Single nucleotide polymorphisms (SNPs) are the most common source of human genetic variation, occurring at a frequency of 1 in every 1000 nucleotide bases of the 3 billion bases human genome. SNPs can be intronic or exonic and can thus affect mRNA transcription, structure, splicing and translation, protein expression, structure, enzymatic activity and stability. SNPs are linked large number of genetic defects and also associated with many diseases. In this study we analyzed structural and dynamical effects of some of the important SNPs from Arylamine N-acetyltransferase 2 (NAT2) and the transcription factor pituitary homeobox protein 2 (PITX2).
NAT2 is an important catalytic enzyme that metabolizes the carcinogenic arylamines, hydrazine drugs and chemicals. This enzyme is highly polymorphic in different human populations. Several polymorphisms of NAT2, including the single amino acid substitutions R64Q, I114T, D122N, L137F, Q145P, R197Q, and G286E, are classified as slow acetylators, whereas the wild-type NAT2 is classified as a fast acetylator. The slow acetylators are often associated with drug toxicity and efficacy as well as cancer susceptibility. The biological functions of these 7 mutations have previously been characterized, but the structural basis behind the reduced catalytic activity and reduced protein level is not clear. We performed multiple molecular dynamics simulations of these mutants as well as NAT2 to investigate the structural and dynamical effects throughout the protein structure, specifically the catalytic triad, cofactor binding site, and the substrate binding pocket. None of these mutations induced unfolding; instead, their effects were confined to the inter-domain, domain 3 and 17-residue insert region, where the flexibility was significantly reduced relative to the wild-type. Structural effects of these mutations propagate through space and cause a change in catalytic triad conformation, cofactor binding site, substrate binding pocket size/shape and electrostatic potential. Our results showed that the dynamical properties of all the mutant structures, especially in inter-domain, domain 3 and 17-residue insert region were affected in the same manner. Similarly, the electrostatic potential of all the mutants were altered and also the functionally important regions such as catalytic triad, cofactor binding site, and substrate binding pocket adopted different orientation and/or conformation relative to the wild-type that may affect the functions of the mutants. Overall, our study may provide the structural basis for reduced catalytic activity and protein level, as was experimentally observed for these polymorphisms.
PITX2 is involved in genetic control of development. Mutations in PITX2, most in the homeodomain, cause the autosomal-dominant disorder Rieger syndrome. The mutants L16Q, K50E and R53P destabilize the structure and disrupt DNA-binding activity. The biological functions of these mutants have been characterized but not the structural basis behind the loss of DNA-binding activity. We performed multiple molecular dynamics simulations at 37º C to investigate the structural and dynamic effects of these mutants. Compared with the wild type (WT), the L16Q mutant induces a kink in the alpha 3 helix, which is stabilized by the hydrogen bond of Q21-R59. The disruption in backbone hydrogen bonds of V47-N51 and W48-R52 leads to a kink formation in the α3 helix of K50E. The R53P mutant alters the relative orientation of helices, which is apparently stabilized by the formation of new hydrogen bonds of T38-Q11, T38-Q12, T38-R2, N39-R2, L40-Q1, L40-R2, and T41-Q4. The hydrophobic core residues F8, L13, L40 and V45 change their positions in all mutants to break the hydrophobic core. Thus, changes in helical orientations and hydrophobic core cause rearrangement of the DNA-binding surface and disrupt DNA-binding activity in the mutants. The structural and molecular dynamics properties of 3 PITX2 homeodomain mutants differ from those of the WT, especially in formation of a kink in the recognition helix, change in the packing of helices and disruption of the hydrophobic core. Overall, our study may provide the structural basis for reduced catalytic activity and protein level, as was experimentally observed for these polymorphisms in NAT2 and the structural basis for the loss of DNA-binding activity for these polymorphisms in PITX2 homeodomain.

1. Watson, J. D., and Crick, F. H. (1953) Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid, Nature 171, 737-738.
2. Goodsell, D. S. (2011) Atomic evidence: the foundations of structural molecular biology, Sci Prog 94, 414-430.
3. Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H., Shindyalov, I. N., and Bourne, P. E. (2000) The Protein Data Bank, Nucleic Acids Res 28, 235-242.
4. Karplus, M., and Kuriyan, J. (2005) Molecular dynamics and protein function, Proc Natl Acad Sci U S A 102, 6679-6685.
5. Weiss, S. (2000) Measuring conformational dynamics of biomolecules by single molecule fluorescence spectroscopy, Nat Struct Biol 7, 724-729.
6. Schuler, B. (2005) Single-molecule fluorescence spectroscopy of protein folding, Chemphyschem 6, 1206-1220.
7. Besteman, K., Hage, S., Dekker, N. H., and Lemay, S. G. (2007) Role of tension and twist in single-molecule DNA condensation, Phys Rev Lett 98, 058103.
8. Eisenmesser, E. Z., Bosco, D. A., Akke, M., and Kern, D. (2002) Enzyme dynamics during catalysis, Science 295, 1520-1523.
9. MP Allen, D. T. (1987) Computer Simulation of Liquids Oxford Science Publications, Oxford, UK.
10. McCammon, J. A., Gelin, B. R., and Karplus, M. (1977) Dynamics of folded proteins, Nature 267, 585-590.
11. Simmerling, C., Strockbine, B., and Roitberg, A. E. (2002) All-atom structure prediction and folding simulations of a stable protein, J Am Chem Soc 124, 11258-11259.
12. Freddolino, P. L., Liu, F., Gruebele, M., and Schulten, K. (2008) Ten-microsecond molecular dynamics simulation of a fast-folding WW domain, Biophys J 94, L75-77.
13. Snow, C. D., Zagrovic, B., and Pande, V. S. (2002) The Trp cage: folding kinetics and unfolded state topology via molecular dynamics simulations, J Am Chem Soc 124, 14548-14549.
14. Legge, F. S., Budi, A., Treutlein, H., and Yarovsky, I. (2006) Protein flexibility: multiple molecular dynamics simulations of insulin chain B, Biophys Chem 119, 146-157.
15. Rueda, M., Ferrer-Costa, C., Meyer, T., Perez, A., Camps, J., Hospital, A., Gelpi, J. L., and Orozco, M. (2007) A consensus view of protein dynamics, Proc Natl Acad Sci U S A 104, 796-801.
16. Rutherford, K., and Daggett, V. (2009) The V119I polymorphism in protein L-isoaspartate O-methyltransferase alters the substrate-binding interface, Protein Eng Des Sel 22, 713-721.
17. Rutherford, K., and Daggett, V. (2009) A hotspot of inactivation: The A22S and V108M polymorphisms individually destabilize the active site structure of catechol O-methyltransferase, Biochemistry 48, 6450-6460.
18. Achary, M. S., Reddy, A. B., Chakrabarti, S., Panicker, S. G., Mandal, A. K., Ahmed, N., Balasubramanian, D., Hasnain, S. E., and Nagarajaram, H. A. (2006) Disease-causing mutations in proteins: structural analysis of the CYP1B1 mutations causing primary congenital glaucoma in humans, Biophys J 91, 4329-4339.
19. Domene, C., and Illingworth, C. J. (2012) Effects of point mutations in pVHL on the binding of HIF-1alpha, Proteins 80, 733-746.
20. Bockmann, R. A., and Grubmuller, H. (2002) Nanoseconds molecular dynamics simulation of primary mechanical energy transfer steps in F1-ATP synthase, Nat Struct Biol 9, 198-202.
21. de Groot, B. L., Vriend, G., and Berendsen, H. J. (1999) Conformational changes in the chaperonin GroEL: new insights into the allosteric mechanism, J Mol Biol 286, 1241-1249.
22. Frauenfelder, H., Sligar, S. G., and Wolynes, P. G. (1991) The energy landscapes and motions of proteins, Science 254, 1598-1603.
23. Jung-Hwan Oh, B.-W. Y. (2008) J Korean Assoc Oral Maxillofac Surg 34, 450-455.
24. Krawczak, M., Ball, E. V., and Cooper, D. N. (1998) Neighboring-nucleotide effects on the rates of germ-line single-base-pair substitution in human genes, Am J Hum Genet 63, 474-488.
25. Stone, E. A., and Sidow, A. (2005) Physicochemical constraint violation by missense substitutions mediates impairment of protein function and disease severity, Genome Res 15, 978-986.
26. Rutherford, K., Parson, W. W., and Daggett, V. (2008) The histamine N-methyltransferase T105I polymorphism affects active site structure and dynamics, Biochemistry 47, 893-901.
27. Rutherford, K., and Daggett, V. (2008) Four human thiopurine s-methyltransferase alleles severely affect protein structure and dynamics, J Mol Biol 379, 803-814.
28. Anderson, P. C., and Daggett, V. (2008) Molecular basis for the structural instability of human DJ-1 induced by the L166P mutation associated with Parkinson's disease, Biochemistry 47, 9380-9393.
29. Cuesta-Lopez, S., Falo, F., and Sancho, J. (2007) Computational diagnosis of protein conformational diseases: short molecular dynamics simulations reveal a fast unfolding of r-LDL mutants that cause familial hypercholesterolemia, Proteins 66, 87-95.
30. Chen, W., van der Kamp, M. W., and Daggett, V. (2010) Diverse effects on the native beta-sheet of the human prion protein due to disease-associated mutations, Biochemistry 49, 9874-9881.
31. van der Kamp, M. W., and Daggett, V. (2010) Pathogenic mutations in the hydrophobic core of the human prion protein can promote structural instability and misfolding, J Mol Biol 404, 732-748.
32. Duboule, D. (1994) Guidebook to the homeodomain genes., Oxford University Press, Oxford.
33. Semenza, G. (1989) Homeodomain revisited: a lesson from disease-causing mutations, Oxford University Press; Oxford.
34. Engelkamp, D., and van Heyningen, V. (1996) Transcription factors in disease, Curr Opin Genet Dev 6, 334-342.
35. Jimenez-Sanchez, G., Childs, B., and Valle, D. (2001) Human disease genes, Nature 409, 853-855.
36. Levine, M., and Hoey, T. (1988) Homeobox proteins as sequence-specific transcription factors, Cell 55, 537-540.
37. Kaufman, T. C., Seeger, M. A., and Olsen, G. (1990) Molecular and genetic organization of the antennapedia gene complex of Drosophila melanogaster, Adv Genet 27, 309-362.
38. Gehring, W. J., Affolter, M., and Burglin, T. (1994) Homeodomain proteins, Annu Rev Biochem 63, 487-526.
39. Muragaki, Y., Mundlos, S., Upton, J., and Olsen, B. R. (1996) Altered growth and branching patterns in synpolydactyly caused by mutations in HOXD13, Science 272, 548-551.
40. Semenza, G. (1998) Homeodomain proteins. In: Transcription factors and human diseases, Oxford University Press.
41. Coulier, F., Burtey, S., Chaffanet, M., Birg, F., and Birnbaum, D. (2000) Ancestrally-duplicated paraHOX gene clusters in humans, Int J Oncol 17, 439-444.
42. D'Elia, A. V., Tell, G., Paron, I., Pellizzari, L., Lonigro, R., and Damante, G. (2001) Missense mutations of human homeoboxes: A review, Hum Mutat 18, 361-374.
43. Goodman, F. R., and Scambler, P. J. (2001) Human HOX gene mutations, Clin Genet 59, 1-11.
44. Zhao, Y., and Westphal, H. (2002) Homeobox genes and human genetic disorders, Curr Mol Med 2, 13-23.
45. Wang, Z., and Moult, J. (2001) SNPs, protein structure, and disease, Hum Mutat 17, 263-270.
46. W. F. van Gunsteren, P. K. W., and A. J. Wilkinson (1997) Computer Simulation of Biomolecular Systems, theoretical and experimental applications, Vol. 3, Kluwer/ESCOM.
47. Leach., A. R. (1996) Molecular Modelling: Principles and Applications., Addison Wesley Longman Limited, Essex.
48. Jensen, F. (2001) Computational Chemistry., John Wiley and Sons Ltd, Sussex, England.
49. W. F. van Gunsteren, H. J. C. B. (1990) Computer simulation of molecular dynamics: Methodology, applications, and perspectives in chemistry, Angew. Chem. Int. Ed. Engl 29, 992–1023.
50. Hansson, T., Oostenbrink, C., and van Gunsteren, W. (2002) Molecular dynamics simulations, Curr Opin Struct Biol 12, 190-196.
51. M.E. Tuckerman, G. J. M. (2000) Understanding modern molecular dynamics: Techniques and applications., J. Phys. Chem. B 59–178.
52. van Gunsteren, W. F., Bakowies, D., Baron, R., Chandrasekhar, I., Christen, M., Daura, X., Gee, P., Geerke, D. P., Glattli, A., Hunenberger, P. H., Kastenholz, M. A., Oostenbrink, C., Schenk, M., Trzesniak, D., van der Vegt, N. F., and Yu, H. B. (2006) Biomolecular modeling: Goals, problems, perspectives, Angew Chem Int Ed Engl 45, 4064-4092.
53. M. Born, R. O. (1927) Zur Quantentheorie der Molekeln, Ann. Phys. Leipzig.
54. McCammon, J. A., Wolynes, P. G., and Karplus, M. (1979) Picosecond dynamics of tyrosine side chains in proteins, Biochemistry 18, 927-942.
55. Lennard-Jones, J. E. (1925) On the forces between atoms and ions., Proc. Roy. Soc. London Ser. A - Math. and Phys. Sc. 4, 584-597.
56. B. R. Brooks, R. E. B., B. D. Olafson, D. J. States, S. Swaminathan, and M. Karplus. (1983) CHARMM: A program for macromolecular energy, minimization, and dynamics calculations, J. Comp. Chem 4, 187-217.
57. W. F. Gunsteren, H. J. C. B. (1987) Gromos-87 manual, BIOMOS b.v., Nijenborgh 4,9747 AG Groningen, The Netherlands.
58. S. J. Weiner, P. A. K., D. T. Nguyen, D. A. Case. (1986) An all atom force field for simulations of proteins and nucleic acids, J. Comp. Chem 7, 230-252.
59. W. L. Jorgensen, J. T.-R. (1988) The OPLS potential functions for proteins- energy minimizations for crystals of cyclic peptides and crambin, J. Am. Chem. Soc. 110, 1657-1666.
60. R. Lavery, K. Z., H. Sklenar. (1995) JUMNA (junction minimization of nucleicacids), Comput. Phys. Commun. 91, 135-158.
61. Vergoten, G., Mazur, I., Lagant, P., Michalski, J. C., and Zanetta, J. P. (2003) The SPASIBA force field as an essential tool for studying the structure and dynamics of saccharides, Biochimie 85, 65-73.
62. N. L. Allinger, Y. H. Y., J. H. Lii. (1989) Molecular mechanics. the MM3 force fields for hydrocarbons, J. Am. Chem. Soc. 111, 8551–8566.
63. Van Der Spoel, D., Lindahl, E., Hess, B., Groenhof, G., Mark, A. E., and Berendsen, H. J. (2005) GROMACS: fast, flexible, and free, J Comput Chem 26, 1701-1718.
64. H.J.C. Berendsen, D. v. d. S., R. van Drunen. (1995) Gromacs: A message-passing parallel molecular dynamics implementation, Comp. Phys. Comm. 91, 43-56.
65. Hockney, R. W. (1970) The potential calculation and some applications, Meth Comput. Phys. 9, 136-211.
66. M. P. Allen, D. J. T. (1987) computer simulation of liquids, Clarendon press, Oxford.
67. Verlet, L. (1967) Computer “experiments” on classical fluids.i.thermodynamic properties of lennard-jones molecules, Phys. Rev. 159, 98-103.
68. Gear, C. W. (1971) Numerical initial value problems in ordinary differential equations, Prentice Hall, Englewood Cliffs, NJ.
69. Beeman, D. (1976) Some multistep methods for use in molecular dynamics calculations, J.Comput. Phys. Commun. 20, 130-139.
70. D. Janezic, B. O. (1993) Implicit runge-kutta methods for molecular dynamics integration, J. Chem. Inf. Comput. Sci. 33, 252-257.
71. Perutz, M. F. (1978) Electrostatic effects in proteins, Science 201, 1187-1191.
72. Orozco, M., and Luque, F. J. (2001) Theoretical methods for the description of the solvent effect in biomolecular systems. (Chem. Rev. 2000, 100, 4187-4226. Published on the web oct 21, 2000.), Chem Rev 101, 203-204.
73. W. L. Jorgensen, J. C., J. D. Madura, R.W. Impey, M. L. Klein. (1983) Comparison of simple potential functions for simulating liquid water, J. Chem. Phys. 79, 926-935.
74. M. Berkowitz, J. A. M. (1982) Molecular dynamics with stochastic boundary conditions, Chem. Phys. Lett. 90, 215-217.
75. W. Weber, P. H. H. u., J.A. McCammon. (2000) Molecular dynamics simulations of a polyalanine octapeptide under Ewald boundary conditions: Influenceof artificial periodicity on peptide conformation, J. Phys. Chem. B 104, 3668-3675.
76. H. J. C. Berendsen, J. P. M. P., A. DiNola, J. R. Haak. (1984) Molecular dynamics with coupling to an external bath, J. Chem. Phys. 81, 3684–3690.
77. Saito, M. (1994) Molecular dynamics simulations of proteins in solution - artefacts by the cutoff approximation, J. Chem. Phys. 101, 4055–4061.
78. Saito, M. (1995) Molecular dynamics simulations of proteins in solution with all degrees of freedom and long-range Coulomb interactions, J. Phys. Chem. B 99, 17043–17048.
79. T. Darden, D. Y., L. Pedersen. (1993) Particle mesh ewald - an N log N method for Ewald sums in large systems, J. Chem. Phys. 98, 10089-10092.
80. U. Essmann, L. P., M. L. Berkowitz, T. Darden, H. Lee, L. G. Pedersen. (1995) The smooth particle mesh Ewald method, J. Chem. Phys. 103.
81. Riddle, B., and Jencks, W. P. (1971) Acetyl-coenzyme A: arylamine N-acetyltransferase. Role of the acetyl-enzyme intermediate and the effects of substituents on the rate, J Biol Chem 246, 3250-3258.
82. Pompeo, F., Brooke, E., Kawamura, A., Mushtaq, A., and Sim, E. (2002) The pharmacogenetics of NAT: structural aspects, Pharmacogenomics 3, 19-30.
83. Windmill, K. F., Gaedigk, A., Hall, P. M., Samaratunga, H., Grant, D. M., and McManus, M. E. (2000) Localization of N-acetyltransferases NAT1 and NAT2 in human tissues, Toxicol Sci 54, 19-29.
84. Hein, D. W. (2002) Molecular genetics and function of NAT1 and NAT2: role in aromatic amine metabolism and carcinogenesis, Mutat Res 506-507, 65-77.
85. Evans, D. A., and White, T. A. (1964) Human Acetylation Polymorphism, J Lab Clin Med 63, 394-403.
86. Boukouvala, S., and Fakis, G. (2005) Arylamine N-acetyltransferases: what we learn from genes and genomes, Drug Metab Rev 37, 511-564.
87. Zang, Y., Doll, M. A., Zhao, S., States, J. C., and Hein, D. W. (2007) Functional characterization of single-nucleotide polymorphisms and haplotypes of human N-acetyltransferase 2, Carcinogenesis 28, 1665-1671.
88. Brockton, N., Little, J., Sharp, L., and Cotton, S. C. (2000) N-acetyltransferase polymorphisms and colorectal cancer: a HuGE review, Am J Epidemiol 151, 846-861.
89. Grant, D. M., Hughes, N. C., Janezic, S. A., Goodfellow, G. H., Chen, H. J., Gaedigk, A., Yu, V. L., and Grewal, R. (1997) Human acetyltransferase polymorphisms, Mutat Res 376, 61-70.
90. Walraven, J. M., Zang, Y., Trent, J. O., and Hein, D. W. (2008) Structure/function evaluations of single nucleotide polymorphisms in human N-acetyltransferase 2, Curr Drug Metab 9, 471-486.
91. Alberg, A. J., Daudt, A., Huang, H. Y., Hoffman, S. C., Comstock, G. W., Helzlsouer, K. J., Strickland, P. T., and Bell, D. A. (2004) N-acetyltransferase 2 (NAT2) genotypes, cigarette smoking, and the risk of breast cancer, Cancer Detect Prev 28, 187-193.
92. Risch, A., Wallace, D. M., Bathers, S., and Sim, E. (1995) Slow N-acetylation genotype is a susceptibility factor in occupational and smoking related bladder cancer, Hum Mol Genet 4, 231-236.
93. Le Marchand, L., Hankin, J. H., Wilkens, L. R., Pierce, L. M., Franke, A., Kolonel, L. N., Seifried, A., Custer, L. J., Chang, W., Lum-Jones, A., and Donlon, T. (2001) Combined effects of well-done red meat, smoking, and rapid N-acetyltransferase 2 and CYP1A2 phenotypes in increasing colorectal cancer risk, Cancer Epidemiol Biomarkers Prev 10, 1259-1266.
94. Sillanpaa, P., Hirvonen, A., Kataja, V., Eskelinen, M., Kosma, V. M., Uusitupa, M., Vainio, H., and Mitrunen, K. (2005) NAT2 slow acetylator genotype as an important modifier of breast cancer risk, Int J Cancer 114, 579-584.
95. van der Hel, O. L., Peeters, P. H., Hein, D. W., Doll, M. A., Grobbee, D. E., Kromhout, D., and Bueno de Mesquita, H. B. (2003) NAT2 slow acetylation and GSTM1 null genotypes may increase postmenopausal breast cancer risk in long-term smoking women, Pharmacogenetics 13, 399-407.
96. Zhu, J., Chang, P., Bondy, M. L., Sahin, A. A., Singletary, S. E., Takahashi, S., Shirai, T., and Li, D. (2003) Detection of 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine-DNA adducts in normal breast tissues and risk of breast cancer, Cancer Epidemiol Biomarkers Prev 12, 830-837.
97. Costa, S., Pinto, D., Morais, A., Vasconcelos, A., Oliveira, J., Lopes, C., and Medeiros, R. (2005) Acetylation genotype and the genetic susceptibility to prostate cancer in a southern European population, Prostate 64, 246-252.
98. Hein, D. W., Leff, M. A., Ishibe, N., Sinha, R., Frazier, H. A., Doll, M. A., Xiao, G. H., Weinrich, M. C., and Caporaso, N. E. (2002) Association of prostate cancer with rapid N-acetyltransferase 1 (NAT1*10) in combination with slow N-acetyltransferase 2 acetylator genotypes in a pilot case-control study, Environ Mol Mutagen 40, 161-167.
99. Deguchi, T. (1992) Sequences and expression of alleles of polymorphic arylamine N-acetyltransferase of human liver, J Biol Chem 267, 18140-18147.
100. Hein, D. W., Fretland, A. J., and Doll, M. A. (2006) Effects of single nucleotide polymorphisms in human N-acetyltransferase 2 on metabolic activation (O-acetylation) of heterocyclic amine carcinogens, Int J Cancer 119, 1208-1211.
101. Fretland, A. J., Leff, M. A., Doll, M. A., and Hein, D. W. (2001) Functional characterization of human N-acetyltransferase 2 (NAT2) single nucleotide polymorphisms, Pharmacogenetics 11, 207-215.
102. Hein, D. W., Ferguson, R. J., Doll, M. A., Rustan, T. D., and Gray, K. (1994) Molecular genetics of human polymorphic N-acetyltransferase: enzymatic analysis of 15 recombinant wild-type, mutant, and chimeric NAT2 allozymes, Hum Mol Genet 3, 729-734.
103. Zang, Y., Zhao, S., Doll, M. A., States, J. C., and Hein, D. W. (2004) The T341C (Ile114Thr) polymorphism of N-acetyltransferase 2 yields slow acetylator phenotype by enhanced protein degradation, Pharmacogenetics 14, 717-723.
104. Zang, Y., Zhao, S., Doll, M. A., Christopher States, J., and Hein, D. W. (2007) Functional characterization of the A411T (L137F) and G364A (D122N) genetic polymorphisms in human N-acetyltransferase 2, Pharmacogenet Genomics 17, 37-45.
105. Lau, E. Y., Felton, J. S., and Lightstone, F. C. (2006) Insights into the o-acetylation reaction of hydroxylated heterocyclic amines by human arylamine N-acetyltransferases: a computational study, Chem Res Toxicol 19, 1182-1190.
106. Rodrigues-Lima, F., and Dupret, J. M. (2002) 3D model of human arylamine N-acetyltransferase 2: structural basis of the slow acetylator phenotype of the R64Q variant and analysis of the active-site loop, Biochem Biophys Res Commun 291, 116-123.
107. Walraven, J. M., Trent, J. O., and Hein, D. W. (2007) Computational and experimental analyses of mammalian arylamine N-acetyltransferase structure and function, Drug Metab Dispos 35, 1001-1007.
108. Wu, H., Dombrovsky, L., Tempel, W., Martin, F., Loppnau, P., Goodfellow, G. H., Grant, D. M., and Plotnikov, A. N. (2007) Structural basis of substrate-binding specificity of human arylamine N-acetyltransferases, J Biol Chem 282, 30189-30197.
109. Dupret, J. M., and Grant, D. M. (1992) Site-directed mutagenesis of recombinant human arylamine N-acetyltransferase expressed in Escherichia coli. Evidence for direct involvement of Cys68 in the catalytic mechanism of polymorphic human NAT2, J Biol Chem 267, 7381-7385.
110. Wang, H., Vath, G. M., Gleason, K. J., Hanna, P. E., and Wagner, C. R. (2004) Probing the mechanism of hamster arylamine N-acetyltransferase 2 acetylation by active site modification, site-directed mutagenesis, and pre-steady state and steady state kinetic studies, Biochemistry 43, 8234-8246.
111. Wang, H., Liu, L., Hanna, P. E., and Wagner, C. R. (2005) Catalytic mechanism of hamster arylamine N-acetyltransferase 2, Biochemistry 44, 11295-11306.
112. Oda, A., Kobayashi, K., and Takahashi, O. (2010) Computational study of the three-dimensional structure of N-acetyltransferase 2-acetyl coenzyme a complex, Biol Pharm Bull 33, 1639-1643.
113. Darden, T., D. York, L. Pedersen. (1993) Particle mesh Ewald: an N-log(N) method for Ewald sums in larger systems, J Chem Phys 98, 10089-10092.
114. Hess, B., Bekker, H., Berendsen, H.J.C., Fraaije, J.G.E.M. (1997) LINCS: a linear constraint solver for molecular simulations., J. Comp. Chem. 18, 1463-1472.
115. Miyamoto, S., Kollman, P.A. (1992) Settle: an analytical version of the SHAKE and RATTLE algorithm for rigid water models., J. Comp. Chem. 13, 952-962.
116. Hubbard, S. J., J.M. Thornton. (1993) NACCESS, Department of Biochemistry and MOlecular biology, University College of London.
117. McDonald, I. K., and Thornton, J. M. (1994) Satisfying hydrogen bonding potential in proteins, J Mol Biol 238, 777-793.
118. Wade, R. C., R.R. Gabdoulline, F. De Rienzo. (2001) Protein interaction property similarity analysis, Intl. J. Quant. Chem 83, 122-127.
119. Richter, S., Wenzel, A., Stein, M., Gabdoulline, R. R., and Wade, R. C. (2008) webPIPSA: a web server for the comparison of protein interaction properties, Nucleic Acids Res 36, W276-280.
120. Baker, N. A., Sept, D., Joseph, S., Holst, M. J., and McCammon, J. A. (2001) Electrostatics of nanosystems: application to microtubules and the ribosome, Proc Natl Acad Sci U S A 98, 10037-10041.
121. Hodgkin, E. E., W.G. Richards (1987) Molecular similarity based on electrostatic potential and electric field, Int. J. Quant. Chem. Quant. Biol. Symp. 14, 105-110.
122. Goodfellow, G. H., Dupret, J. M., and Grant, D. M. (2000) Identification of amino acids imparting acceptor substrate selectivity to human arylamine acetyltransferases NAT1 and NAT2, Biochem J 348 Pt 1, 159-166.
123. Butcher, N. J., Arulpragasam, A., and Minchin, R. F. (2004) Proteasomal degradation of N-acetyltransferase 1 is prevented by acetylation of the active site cysteine: a mechanism for the slow acetylator phenotype and substrate-dependent down-regulation, J Biol Chem 279, 22131-22137.
124. Hein, D. W. (2009) N-acetyltransferase SNPs: emerging concepts serve as a paradigm for understanding complexities of personalized medicine, Expert Opin Drug Metab Toxicol 5, 353-366.
125. Semina, E. V., Reiter, R., Leysens, N. J., Alward, W. L., Small, K. W., Datson, N. A., Siegel-Bartelt, J., Bierke-Nelson, D., Bitoun, P., Zabel, B. U., Carey, J. C., and Murray, J. C. (1996) Cloning and characterization of a novel bicoid-related homeobox transcription factor gene, RIEG, involved in Rieger syndrome, Nat Genet 14, 392-399.
126. Gehring, W. J., Qian, Y. Q., Billeter, M., Furukubo-Tokunaga, K., Schier, A. F., Resendez-Perez, D., Affolter, M., Otting, G., and Wuthrich, K. (1994) Homeodomain-DNA recognition, Cell 78, 211-223.
127. Kumar, J., and Moses, K. (1997) Transcription factors in eye development: a gorgeous mosaic?, Genes Dev 11, 2023-2028.
128. McGinnis, W., and Krumlauf, R. (1992) Homeobox genes and axial patterning, Cell 68, 283-302.
129. Gage, P. J., and Camper, S. A. (1997) Pituitary homeobox 2, a novel member of the bicoid-related family of homeobox genes, is a potential regulator of anterior structure formation, Hum Mol Genet 6, 457-464.
130. Mucchielli, M. L., Mitsiadis, T. A., Raffo, S., Brunet, J. F., Proust, J. P., and Goridis, C. (1997) Mouse Otlx2/RIEG expression in the odontogenic epithelium precedes tooth initiation and requires mesenchyme-derived signals for its maintenance, Dev Biol 189, 275-284.
131. Hjalt, T. A., Semina, E. V., Amendt, B. A., and Murray, J. C. (2000) The Pitx2 protein in mouse development, Dev Dyn 218, 195-200.
132. Rieger, H. (1935) Dysgenesis mesodemalis corneal et iridis, Z. Augenheilk 86, 333.
133. Perveen, R., Lloyd, I. C., Clayton-Smith, J., Churchill, A., van Heyningen, V., Hanson, I., Taylor, D., McKeown, C., Super, M., Kerr, B., Winter, R., and Black, G. C. (2000) Phenotypic variability and asymmetry of Rieger syndrome associated with PITX2 mutations, Invest Ophthalmol Vis Sci 41, 2456-2460.
134. Xia, K., Wu, L., Liu, X., Xi, X., Liang, D., Zheng, D., Cai, F., Pan, Q., Long, Z., Dai, H., Hu, Z., Tang, B., Zhang, Z., and Xia, J. (2004) Mutation in PITX2 is associated with ring dermoid of the cornea, J Med Genet 41, e129.
135. Chaney, B. A., Clark-Baldwin, K., Dave, V., Ma, J., and Rance, M. (2005) Solution structure of the K50 class homeodomain PITX2 bound to DNA and implications for mutations that cause Rieger syndrome, Biochemistry 44, 7497-7511.
136. Qian, Y. Q., Billeter, M., Otting, G., Muller, M., Gehring, W. J., and Wuthrich, K. (1989) The structure of the Antennapedia homeodomain determined by NMR spectroscopy in solution: comparison with prokaryotic repressors, Cell 59, 573-580.
137. Kornberg, T. B. (1993) Understanding the homeodomain, J Biol Chem 268, 26813-26816.
138. Kissinger, C. R., Liu, B. S., Martin-Blanco, E., Kornberg, T. B., and Pabo, C. O. (1990) Crystal structure of an engrailed homeodomain-DNA complex at 2.8 A resolution: a framework for understanding homeodomain-DNA interactions, Cell 63, 579-590.
139. Banerjee-Basu, S., and Baxevanis, A. D. (1999) Threading analysis of the Pitx2 homeodomain: predicted structural effects of mutations causing Rieger syndrome and iridogoniodysgenesis, Hum Mutat 14, 312-319.
140. Amendt, B. A., Sutherland, L. B., Semina, E. V., and Russo, A. F. (1998) The molecular basis of Rieger syndrome. Analysis of Pitx2 homeodomain protein activities, J Biol Chem 273, 20066-20072.
141. Saadi, I., Semina, E. V., Amendt, B. A., Harris, D. J., Murphy, K. P., Murray, J. C., and Russo, A. F. (2001) Identification of a dominant negative homeodomain mutation in Rieger syndrome, J Biol Chem 276, 23034-23041.
142. Amendt, B. A., Semina, E. V., and Alward, W. L. (2000) Rieger syndrome: a clinical, molecular, and biochemical analysis, Cell Mol Life Sci 57, 1652-1666.
143. Kozlowski, K., and Walter, M. A. (2000) Variation in residual PITX2 activity underlies the phenotypic spectrum of anterior segment developmental disorders, Hum Mol Genet 9, 2131-2139.
144. H. J. C. Berendsen, J. P. M. P., W. F. van Gunsteren, A. DiNola, and J. R. Haak. (1984) Molecular dynamics with coupling to an external bath, J. Chem. Phys. 81, 3684-3690.
145. Parrinello M, R. A. (1981) Polymorphic transitions in single crystals: A new molecular dynamics method, J Appl Phys 52, 7182-7190.
146. DeLano, W. (2002) The PyMOL Molecular Graphics System.
147. Rajasekaran, M., Abirami, S., and Chen, C. (2011) Effects of single nucleotide polymorphisms on human N-acetyltransferase 2 structure and dynamics by molecular dynamics simulation, PLoS One 6, e25801.
148. Anderson, P. C., and Daggett, V. (2009) The R46Q, R131Q and R154H polymorphs of human DNA glycosylase/beta-lyase hOgg1 severely distort the active site and DNA recognition site but do not cause unfolding, J Am Chem Soc 131, 9506-9515.
149. Cordomi, A., Ramon, E., Garriga, P., and Perez, J. J. (2008) Molecular dynamics simulations of rhodopsin point mutants at the cytoplasmic side of helices 3 and 6, J Biomol Struct Dyn 25, 573-587.
150. Blaise, M. C., Bhattacharyya, D., Sowdhamini, R., and Pradhan, N. (2005) Structural consequences of D481N/K483Q mutation at glycine binding site of NMDA ionotropic glutamate receptors: a molecular dynamics study, J Biomol Struct Dyn 22, 399-410.
151. Hao, G. F., and Yang, G. F. (2010) The role of Phe82 and Phe351 in auxin-induced substrate perception by TIR1 ubiquitin ligase: a novel insight from molecular dynamics simulations, PLoS One 5, e10742.
152. Biocca, S., Falconi, M., Filesi, I., Baldini, F., Vecchione, L., Mango, R., Romeo, F., Federici, G., Desideri, A., and Novelli, G. (2009) Functional analysis and molecular dynamics simulation of LOX-1 K167N polymorphism reveal alteration of receptor activity, PLoS One 4, e4648.
153. Roy, S., and Thakur, A. R. (2010) 20ns molecular dynamics simulation of the antennapedia homeodomain-DNA complex: water interaction and DNA structure analysis, J Biomol Struct Dyn 27, 443-456.
154. Zhao, X., Huang, X. R., and Sun, C. C. (2006) Molecular dynamics analysis of the engrailed homeodomain-DNA recognition, J Struct Biol 155, 426-437.
155. Gutmanas, A., and Billeter, M. (2004) Specific DNA recognition by the Antp homeodomain: MD simulations of specific and nonspecific complexes, Proteins 57, 772-782.
156. Yang, S. Y., Yang, X. L., Yao, L. F., Wang, H. B., and Sun, C. K. (2011) Effect of CpG methylation on DNA binding protein: molecular dynamics simulations of the homeodomain PITX2 bound to the methylated DNA, J Mol Graph Model 29, 920-927.
157. Chaney, B. A. (2005) DNA Recognition by the K50 Class Homeodomain PITX2: Solution Structure, Molecular Dynamics, and Implications for Mutations That Cause Rieger Syndrome, PhD diss., University of Cincinnati.
158. Duboule, D. (1994) Guidebook to the Homeobox Genes, Oxford University Press, New York.
159. Ades, S. E., and Sauer, R. T. (1994) Differential DNA-binding specificity of the engrailed homeodomain: the role of residue 50, Biochemistry 33, 9187-9194.
160. Connolly, J. P., Augustine, J. G., and Francklyn, C. (1999) Mutational analysis of the engrailed homeodomain recognition helix by phage display, Nucleic Acids Res 27, 1182-1189.
161. Pomerantz, J. L., and Sharp, P. A. (1994) Homeodomain determinants of major groove recognition, Biochemistry 33, 10851-10858.
162. Chi, Y. I. (2005) Homeodomain revisited: a lesson from disease-causing mutations, Hum Genet 116, 433-444.
163. Boncinelli, E. (1997) Homeobox genes and disease, Curr Opin Genet Dev 7, 331-337.
164. Nakamura, T., Largaespada, D. A., Lee, M. P., Johnson, L. A., Ohyashiki, K., Toyama, K., Chen, S. J., Willman, C. L., Chen, I. M., Feinberg, A. P., Jenkins, N. A., Copeland, N. G., and Shaughnessy, J. D., Jr. (1996) Fusion of the nucleoporin gene NUP98 to HOXA9 by the chromosome translocation t(7;11)(p15;p15) in human myeloid leukaemia, Nat Genet 12, 154-158.
165. Borrow, J., Shearman, A. M., Stanton, V. P., Jr., Becher, R., Collins, T., Williams, A. J., Dube, I., Katz, F., Kwong, Y. L., Morris, C., Ohyashiki, K., Toyama, K., Rowley, J., and Housman, D. E. (1996) The t(7;11)(p15;p15) translocation in acute myeloid leukaemia fuses the genes for nucleoporin NUP98 and class I homeoprotein HOXA9, Nat Genet 12, 159-167.
166. Ring, H. Z., Kwok, P. Y., and Cotton, R. G. (2006) Human Variome Project: an international collaboration to catalogue human genetic variation, Pharmacogenomics 7, 969-972.
167. Hudson, T. J., Anderson, (2010) International network of cancer genome projects, Nature 464, 993-998.
168. Durbin, R. M. (2010) A map of human genome variation from population-scale sequencing, Nature 467, 1061-1073.
169. Smigielski, E. M., Sirotkin, K., Ward, M., and Sherry, S. T. (2000) dbSNP: a database of single nucleotide polymorphisms, Nucleic Acids Res 28, 352-355.
170. Stenson, P. D., Mort, M., Ball, E. V., Howells, K., Phillips, A. D., Thomas, N. S., and Cooper, D. N. (2009) The Human Gene Mutation Database: 2008 update, Genome Med 1, 13.
171. Bairoch, A., Apweiler, R., Wu, C. H., Barker, W. C., Boeckmann, B., Ferro, S., Gasteiger, E., Huang, H., Lopez, R., Magrane, M., Martin, M. J., Natale, D. A., O'Donovan, C., Redaschi, N., and Yeh, L. S. (2005) The Universal Protein Resource (UniProt), Nucleic Acids Res 33, D154-159.
172. Weber, W. W., and Hein, D. W. (1985) N-acetylation pharmacogenetics, Pharmacol Rev 37, 25-79.
173. Cascorbi, I., Brockmoller, J., Bauer, S., Reum, T., and Roots, I. (1996) NAT2*12A (803A-->G) codes for rapid arylamine n-acetylation in humans, Pharmacogenetics 6, 257-259.
174. Hein, D. W., Doll, M. A., Fretland, A. J., Leff, M. A., Webb, S. J., Xiao, G. H., Devanaboyina, U. S., Nangju, N. A., and Feng, Y. (2000) Molecular genetics and epidemiology of the NAT1 and NAT2 acetylation polymorphisms, Cancer Epidemiol Biomarkers Prev 9, 29-42.
175. Tassabehji, M., Read, A. P., Newton, V. E., Patton, M., Gruss, P., Harris, R., and Strachan, T. (1993) Mutations in the PAX3 gene causing Waardenburg syndrome type 1 and type 2, Nat Genet 3, 26-30.
176. Wang, H., Antinozzi, P. A., Hagenfeldt, K. A., Maechler, P., and Wollheim, C. B. (2000) Molecular targets of a human HNF1 alpha mutation responsible for pancreatic beta-cell dysfunction, EMBO J 19, 4257-4264.
177. Ferda Percin, E., Ploder, L. A., Yu, J. J., Arici, K., Horsford, D. J., Rutherford, A., Bapat, B., Cox, D. W., Duncan, A. M., Kalnins, V. I., Kocak-Altintas, A., Sowden, J. C., Traboulsi, E., Sarfarazi, M., and McInnes, R. R. (2000) Human microphthalmia associated with mutations in the retinal homeobox gene CHX10, Nat Genet 25, 397-401.