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. 1994 Jan;3(1):22–29. doi: 10.1002/pro.5560030104

Hydrogen bond interactions of G proteins with the guanine ring moiety of guanine nucleotides.

G Weng 1, C X Chen 1, V Balogh-Nair 1, R Callender 1, D Manor 1
PMCID: PMC2142473  PMID: 8142894

Abstract

We have utilized Raman difference spectroscopy to investigate hydrogen bonding interactions of the guanine moiety in guanine nucleotides with the binding site of two G proteins, EF-Tu (elongation factor Tu from Escherichia coli) and the c-Harvey ras protein, p21 (the gene product of the human c-H-ras proto-oncogene). Raman spectra of proteins complexed with GDP (guanosine 5' diphosphate), IDP (inosine 5' diphosphate), 6-thio-GDP, and 6-18O-GDP were measured, and the various difference spectra were determined. These were compared to the difference spectra obtained in solution, revealing vibrational features of the nucleotide that are altered upon binding. Specifically, we observed significant frequency shifts in the vibrational modes associated with the 6-keto and 2-amino positions of the guanine group of GDP and IDP that result from hydrogen bonding interactions between these groups and the two proteins. These shifts are interpreted as being proportional to the local energy of interaction (delta H) between the two groups and protein residues at the nucleotide binding site. Consistent with the tight binding between the nucleotides and the two proteins, the shifts indicate that the enthalpic interactions are stronger between these two polar groups and protein than with water. In general, the spectral shifts provide a rationale for the stronger binding of GDP and IDP with p21 compared to EF-Tu. Despite the structural similarity of the binding sites of EF-Tu and p21, the strengths of the observed hydrogen bonds at the 6-keto and 2-amino positions vary substantially, by up to a factor of 2.(ABSTRACT TRUNCATED AT 250 WORDS)

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Selected References

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  1. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
  2. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: a conserved switch for diverse cell functions. Nature. 1990 Nov 8;348(6297):125–132. doi: 10.1038/348125a0. [DOI] [PubMed] [Google Scholar]
  3. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature. 1991 Jan 10;349(6305):117–127. doi: 10.1038/349117a0. [DOI] [PubMed] [Google Scholar]
  4. Breter H. J., Mertes H. The quantitative determination of metabolites of 6-mercaptopurine in biological materials. VII. Chemical synthesis by phosphorylation of 6-thioguanosine 5'-monophosphate, 5'-diphosphate and 5'-triphosphate, and their purification and identification by reversed-phase/ion-pair high-performance liquid chromatography and by various enzymatic assays. Biochim Biophys Acta. 1990 Feb 26;1033(2):124–132. doi: 10.1016/0304-4165(90)90002-e. [DOI] [PubMed] [Google Scholar]
  5. Delaria K., Jurnak F. Preparation of Escherichia coli elongation factor Tu-guanosine 5'-triphosphate analogs. Anal Biochem. 1989 Feb 15;177(1):188–193. doi: 10.1016/0003-2697(89)90038-9. [DOI] [PubMed] [Google Scholar]
  6. Deng H., Burgner J., Callender R. Raman spectroscopic studies of NAD coenzymes bound to malate dehydrogenases by difference techniques. Biochemistry. 1991 Sep 10;30(36):8804–8811. doi: 10.1021/bi00100a011. [DOI] [PubMed] [Google Scholar]
  7. Deng H., Zheng J., Burgner J., Callender R. Molecular properties of pyruvate bound to lactate dehydrogenase: a Raman spectroscopic study. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4484–4488. doi: 10.1073/pnas.86.12.4484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Deng H., Zheng J., Sloan D., Burgner J., Callender R. Classical Raman spectroscopic studies of NADH and NAD+ bound to lactate dehydrogenase by difference techniques. Biochemistry. 1989 Feb 21;28(4):1525–1533. doi: 10.1021/bi00430a016. [DOI] [PubMed] [Google Scholar]
  9. Eccleston J. F., Trentham D. R. The interaction of chromophoric nucleotides with subfragment 1 of myosin. Biochem J. 1977 Apr 1;163(1):15–29. doi: 10.1042/bj1630015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Grand R. J., Owen D. The biochemistry of ras p21. Biochem J. 1991 Nov 1;279(Pt 3):609–631. doi: 10.1042/bj2790609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hall A. The cellular functions of small GTP-binding proteins. Science. 1990 Aug 10;249(4969):635–640. doi: 10.1126/science.2116664. [DOI] [PubMed] [Google Scholar]
  12. John J., Sohmen R., Feuerstein J., Linke R., Wittinghofer A., Goody R. S. Kinetics of interaction of nucleotides with nucleotide-free H-ras p21. Biochemistry. 1990 Jun 26;29(25):6058–6065. doi: 10.1021/bi00477a025. [DOI] [PubMed] [Google Scholar]
  13. Kaziro Y., Itoh H., Kozasa T., Nakafuku M., Satoh T. Structure and function of signal-transducing GTP-binding proteins. Annu Rev Biochem. 1991;60:349–400. doi: 10.1146/annurev.bi.60.070191.002025. [DOI] [PubMed] [Google Scholar]
  14. Kjeldgaard M., Nyborg J. Refined structure of elongation factor EF-Tu from Escherichia coli. J Mol Biol. 1992 Feb 5;223(3):721–742. doi: 10.1016/0022-2836(92)90986-t. [DOI] [PubMed] [Google Scholar]
  15. Lane M. J., Thomas G. J., Jr Kinetics of hydrogen-deuterium exchange in guanosine 5'-monophosphate and guanosine 3':5'-monophosphate determined by laser-Raman spectroscopy. Biochemistry. 1979 Sep 4;18(18):3839–3846. doi: 10.1021/bi00585a002. [DOI] [PubMed] [Google Scholar]
  16. Manor D., Weng G. Z., Deng H., Cosloy S., Chen C. X., Balogh-Nair V., Delaria K., Jurnak F., Callender R. An isotope edited classical Raman difference spectroscopic study of the interactions of guanine nucleotides with elongation factor Tu and H-ras p21. Biochemistry. 1991 Nov 12;30(45):10914–10920. doi: 10.1021/bi00109a015. [DOI] [PubMed] [Google Scholar]
  17. Masters S. B., Stroud R. M., Bourne H. R. Family of G protein alpha chains: amphipathic analysis and predicted structure of functional domains. Protein Eng. 1986 Oct-Nov;1(1):47–54. [PubMed] [Google Scholar]
  18. Medeiros G. C., Thomas G. J., Jr On the tautomeric structure of inosine. Biochim Biophys Acta. 1971 Apr 29;238(1):1–4. doi: 10.1016/0005-2787(71)90002-5. [DOI] [PubMed] [Google Scholar]
  19. Miller D. L., Weissbach H. Elongation factor Tu and the aminoacyl-tRNA-EFTu-GTP complex. Methods Enzymol. 1974;30:219–232. doi: 10.1016/0076-6879(74)30024-9. [DOI] [PubMed] [Google Scholar]
  20. Pai E. F., Kabsch W., Krengel U., Holmes K. C., John J., Wittinghofer A. Structure of the guanine-nucleotide-binding domain of the Ha-ras oncogene product p21 in the triphosphate conformation. Nature. 1989 Sep 21;341(6239):209–214. doi: 10.1038/341209a0. [DOI] [PubMed] [Google Scholar]
  21. Pai E. F., Krengel U., Petsko G. A., Goody R. S., Kabsch W., Wittinghofer A. Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis. EMBO J. 1990 Aug;9(8):2351–2359. doi: 10.1002/j.1460-2075.1990.tb07409.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Psoda A., Shugar D. Spectral studies on tautomeric forms of inosine. Biochim Biophys Acta. 1971 Nov 19;247(4):507–513. doi: 10.1016/0005-2787(71)90686-1. [DOI] [PubMed] [Google Scholar]
  23. Sigal I. S., Gibbs J. B., D'Alonzo J. S., Temeles G. L., Wolanski B. S., Socher S. H., Scolnick E. M. Mutant ras-encoded proteins with altered nucleotide binding exert dominant biological effects. Proc Natl Acad Sci U S A. 1986 Feb;83(4):952–956. doi: 10.1073/pnas.83.4.952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Trahey M., McCormick F. A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. Science. 1987 Oct 23;238(4826):542–545. doi: 10.1126/science.2821624. [DOI] [PubMed] [Google Scholar]
  25. Wittinghofer A., Warren W. F., Leberman R. Structural requirements of the GDP binding site of elongation factor Tu. FEBS Lett. 1977 Mar 15;75(1):241–243. doi: 10.1016/0014-5793(77)80095-1. [DOI] [PubMed] [Google Scholar]
  26. Yue K. T., Yang J. P., Martin C. L., Lee S. K., Sloan D. L., Callender R. H. Raman study of reduced nicotinamide adenine dinucleotide bound to liver alcohol dehydrogenase. Biochemistry. 1984 Dec 18;23(26):6480–6483. doi: 10.1021/bi00321a032. [DOI] [PubMed] [Google Scholar]

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