Molecular Mechanism of Amino Acid Discrimination for the Editing Reaction of E.coli Leucyl-tRNA Synthetase
Keun Woo Lee;
Certain tRNA synthetases have developed highly accurate molecular machinery to discriminate their cognate amino acids. Those aaRSs achieve their goal via editing reaction in the Connective Polypeptide 1 (CP1). Recently mutagenesis studies have revealed the critical importance of residues in the CP1 domain for editing activity and X-ray structures have shown binding mode of noncognate amino acids in the editing domain. To pursue molecular mechanism for amino acid discrimination, molecular modeling studies were performed. Our results suggest that aaRS bind the noncognate amino acid more tightly than the cognate one. Finally, by comparing binding conformations of the amino acids in three systems, the amino acid binding mode was elucidated and a discrimination mechanism proposed. The results strongly reveal that the conserved threonines are responsible for amino acid discrimination. This is achieved through side chain interactions between T252 and T247/T248 as well as between those threonines and the incoming amino acids.
Carter Jr., C. W.,"Cognition, mechanism, and evolutionary relationships
in aminoacyl-tRNAsynthetases",Annu. Rev. Biochem,vol. 62, pp.
Eldred, E. W., Schimmel, P.,"Rapid deacylation by isoleucyl transfer
ribonucleic acid synthetase of isoleucine-specific transfer ribonucleic acid
aminoacylated with valine", J. Biol. Chem.,vol. 247, pp. 2961-2964,
Fersht, A. R., Kaethner, M. M., "Mechanism of aminoacylation of tRNA.
Proof of the aminoacyladenylate pathway for the isoleucyl- and
tyrosyl-tRNAsynthetases from Escherichia coli K12", Biochemistry,vol.
15, pp. 818-823,1976.
Giege, R., Sissler, M., Florentz, C.,"Universal rules and idiosyncratic
features in tRNA identity",Nucleic Acids Res,vol. 26, pp.
Hale, S. P., Schimmel, P.,"Protein synthesis editing by a DNA
aptamer",Proc. Natl. Acad. Sci. USA,vol. 93, pp. 2755-2758, 1996.
 Fersht, A. R., Kaethner, M. M.,"Enzyme hyperspecificity. Rejection of
threonine by the valyltRNAsynthetase by misacylation and hydrolytic
editing",Biochemistry,vol. 15, pp. 3342-3346, 1976.
 Fersht, A. R.,"Editing mechanisms in protein synthesis. Rejection of
valine by the isoleucyl-tRNAsynthetase",Biochemistry,vol. 16, pp.
 Fersht, A. R.,"Enzyme Structure and Mechanism", Freeman, San
 Fersht, A. R., Dingwall C.,"Establishing the misacylation/deacylation of
the tRNA pathway for the editing mechanism of prokaryotic and
eukaryotic valyl-tRNAsynthetases",Biochemistry,vol. 18, pp. 1238-1245,
 Fersht, A. R.,"Sieves in sequence",Science,vol. 280, pp. 541, 1998.
 Nureki, O., Vassylyev D. G., Tateno, M., Shimada, A., Nakama, T., Fukai,
S., Konno, M., Hendrickson, T.L., Schimmel, P., Yokoyama, S.,"Enzyme
structure with two catalytic sites for double-sieve selection of
substrate",Science,vol. 280, pp. 578-582, 1998.
 Silvian, L. F., Wang, J., Steitz, T. A.,"Insights into editing from an
ile-tRNAsynthetase structure with tRNAile and mupirocin",Science,vol.
285, pp. 1074-1077, 1999.
 Fukai, S., Nureki, O., Sekine, S., Shimada, A., Tao, J., Vassylyev, D. G.,
Yokoyama, S.,"Structural basis for double-sieve discrimination of L-Val
from L-Ile and L-threonine by the complex of tRNAVal and
valyl-tRNAsynthetase",Cell,vol. 103, pp. 793-893, 2000.
 Cusack, S., Yaremchuk, A., Tukalo, M.,"The 2 Å crystal structure of
leucyl-tRNAsynthetase and its complex with a leucyl-adenylate
analogue",EMBO J.,vol. 19, pp. 2351-2631, 2000.
 Lincecum Jr., T. L., Tukalo, M,, Yaremchuk, A., Mursinna, R. S.,
Williams, A. M., Sproat, B. S., Van Den Eynde, W., Link, A., Van
Calenbergh, S., Grotli, M., Martinis, S. A., Cusack, S.,"Structural and
mechanistic basis of pre- and posttransfer editing by
leucyl-tRNAsynthetase",Mol. Cell,vol. 11, pp. 951-963, 2003.
 Bishop, A. C., Nomanbhoy, T. K., Schimmel, P., "locking site-to-site
translocation of a misactivated amino acid by mutation of a class I
tRNAsynthetase", Proc. Natl. Acad. Sci. USA,vol. 99, pp. 585-590, 2002.
 Hendrickson, T. L., Nomanbhoy, T. K., Schimmel, P.,"Errors from
selective disruption of the editing center in a
tRNAsynthetase",Biochemistry,vol. 39, pp. 8180-8186,2000.
 Mursinna, R. S., Lincecum, T. L., Martinis, S. A.,"A conserved threonine
within Escherichia coli leucyl-tRNAsynthetase prevents hydrolytic
editing of leucyl-tRNALeu",Biochemistry,vol. 40, pp. 5376-5381, 2001.
 Mursinna, R. S., Martinis, S. A., "Rational design to block amino acid
editing of a tRNAsynthetase",J. Am. Chem. Soc.,vol. 124, pp. 7286-7287,
 Mursinna, R. S., Lee, K. W., Briggs, J. M., Martinis, S. A.,"Molecular
dissection of a critical specificity determinant within the amino acid
editing domain of leucyl-tRNAsynthetase",Biochemistry,vol. 43, pp.
 Lee, K. W., Briggs, J. M.,"Molecular modeling study of the editing active
site of Escherichia coli leucyl-tRNAsynthetase: two amino acid binding
sites in the editing domain",Proteins: Struct. Funct. Bioinformatics,vol.
54,pp. 693-704, 2004.
 Brooks, B. R., Bruccoleri, R. E., Olafson, B. D., States, D. J.,
Swaminathan, S., Karplus, M.,"CHARMM: A program for
macromolecular energy, minimisation and dynamics calculations",J.
Comp. Chem.,vol. 4, pp. 187-217, 1983.
 Kale, L., Skeel, R., Bhandarkar, M., Brunner, R., Gursoy, A., Krawetz, N.,
Phillips, J., Shinozaki, A., Varadarajan, K., Schulten, K.,"NAMD2:
Greater Scalability for Parallel Molecular Dynamics",J. Comp. Phys.,vol.
151, pp. 283-312,1999.
 Berendsen, H. J. C., Postma, J. P. M., van Gunsteren, W. F., DiNola, A.,
Haak, J. R.,"Molecular dynamics with coupling to an external bath",J.
Chem. Phys.,vol. 81, pp. 3684-3690,1984.
 Amadei, A., Linssen, A. B. M., Berendsen, H. J. C., "Essential dynamics
of proteins",Proteins: Struct. Funct. Genetics,vol. 17, pp. 412-425,1993.
 Vriend, G.,"WHAT IF: A molecular modeling and drug design
program",J. Mol. Graph.,vol. 8, pp. 52-56, 1990.
 Aqvist, J., Medina, C., Samuel, J. E.,"A new method for predicting
binding affinity in computer-aided drug design",Protein Eng.,vol. 7, pp.
 Hansson, T., Åqvist, J.,"Estimation of binding free energies for HIV
proteinase inhibitors by molecular dynamics simulations",Protein
Eng.,vol. 8, pp. 1137-1144,1995.
 Aqvist, J.,"Calculation of absolute binding free energies for charged
ligands and effects of long-range electrostatic interactions",J. Comp.
Chem.,vol. 17, pp. 1587-1597,1996.
 Srinivasan, J., Cheatham, T. E., Cieplak, P., Kollman, P. A., Case, D.
A.,"Continuum Solvent Studies of the Stability of DNA, RNA, and
Phosphoramidate-DNA Helices",J. Am. Chem. Soc.,vol. 120, pp.
 Kollman, P. A., Massova, I., Reyes, C., Kuhn, B., Huo, S., Chong, L., Lee,
M., Lee, T., Duan, Y., Wang, W., Donini, O., Cieplak, P., Srinivasan, J.,
Case, D. A., Cheatham, T. E. III.,"Calculating structures and free energies
of complex molecules: combining molecular mechanics and continuum
models",Acc. Chem. Res.,vol. 33, pp. 889-897, 2000.
Loftfield, R. B.,"The Frequency of errors in protein
biosynthesis",Biochem. J.,vol. 89, pp. 82-92, 1963.
Martinis, S.A., and Schimmel, P.,"Escherichia coli and Salmonella
Cellular and Molecular Biology", 2nd Ed., ASM, Neidhardt FC,
Washington DC, 1996.
Pauling, L., Festschrift f├╝r Prof. Dr. Arthur Stoll, BirkhauserVerlag, Basel
Schmidt, E., Schimmel, P.,"Insights into editing from an
ile-tRNAsynthetase structure with tRNAile and mupirocin",Science,vol.
285, pp. 1074-1077, 1994.