Mechanism of nucleotide sensing in group II chaperonins

Abstract

Group II chaperonins mediate protein folding in an ATP-dependent manner in eukaryotes and archaea. The binding of ATP and subsequent hydrolysis promotes the closure of the multi-subunit rings where protein folding occurs. The mechanism by which local changes in the nucleotide-binding site are communicated between individual subunits is unknown. The crystal structure of the archaeal chaperonin from Methanococcus maripaludis in several nucleotides bound states reveals the local conformational changes associated with ATP hydrolysis. Residue Lys-161, which is extremely conserved among group II chaperonins, forms interactions with the γ-phosphate of ATP but shows a different orientation in the presence of ADP. The loss of the ATP γ-phosphate interaction with Lys-161 in the ADP state promotes a significant rearrangement of a loop consisting of residues 160-169. We propose that Lys-161 functions as an ATP sensor and that 160-169 constitutes a nucleotide-sensing loop (NSL) that monitors the presence of the γ-phosphate. Functional analysis using NSL mutants shows a significant decrease in ATPase activity, suggesting that the NSL is involved in timing of the protein folding cycle.

Figure 1

(A) Rearrangement of the NSL between the Cpn–AMP–PNP (magenta) and Cpn–ADP (cyan) states. The loop assumes an α-helical structure in the Cpn–ADP state, extending helix-7. (B) Change in the interactions between residues Gly-160 and Lys-161, located in NSL, and the phosphate groups of the AMP–PNP and ADP states. In the Cpn–AMP–PNP structure, the residues Gly-160 and Lys-161 make direct contacts with the α- and γ-phosphates, respectively, whereas these interactions are not present in Cpn–ADP structure. (C) Additional electrostatic interactions of the residue Lys-161 in the AMP–PNP state. The position assumed by Lys-161 in the Cpn–AMP–PNP state caused by the presence of γ-phosphate promotes an interaction of this residue via salt bridges with the two catalytic residues Asp-60 and Asp-386.

Figure 2

Sequence alignment of the NSL region of all group II chaperonins present in the Protein Data Bank (M. maripaludis; Thermococcus strain KS-1 subunit α; Thermoplasma acidophilum (Thermosome) subunits α and β; and the eight CCT subunits from Bos taurus). The two residues of the NSL of Cpn that interact directly with nucleotide are located at position 160 and 161 (regions coloured in green and blue, respectively). Gly-160 (Cpn sequence) is not highly conserved among the sequences; however, the interaction with nucleotide occurs via the main-chain carbonyl group and the side chain properties do not directly affect this contact. Interestingly, all of the sequences at position 160 have a residue with a short side chain (glycine, serine or threonine). A short side chain might confer the flexibility necessary for the NSL to assume the different conformations observed in the AMP–PNP and ADP states. In contrast with site 160, position 161 shows an extremely conserved lysine residue among the group II chaperonin sequences. The ATPase sensor mechanism proposed for Lys-161 appears to be a general mechanism for the entire class.

Figure 3

(A) Secondary structural rearrangements in the nucleotide-binding site related to NSL motion. The lower region of the nucleotide-binding site possesses a β-hairpin unit formatted from an antiparallel arrangement of strand-16 and strand-17. This β-hairpin structural motif undergoes a rigid body motion (indicated with the orange arrow) between the Cpn–AMP–PNP and Cpn–ADP states, except for the residue Asn-474, which shows a change in size chain direction (indicated with the red arrow). (B) A molecular surface representation of the Cpn–AMP–PNP (magenta) and Cpn–ADP (cyan) states. A rearrangement at the interface between the β-hairpin motif and the NSL promotes an opening of the nucleotide-binding site entrance in the Cpn–ADP state.

Figure 4

(A) PK digestion of Cpn-WT, Cpn-G160S, Cpn-K161A and Cpn-E164A mutants, analysed by SDS–PAGE followed by Coomassie blue staining. PK leads to full digestion of the open Cpn lids. Incubation with ATP–AlF_x locks the complex closed leading to complete PK protection of lids (compare ATP free and +ATP AlF_x lanes). (B) ATP hydrolysis by Cpn-WT, Cpn-G160S, Cpn-K161A and Cpn-E164A measured at 1 and 2 mM of α-[³²P]-ATP. (C) ATP hydrolysis by Cpn-G160S and the catalytic dead mutant Cpn-D386A measured at 1 mM α-[³²P]-ATP. The mean with the error bars representing standard error of the mean (s.e.m.) is shown. (D) Filter-binding assays for Cpn-WT, Cpn-G160S, Cpn-K161A and Cpn-E164A. Shown is the mean, with the error bars representing s.e.m. Data shown in (C, D) are the result of three independent repeats.

Figure 5

Overview of double ring Cpn–AMP–PNP and Cpn–ADP arrangements show that the NSL (coloured in magenta and cyan, respectively) is located between adjacent subunits. The interaction between Asp-45 and Arg-511 from adjacent subunit serves as hinge for intra-ring motion between the open and closed states of Cpn (Pereira et al, 2010; Zhang et al, 2010). The Cpn–AMP–PNP state demonstrates additional contacts between NSL residues and residues present on the equatorial domain of neighbouring subunits. The residue Glu-164 of NSL makes a salt bridge and a hydrogen bond with the residues Arg-511 and Gln-125. The residue Lys-167 makes an additional hydrogen bond with Gln-125. All these interactions are not present in Cpn–ADP state. Conformational changes in the NSL can be communicated to a lateral subunit by changes in intra-subunits contacts. ATPase assays using NSL residue mutant (Cpn-G160S, Cpn-K161A and Cpn-E164A) shows a significant difference on ATP hydrolysis activity compared with Cpn-WT demonstrating the influence of this region on enzyme activity. The hydrogen bonds are shown in broken lines and the distances are given in Angstroms.

Figure 6

(A) The AlF_x-binding site was identified using a cross-validated σ_A weighted electron density map (mF_o–DF_c) contoured at 7.0 σ (coloured in green). A 2mF_o–DF_c electron density map around the ADP nucleotide contoured at 1.5 σ is shown in blue. (B) The presence of AlF_x at the γ-phosphate-binding site promotes a similar structural arrangement in the NSL as observed for the Cpn–AMP–PNP state. The interactions between the oxygen atoms of the γ-phosphate of the AMP–PNP nucleotide are replaced by fluorine contacts in the Cpn–ADP–AlF_x structure. The interaction between Lys-161 and the γ-phosphate is conserved in the Cpn–ADP–AlF_x structure and allows the NSL to assume a similar conformation to Cpn–AMP–PNP.