Review
GHKL, an emergent ATPase/kinase superfamily

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Abstract

An interesting recent development is the recognition of a novel ATP-binding superfamily that includes diverse protein families such as DNA topoisomerase II, molecular chaperones Hsp90, DNA-mismatch-repair enzymes MutL and histidine kinases. The most singular unifying feature of this superfamily is the unconventional Bergerat ATP-binding fold. The far-reaching significance of this commonality is still in the process of being explored.

Section snippets

Signature sequences

The four conserved motifs common to the GHKL superfamily of proteins are Motif I (uubEuuaNouDA), Motif II (uxuxDNGxGuxbaauxxuu), Motif III (uGxxGxouxSxxxuoxbuTuxT) and Motif IV (TxnGT). The first three motifs correspond to the N, G1 and G2 conserved boxes in histidine kinases8 (u, conserved bulky hydrophobic residues; o, small residues; b, basic residues; a, acidic residues)1, 9, 10. Bergerat et al.10 first proposed that these three motifs define a new ATP-binding fold, henceforth referred to

The family members

Histidine kinases are to prokaryotes what serine/threonine and tyrosine kinases are to eukaryotes: signal sensors, processors and transducers8. Recently, they have been discovered also in lower eukaryotes15. Histidine kinases usually function paired with a response-regulator protein, and signaling circuits that involve such a conjugate pair are called two-component systems or His–Asp phosphorelay systems16, 17. Typically, a histidine kinase uses ATP to autophosphorylate a conserved histidine.

Topological similarity of the ATP-binding domains

A high degree of resemblance exists among the nucleotide-binding domains of the four family members (Fig. 1a,b)1, 2, 6, 7. The similarity is highest between those of MutL and GyrB1, and differences observed are minor except for some transpositions. For example, a β strand and an α helix N-terminal to the Bergerat ATP-binding fold in the histidine kinases EnvZ and CheA are transposed to the C-terminal end of the core region in GyrB, Hsp90 and MutL. It is interesting that although there is little

The novel ATP-binding Bergerat fold

A comparison of the solved structures of the nucleotide-complexed ATP-binding domains of the GHKL superfamily1, 2, 4, 6, 11, 12, 13 has identified the core elements of the Bergerat ATP-binding fold. It is an α/β sandwich consisting of a four-stranded mixed β sheet and three α helices (Fig. 2a). On the helix face, helices α1 and α3 are amphipathic and pack parallel to the β strands. Helix α2 is oriented transversely with respect to the other two helices, and together they form the lining of the

Distinctive features of the ATP-binding domains of individual members

The most significant variations observed within the Bergerat ATP-binding core localize to the ATP-lid that has a strategic location with regard to the ATP-binding pocket. The composition and conformation of the ATP-lid clearly distinguishes the four different classes of proteins in the GHKL superfamily (Fig. 3a,b). The F-box motif that occurs between helix α2 and the G2 box is characteristic for histidine kinases (Fig. 3a).

Approximately 20 residues that precede the G2 box are conserved in the

Roles of residues from adjacent domains in ATP-binding and catalysis

The conserved glutamate in the N box of the members of the ATPase superfamily serves as a general base for water activation in ATP hydrolysis1, 4, 5, 28. In the MutL ADPnP-bound crystal structure, this glutamate (Glu29) is hydrogen bonded to a water molecule that is in-line for a nucleophilic attack on the γ phosphate. The water molecule is also hydrogen bonded with a conserved lysine (Lys307). Interestingly, the latter residue is on a highly conserved loop (L3) in the MutL family, which occurs

ATP binding and hydrolysis

Members of the GHKL superfamily share a common theme in their domain organization: a highly conserved ATP-binding domain is linked to a comparatively divergent dimerization domain. In members of the GHL superfamily the ATP-binding domain is N-terminal to the dimerization domain, whereas in histidine kinases the arrangement is reversed. In the Hsp90 chaperones a charged linker region of variable length connects the two conserved domains.

The eukaryotic enzymes of all four families exist as

Evolution

Gyrase, Hsp90, MutL and histidine kinases perform very different functions in the cell. These diverse protein families were not considered to be even remotely related to each other. Yet, with the recent resolution of their structures, hidden relationships have become unveiled. The Bergerat fold represents the major connecting link. Another unifying feature is the presence of a rare left-handed β–α–β connectivity. In the case of the ATPases, this connection exists in the domain adjoining the

Conclusion

Gyrase, Hsp90, histidine kinase and MutL families have all integrated a Bergerat ATP-binding fold within their respective structures. They utilize the conformational energy of ATP-binding or the energy of its hydrolysis, or both, to drive their various functions in the cell. In view of the diverse nature of proteins in which this fold has been found, it seems unlikely that the ATPase/kinase superfamily is going to be restricted to its current four members. Indeed, this ATP module is also

Acknowledgements

We thank Ling Qin for helpful discussions and careful reading of the manuscript. We thank W. Yang for granting us permission to reproduce Fig. 1a. We are grateful to T. Tanaka for his generous help in creating Fig. 1b. This work was supported by grant GM19043 from the NIH.

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