity in parallel with sealing off the entrance hole.¹⁴ The ATP-dependent and GroES-dependent substrate binding/release cycle has been characterized by its positive and negative cooperativity within intra-ring and inter-ring associations, respectively.22., 23. Various conformers of group II chaperonins have been identified by electron microscopy.24., 25., 26., 27., 28., 29., 30. The crystal structure of the apical protrusion region from Thermoplasma acidophilum suggests that it plays roles as a built-in lid and a binding site for substrate-proteins.31., 32. Moreover, the crystal structure of the α₈β₈ hexadecamer from T. acidophilum was shown to adopt a tightly closed conformation, and its apical domain and inter-ring associates were shown to have large differences in structure compared to those of group I.

Several reports have postulated a sequential scheme of the ATP-dependent protein-folding cycle of group II chaperonins, based on biochemical investigation.33., 34., 35., 36., 37., 38., 39., 40. But the diversification of subunit composition and their biochemical characteristics have discouraged any possible generalization of the mechanism. Nonetheless, several general characteristics of group II chaperonins have been identified, and are considered to be crucial for the establishment of their molecular mechanism: (a) the binding of ATP induces significant conformational changes and generates an asymmetric double ring oligomer, in which apical domains undergo a clockwise rotation to close the entrance of the cavity;²⁵ (b) the nucleotide-free or ADP-bound form of CCT and the archaeal chaperonin are able to capture the denatured protein but unable to discharge it without a subsequent addition of ATP;37., 38. and (c) the binding of ADP-Pi to the archaeal chaperonin induces a conformational change from an open to a closed state.33., 35.

The group II chaperonin from the hyperthermophilic archaeum Thermococcus strain KS-1 is composed of α and β subunits. Because its biochemical properties, including refolding activities for the denatured protein, have been well studied, this chaperonin is suitable for structural study in order to clarify its protein-folding mechanism. Each subunit forms a recombinant homo-hexadecamer, and exhibits ATPase and denatured-protein refolding activities.38., 39. Moreover, it has been revealed that the expression of the α subunit is higher at lower temperature in vivo, whereas that of the β subunits is higher at higher temperature, and the thermostability of the β subunit is higher than that of the α subunit.41., 42. The recombinant hexadecamer of a mutant α subunit with G65C and I125T substitutions is deficient in denatured-protein refolding activity, although it is capable of ATP hydrolysis and denatured-protein capture.38., 39., 43. Of two residues, Gly65 and Ile125, the substitution of Gly65 to cysteine has been shown to be crucial for the defect of refolding activities.⁴³ Here, we report the crystal structures of α subunit hexadecamers (a mutant with two amino acid substitutions, G65C and I125T, a mutant with only G65C substitution, and a mutant with only I125T substitution) of the group II chaperonins from Thermococcus KS-1. Comparison with the known atomic structures of GroEL₁₄–GroES₇–(ADP)₇ indicated the effect of Cys65 on the local conformational change in an equatorial domain caused by the release of γ-phosphate.