J. Am. Chem. SOC. 1995,117, 7213-7227
7213
Insight into the Specificity of Thymidylate Synthase from Molecular Dynamics and Free Energy Perturbation Calculations Giulio RastelliJ96 Bert Thomas:& Peter A. Kollman,*g’ and Daniel V. Santi*Jg’ Contribution from the Departments of Biochemistry and Biophysics and Pharmaceutical Chemistry, University of Califomia, San Francisco, Califomia 94143-0448 Received August 30, 1994@
Abstract: Molecular dynamics and free energy perturbation calculations have been used to calculate the relative free energies of binding of 2’-deoxyuridine 5’-monophosphate (dUMP) and 2‘-deoxycytidine 5’-monophosphate (dCMP) to thymidylate synthase (TS)and two asparagine 229 mutants. Calculations qualitatively reproduce experimentally observed dissociation constants of the protein-nucleotide complexes. Furthermore, they provide insight into structural aspects of binding and catalysis of these two nucleotides to the protein. The simulations of the wild-type TS complexes with dUMP and dCMP support the key role of asparagine 229 in causing tighter binding of dUMP than dCMP; repulsion between the base of dCMP and the asparagine 229 side chain reduces the AG of binding to the protein from that found in aqueous solution and causes the displacement of this nucleotide into a position unsuitable for reaction. The free energy calculations of the aspartate 229 mutant of TS interacting with either dUMP or dCMP suggest a synergism between the aspartate 229 side chain and the vicinal histidine 199 in binding. The best agreement between the calculated and the experimental AAG of binding has been obtained when the aspartate side chain is anionic and the histidine 199 is either protonated or in its 6H tautomer. Under these conditions, dUMP and dCMP are both properly positioned for nucleophilic attack. In contrast, calculations with a neutral aspartic acid side chain suggest a strong discriminating power of the neutral 229 side chain in binding the two nucleotides, the preferred one depending on which of the two oxygens of the aspartate is protonated. We speculate that protonation of the aspartate 229 side chain can be the key to rationalizing why the aspartate 229 mutant selectively methylates dCMP. Finally, calculations of the valine 229 mutant demonstrate that substitution of the polar asparagine side chain with a hydrophobic residue does not result in a significant change in the location of the two nucleotides in the active site, except that dUMP seems to be better positioned for nuclephilic attack than dCMP.
Introduction Thymidylate synthase (TS)catalyzes the reductive methylation of 2‘-deoxyuridine 5’-monophosphate (dUMP) by 5,lOmethylene-5,6,7,8-tetrahydrofolate(CHZKfolate) to give 2’deoxythymidine 5’-monophosphate (dTMP) and 7,8-dihydrofolate (Hzfolate). Its mechanism of action,’ substrate specificity: and atomic ~ t r u c t u r e ~have - ~ been extensively studied over the last few decades. Mutation of specific amino acids in TS has proven to be an important tool in elucidating the intrinsic role of particular residues in binding and catalysise6 Recently, the
’ Department of Biochemistry and Biophysics.
Department of Pharmaceutical Chemistry. Current address: Dipartimento di Scienze Farmaceutiche, Universita di Modena, Via Campi 183, 41100 Modena, Italy. Current address: Procept, Inc., Cambridge, MA. Abstract published in Advance ACS Abstracts, June 1, 1995. (1) Santi, D. V.; Danenberg, P. V. In Folates and Pterins; Blakley, R. L., Benkovic, S. J., Eds.; John Wiley and Sons: New York, 1984; pp 345398. (2) Santi, D. V.; McHenry, C. S.; Raines, R. T.; Ivanetich, K. M. Biochemistry 1987,26, 8606-8613. (3) Hardy, L. W.; Finer-Moore, J. S.; Montford, W. R.; Jones, M. 0.; Santi, D. V.; Stroud, R. M. Science 1987, 235, 448-455. (4) (a) Montford, W. R.; Perry, K. M.; Fauman, E. B.; Finer-Moore, J. S.; Maley, G . F.; Hardy, L. W.; Maley, F. ; Stroud, R. M. Biochemistry 1990.29, 6964-6977. (b) Finer-Moore, J. S.; Montford, W. R.; Stroud, R. M. Biochemistry 1990, 29, 6977-6986. (c) Perry, K. M.; Fauman, E. B.; Finer-Moore, J. S.; Montford, W. R.; Maley, G. F.; Maley, F.; Stroud, R. M. Proteins 1990, 8, 315-333. ( 5 ) (a) Matthews, D. A.; Appelt, K.; Oatley, S. J.; Xuong, N. H. J. Mol. Biol. 1990, 214, 923-936. (b) Matthews, D. A,; Villafranca, J. E.; Janson, P. A.; Smith, W. W.; Welsh, K.; Freer, S . J. Mol. Biol. 1990, 214, 937948. (6) Climie, S.; Ruiz-Perez, L.; Gonzalez-Pacanowska, D.; Prapunwattana, P.; Cho, S. W.; Stroud, R.; Santi, D. V. J. B i d . Chem. 1990, 265, 1877618779. t 5
@
mutation of the conserved asparagine 229 to aspartic acid (N229D) in Lactobacillus casei TS was reported to change the TS to an enzyme which methylates 2’-deoxycytidine 5’-monophosphate (dCMP) instead of dUMP.’ Further studies on As11229 mutants revealed that As11229 is not essential in binding and catalysis, but it plays the important role of discriminating between dUMP and dCMP by excluding dCMP from the binding site.8 With the exception of the N229Q mutant, all Asn229 mutants bind dUMP and dCMP almost equally well, with KD values approaching that of the wild-type (wt) TSdUMP binary complex.8 In the crystal structure of the TSdUMP binary complex, the Asn229 side chain forms a cyclic hydrogen bonding network with N3 and 0 4 of the uracil base (Figure l).9 It has been proposed that the discrimination between dUMP and dCMP results from unfavorable interactions between dCMP and the side chain of Asn229 (Figure l);7 rotation of the Asn229 side chain, which could better accommodate dCMP, is presumably too costly since Asn229 is hydrogen bonded to Gln217 and to an ordered water molecule as part of a larger hydrogen bonding network. It is also not clear how the replacement of Am229 with hydrophobic residues unable to participate in similar hydrogen bonds can preserve the tight binding of dUh4P and cause the binding of dCMP to be as tight as that of dUMP. Similar mutagenesis experiments have been performed with Asn177 of Escherichia coli TS.Io Most mutants of As11177 (7) Liu, L.; Santi, D. V. Biochemistry 1992, 31, 5100-5104. (8) (a) Liu, L.; Santi, D. V. Proc. Nati. Acad. Sci. U.S.A. 1993.90, 86048608. (b) Liu, L.; Santi, D. V. Biochemistry 1993, 32, 9263-9267. (9) Finer-Moore, J. S.; Fauman, E, B.; Foster, P. G . ;Perry, K. M.; Santi, D. V.; Stroud, R. J. Mol. Biol. 1993, 232, 1101-1116.
0002-786319511517-7213$09.00/00 1995 American Chemical Society
7214 J. Am. Chem. Soc., Vol. 117, No. 27, 1995 n N229
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Rastelli et al.
H
dUMP+TS
t dCMP + TS 0
MGbi,d
1
dUMP dCMP Figure 1. Schematic diagram of the hydrogen bonding between dUMP and the asparagine 229 side chain in the wild-type TSdUMP complex. Simple static replacement of dUMP with dCMP would cause repulsion with the asparagine side chain.
lowered the activity of E. coli TS by at least 100-fold,” except for N177D, which converted E. coli TS into a dCMP methylase. I 2 In this study we use the free energy perturbation (FEP) methodI3to calculate the relative binding free energies of dUMP and dCMP with wild-type TS and two mutants. The N229D mutant was chosen because of its ability to catalyze the methylation of dCMP. The N229V mutant was chosen to investigate the effect of a hydrophobic residue, valine, on the binding energies of dUMP and dCMP, and because dCMP binds tighter than dUMP to the N229V mutant. These molecular dynamics (MD) simulations provide further insight into the binding modes of dUMP and dCMP to wild-type TS and to the N229D and N229V mutants.
Computational Method All MD calculations were performed using the AMBER all-atom force field and the AMBER 4.0 molecular dynamics program~.’~ During all simulations, all bond lengths were constrained using the SHAKEI5 algorithm with a tolerance of 0.005 A, allowing a time step of 2.0 fs. Solute and solvent were coupled to a constant temperature heat bath with a coupling constant of 0.2 ps to maintain a temperature of 300 K. A residue-based cutoff of 8.0 8, was employed. Pairlists were generated every 25 time steps. The entire substrate was used as the perturbed group in the FEP calculations. No intraperturbed group contributions to the free energy were calculated. The thermodynamic perturbation method was used to calculate the free energy difference^.'^ The thermodynamic cycle relevant to this study is shown in Figure 2. The free energies of complexation, AG1 and AG2, of dUMP and dCMP are experimentally determinable values, but they are difficult to calculate. AG,I corresponds to mutating dUMP into dCMP in water, while AGprotcorresponds to changing dUMP into dCMP while bound to TS in water. Since the thermodynamic cycle is closed and free energy is a state function, AAG,,,d = AGI - AGP = AG,l - AGpmt. While AGpmand AGs,,l represent physically unrealizable processes, these two simulations are computationally manageable. The experimental free energies of binding for dUMP and dCMP to TS (10) In L. casei TS, the pyrimidine binding residue is N229. TSs from other sources have different numbering systems. In E. coli TS, the pyrimidine binding residue is N117. The numbering system for L. casei TS will be used in this paper when refemng to specific residues unless
otherwise stated. (11) Michaels, M. L.; Kim, C. W.; Manhews, D. A.; Miller, J. H. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 3957-3961. (12) Hardy, L. W.; Nalivaika, E. Proc. Narl. Acad. Sci. U.S.A. 1992, 89, 9725-9729. (13) (a) Bash, P. A.; Singh, U.; Langridge, R.; Kollman, P. A. Science 1987,236, 564-568. (b) McCammon, J. A. Science 1987,238,486-491. (c) Jorgensen, W. L.; Briggs, J. M. J. Am. Chem. SOC.1989, 1 1 1 , 41904197. (d) Kollman, P. A. Chem. Rev. 1993, 93, 2395-2417. (14) Pearlman, D. A,; Case, D. A.; Caldwell, J. A.; Seibel, G. L.; Singh, U. C.; Weiner, P.; Kollman, P. A. AMBER 4 . 0 University of California, San Francisco, 1991. (15) van Gunsteren, W. F.; Berendsen, H. J. C. Mol. Phys. 1977, 34, 1311- 1327.
I
AG I
dUMP-TS
AG1
1 dCMP-TS
AG, - AG2 = AG,,, - AG,,,
Figure 2. Thermodynamic cycle for complex formations of dUMP and dCMP with TS. The horizontal free energies are experimentally determined, and the vertical ones are calculated. Table 1. Experimental Free Energies of Binding (AGInd)and Relative Differences between dUMP and dCMP (AAGmd)O
+ + + + +
wt-TS dUMP wt-TS dCMP N229D dUMP N229D dCMP N229V dUMP N229V dCMP
+
ACibind
AAGbld
-8.81 -5.21 -7.64 -7.62 -5.78 -7.28
-3.60 -0.02 1.50
and the selected mutants are reported in Table 1, as calculated from the dissociation constants (KO)of their binary complexes.8 The crystal structure of the casei TSdUMP binary complex was solved to a resolution of 2.55 A.9 At the time this work was carried out, the only crystal structure of a binary complex available was that of the TSdUMP complex. Briefly, it consists of two identical monomers with 316 residues per monomer; components of each monomer contribute to each of the two active sites. One molecule of dUMP is noncovalently bound to each active site. The side chain atoms of Aspl 11 and Aspl 11’ are not included in the X-ray coordinates due to disorder; they have been generated using the AMBER internal coordinate database. Hydrogens were added to the protein using the stored intemal coordinates of the AMBER all-atom data base and then minimized, keeping the heavy atoms of the protein fixed at their original positions. The initial structures of the mutants were generated from the X-ray structure of the binary complex by replacing the Asn229 with Asp or Val. All Lys and Arg residues were positively charged and Glu and Asp residues negatively charged, except where noted. The His residues were treated as unprotonated, since the pK, of histidine is lower than the pH at which the binding assays were performed. The 6H or EH tautomeric forms of histidines were assigned on the basis of favorable interactions with their environments. In the simulation of the N229D mutant, calculations are presented for the 6H, EH, and protonated forms of His199 because His199 could act in synergism with Asp229 to determine the effective binding of dUMP and dCMP to the N229D mutant. In TS and the N229V mutant, the 6H tautomer of His199 was employed. The parameters for the neutral Asp were taken from ref 16 . Two relative configurations of the neutral Asp229 side chain were initially constructed (Figure 3): configuration 1 places OH of Asp229 next to 0 4 of dUMP and 0 of Asp229 next to HN3 (a situation optimal for dUMP but unfavorable for dCMP); configuration 2 places OH of Asp next to HN3 of dUMP and 0 of Asp next to 0 4 (a situation unfavorable for dUMP but optimal for dCMP). Interestingly, each configuration mutally favors the binding of only one nucleotide. Calculations were started by equilibrating the optimal complexes (N229WUMP for configuration 1 and N229WCMP for configuration 2) and perturbing them in opposite directions (Figure 3). Five crystallographic water molecules were maintained in the initial structure of TS; these ordered waters connect the dUMP base with the vicinal residues Glu60, His199, Asn229, and Ser232, and are probably important for maintaining the proper orientation of dUMP. The active site was further solvated with a spherical cap of 161 TIP3P” water
4.
(16) Ferguson, D. M.; Radmer, R. J.; Kollman P. A. J. Med. Chem. 1991, 34, 2654-2659. (17) Jorgensen, W. L.; Chandrasekhar,J.; Madura, J. D.; Impey, R. W.; Klein, M. L. J. Chem.Phys. 1983, 79, 926-935.
J. Am. Chem. SOC., Vol. 117, No. 27, 1995 1215
Specijicity of Thymidylate Synthase
04
II
0
I
s\r
AP
dUMP
dCMP
Yo 0
H
Q,, 05'
2
3'
L
+sc
dCMP dUMP Figure 3. Schematic diagram showing the two relative configurations adopted for dUMP and dCMP interacting with the neutral aspartic acid mutant of TS (N229D). The perturbations have been carried out in the directions of the arrows. molecules within 20 8, of the center of mass of dUMP, and harmonic radial forces (1.5 kcaVmol A2) were applied to these waters to avoid evaporation. dUMP, all residues within 12 A of each atom of dUMP, and all the water molecules were allowed to move during the simulations. This selection resulted in a large moveable zone (127 protein residues) since dUMP is a fairly long molecule. In order to carry out the perturbation of dUMP into dCMP in the binary complex, the initial structure was minimized and equilibrated. The energy minimization was performed by optimizing the water molecules first, keeping the protein and dUMP frozen. Next, all the residues within the belly and all the waters were optimized until the root-mean-square value of the potential gradients was
