P-loop containing nucleoside triphosphate hydrolase
Identifiers
Symbol?
InterProIPR027417

The Walker A and Walker B motifs are protein sequence motifs, known to have highly conserved three-dimensional structures. These were first reported in ATP-binding proteins by Walker and co-workers in 1982.[1]

Of the two motifs, the A motif is the main "P-loop" responsible for binding phosphate, while the B motif is a much less conserved downstream region. The P-loop is best known for its presence in ATP- and GTP-binding proteins, and is also found in a variety of proteins with phosphorylated substrates. Major lineages include:[2][3][4][5]

Walker A motif

Alignment of the H-Ras mutant A59G mutants in complex with GppNHp (green cartoon) and GDP (cyan cartoon). The P-loop main chain is shown in red, the Mg2+ ion as green sphere and the side chains of the amino acids K16 and S17 are shown as sticks.

Walker A motif, also known as the Walker loop, or P-loop, or phosphate-binding loop, is a motif in proteins that is associated with phosphate binding. The motif has the pattern G-x(4)-GK-[TS], where G, K, T and S denote glycine, lysine, threonine and serine residues respectively, and x denotes any amino acid. It is present in many ATP or GTP utilizing proteins; it is the β phosphate of the nucleotide that is bound. The lysine (K) residue in the Walker A motif, together with the main chain NH atoms, are crucial for nucleotide-binding.[6] It is a glycine-rich loop preceded by a beta strand and followed by an alpha helix; these features are typically part of an α/β domain with four strands sandwiched between two helices on each side. The phosphate groups of the nucleotide are also coordinated to a divalent cation such as a magnesium, calcium, or manganese(II) ion.[7]

Apart from the conserved lysine, a feature of the P-loop used in phosphate binding is a compound LRLR nest[8] comprising the four residues xxGK, as above, whose main chain atoms form a phosphate-sized concavity with the NH groups pointing inwards. The synthetic hexapeptide SGAGKT has been shown[9] to bind inorganic phosphate strongly; since such a short peptide does not form an alpha helix, this suggests that it is the nest, rather than being at the N-terminus of a helix, that is the main phosphate binding feature.

Upon nucleotide hydrolysis the loop does not significantly change the protein conformation, but stays bound to the remaining phosphate groups. Walker motif A-binding has been shown to cause structural changes in the bound nucleotide, along the line of the induced fit model of enzyme binding.

Similar folds

PTPs (protein tyrosine phosphatases) that catalyse the hydrolysis of an inorganic phosphate from a phosphotyrosine residue (the reverse of a tyrosine kinase reaction) contain a motif which folds into a P-loop-like structure with an arginine in the place of the conserved lysine. The conserved sequence of this motif is C-x(5)-R-[ST], where C and R denote cysteine and arginine residues respectively.[10]

Pyridoxal phosphate (PLP) utilizing enzymes such as cysteine synthase have also been said to resemble a P-loop.

A-loop

The A-loop (aromatic residue interacting with the adenine ring of ATP) refers to conserved aromatic amino acids, essential for ATP-binding, found in about 25 amino acids upstream of the Walker A motif in a subset of P-loop proteins.[11]

Walker B motif

Walker B motif is a motif in most P-loop proteins situated well downstream of the A-motif. The consensus sequence of this motif was reported to be [RK]-x(3)-G-x(3)-LhhhD, where R, K, G, L and D denote arginine, lysine, glycine, leucine and aspartic acid residues respectively, x represents any of the 20 standard amino acids and h denotes a hydrophobic amino acid.[1] This motif was changed to be hhhhDE, where E denotes a glutamate residue.[6] The aspartate and glutamate also form a part of the DEAD/DEAH motifs found in helicases. The aspartate residue co-ordinates magnesium ions, and the glutamate is essential for ATP hydrolysis.[6] There is considerable variability in the sequence of this motif, with the only invariant features being a negatively charged residue following a stretch of bulky, hydrophobic amino acids.[12]

Evolutionary connections

There is a hypothesis that the Walker A phosphate binding motif can be evolutionarily related to Rossman's fold phosphate binding motif because of the shared principles in the location of the binding loop between the first β-strand and α-helix in the αβα sandwich fold and positioning of the functionally important aspartate on the tip of the second β-strand. [13]

See also

References

  1. 1 2 Walker JE, Saraste M, Runswick MJ, Gay NJ (1982). "Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold". The EMBO Journal. 1 (8): 945–951. doi:10.1002/j.1460-2075.1982.tb01276.x. PMC 553140. PMID 6329717.
  2. ↑ Leipe DD, Wolf YI, Koonin EV, Aravind L (March 2002). "Classification and evolution of P-loop GTPases and related ATPases". Journal of Molecular Biology. 317 (1): 41–72. doi:10.1006/jmbi.2001.5378. PMID 11916378.
  3. ↑ Stryer L, Berg JM, Tymoczko JL (2002). Biochemistry. San Francisco: W.H. Freeman. ISBN 0-7167-4684-0.
  4. ↑ Ramakrishnan C, Dani VS, Ramasarma T (October 2002). "A conformational analysis of Walker motif A [GXXXXGKT (S)] in nucleotide-binding and other proteins". Protein Engineering. 15 (10): 783–798. doi:10.1093/protein/15.10.783. PMID 12468712.
  5. ↑ Saraste M, Sibbald PR, Wittinghofer A (November 1990). "The P-loop--a common motif in ATP- and GTP-binding proteins". Trends in Biochemical Sciences. 15 (11): 430–434. doi:10.1016/0968-0004(90)90281-f. PMID 2126155.
  6. 1 2 3 Hanson PI, Whiteheart SW (July 2005). "AAA+ proteins: have engine, will work". Nature Reviews. Molecular Cell Biology. 6 (7): 519–529. doi:10.1038/nrm1684. PMID 16072036. S2CID 27830342.
  7. ↑ Bugreev DV, Mazin AV (July 2004). "Ca2+ activates human homologous recombination protein Rad51 by modulating its ATPase activity". Proceedings of the National Academy of Sciences of the United States of America. 101 (27): 9988–9993. Bibcode:2004PNAS..101.9988B. doi:10.1073/pnas.0402105101. PMC 454202. PMID 15226506.
  8. ↑ Watson JD, Milner-White EJ (January 2002). "A novel main-chain anion-binding site in proteins: the nest. A particular combination of phi,psi values in successive residues gives rise to anion-binding sites that occur commonly and are found often at functionally important regions". Journal of Molecular Biology. 315 (2): 171–182. doi:10.1006/jmbi.2001.5227. PMID 11779237.
  9. ↑ Bianchi A, Giorgi C, Ruzza P, Toniolo C, Milner-White EJ (May 2012). "A synthetic hexapeptide designed to resemble a proteinaceous P-loop nest is shown to bind inorganic phosphate". Proteins. 80 (5): 1418–1424. doi:10.1002/prot.24038. PMID 22275093. S2CID 5401588.
  10. ↑ Zhang M, Stauffacher CV, Lin D, Van Etten RL (August 1998). "Crystal structure of a human low molecular weight phosphotyrosyl phosphatase. Implications for substrate specificity". The Journal of Biological Chemistry. 273 (34): 21714–21720. doi:10.1074/jbc.273.34.21714. PMID 9705307.
  11. ↑ Ambudkar SV, Kim IW, Xia D, Sauna ZE (February 2006). "The A-loop, a novel conserved aromatic acid subdomain upstream of the Walker A motif in ABC transporters, is critical for ATP binding". FEBS Letters. 580 (4): 1049–1055. doi:10.1016/j.febslet.2005.12.051. PMID 16412422.
  12. ↑ Koonin EV (June 1993). "A common set of conserved motifs in a vast variety of putative nucleic acid-dependent ATPases including MCM proteins involved in the initiation of eukaryotic DNA replication". Nucleic Acids Research. 21 (11): 2541–2547. doi:10.1093/nar/21.11.2541. PMC 309579. PMID 8332451.
  13. ↑ Longo LM, Jabłońska J, Vyas P, Kanade M, Kolodny R, Ben-Tal N, Tawfik DS (December 2020). Deane CM, Boudker O (eds.). "On the emergence of P-Loop NTPase and Rossmann enzymes from a Beta-Alpha-Beta ancestral fragment". eLife. 9: e64415. doi:10.7554/eLife.64415. PMC 7758060. PMID 33295875.
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