Multi-targeted antifolates aimed at avoiding drug resistance form covalent closed inhibitory complexes with human and Escherichia coli thymidylate synthases.

Crystal structures of four pyrrolo(2,3-d)pyrimidine-based antifolate compounds, developed as inhibitors of thymidylate synthase (TS) in a strategy to circumvent drug-resistance, have been determined in complexes with their in vivo target, human thymidylate synthase, and with the structurally best-characterized Escherichia coli enzyme, to resolutions of 2.2-3.0 A. The 2.9 A crystal structure of a complex of human TS with one of the inhibitors, the multi-targeted antifolate LY231514, demonstrates that this compound induces a "closed" enzyme conformation and leads to formation of a covalent bond between enzyme and substrate. This structure is one of the first liganded human TS structures, and its solution was aided by mutation to facilitate crystallization. Structures of three other pyrrolo(2,3-d)pyrimidine-based antifolates in complex with Escherichia coli TS confirm the orientation of this class of inhibitors in the active site. Specific interactions between the polyglutamyl moiety and a positively charged groove on the enzyme surface explain the marked increase in affinity of the pyrrolo(2,3-d)pyrimidine inhibitors once they are polyglutamylated, as mediated in vivo by the cellular enzyme folyl polyglutamate synthetase.

Predicting and harnessing protein flexibility in the design of species-specific inhibitors of thymidylate synthase.

BACKGROUND: Protein plasticity in response to ligand binding abrogates the notion of a rigid receptor site. Thus, computational docking alone misses important prospective drug design leads. Bacterial-specific inhibitors of an essential enzyme, thymidylate synthase (TS), were developed using a combination of computer-based screening followed by in-parallel synthetic elaboration and enzyme assay [Tondi et al. (1999) Chem. Biol. 6, 319-331]. Specificity was achieved through protein plasticity and despite the very high sequence conservation of the enzyme between species. RESULTS: The most potent of the inhibitors synthesized, N,O-didansyl-L-tyrosine (DDT), binds to Lactobacillus casei TS (LcTS) with 35-fold higher affinity and to Escherichia coli TS (EcTS) with 24-fold higher affinity than to human TS (hTS). To reveal the molecular basis for this specificity, we have determined the crystal structure of EcTS complexed with DDT and 2'-deoxyuridine-5'-monophosphate (dUMP). The 2.0 A structure shows that DDT binds to EcTS in a conformation not predicted by molecular docking studies and substantially differently than other TS inhibitors. Binding of DDT is accompanied by large rearrangements of the protein both near and distal to the enzyme's active site with movement of C alpha carbons up to 6 A relative to other ternary complexes. This protein plasticity results in novel interactions with DDT including the formation of hydrogen bonds and van der Waals interactions to residues conserved in bacterial TS but not hTS and which are hypothesized to account for DDT's specificity. The conformation DDT adopts when bound to EcTS explains the activity of several other LcTS inhibitors synthesized in-parallel with DDT suggesting that DDT binds to the two enzymes in similar orientations. CONCLUSIONS: Dramatic protein rearrangements involving both main and side chain atoms play an important role in the recognition of DDT by EcTS and highlight the importance of incorporating protein plasticity in drug design. The crystal structure of the EcTS/dUMP/DDT complex is a model system to develop more selective TS inhibitors aimed at pathogenic bacterial species. The crystal structure also suggests a general formula for identifying regions of TS and other enzymes that may be treated as flexible to aid in computational methods of drug discovery.

Crystal structure of the HIV-1 integrase catalytic core and C-terminal domains: a model for viral DNA binding.

Insolubility of full-length HIV-1 integrase (IN) limited previous structure analyses to individual domains. By introducing five point mutations, we engineered a more soluble IN that allowed us to generate multidomain HIV-1 IN crystals. The first multidomain HIV-1 IN structure is reported. It incorporates the catalytic core and C-terminal domains (residues 52-288). The structure resolved to 2.8 A is a Y-shaped dimer. Within the dimer, the catalytic core domains form the only dimer interface, and the C-terminal domains are located 55 A apart. A 26-aa alpha-helix, alpha6, links the C-terminal domain to the catalytic core. A kink in one of the two alpha6 helices occurs near a known proteolytic site, suggesting that it may act as a flexible elbow to reorient the domains during the integration process. Two proteins that bind DNA in a sequence-independent manner are structurally homologous to the HIV-1 IN C-terminal domain, suggesting a similar protein-DNA interaction in which the IN C-terminal domain may serve to bind, bend, and orient viral DNA during integration. A strip of positively charged amino acids contributed by both monomers emerges from each active site of the dimer, suggesting a minimally dimeric platform for binding each viral DNA end. The crystal structure of the isolated catalytic core domain (residues 52-210), independently determined at 1.6-A resolution, is identical to the core domain within the two-domain 52-288 structure.

Inactivity of N229A thymidylate synthase due to water-mediated effects: isolating a late stage in methyl transfer.

Mutation of thymidylate synthase N229(177) to alanine results in an essentially inactive enzyme, yet it leads to formation of a stable ternary complex. The kinetics of N229(177)A show that kcat for Escherichia coli is reduced by 200-fold while the Km for dUMP is increased 200-fold and the Km for folate increased by tenfold versus the wild-type enzyme. The crystal structures of N229(177)A in complex with dUMP and CB3717, and in complex with dUMP alone are determined at 2.4 A, and 2.5 A resolution. These structures identify the covalently bound ternary complex and show how N229(177)A traps an intermediate, and so becomes inactive in a later step of the reaction. Since the smaller alanine side-chain at N229(177)A does not directly sterically impair binding of ligands, the structures implicate, and place quantitative limits on the involvement of the structured water network in the active site of thymidylate synthase in both catalysis and in determining the binding affinity for dUMP (in contrast, the N229(177)V mutation in Lactobacillus casei has minimal effect on activity).

Contributions of orientation and hydrogen bonding to catalysis in Asn229 mutants of thymidylate synthase.

We have determined structures of binary and ternary complexes of five Asn229 variants of thymidylate synthase (TS) and related their structures to the kinetic constants measured previously. Asn229 forms two hydrogen bonds to the pyrimidine ring of the substrate 2'-deoxyuridine-5'-monophosphate (dUMP). These hydrogen bonds constrain the orientation of dUMP in binary complexes with dUMP, and in ternary complexes with dUMP and the TS cofactor, 5,10-methylene-5,6,7,8-tetrahydrofolate. In N229 mutants, where these hydrogen bonds cannot be made, dUMP binds in a misoriented or more disordered fashion. Most N229 mutants exhibit no activity for the dehalogenation of 5-bromo-dUMP, which requires correct orientation of dUMP against Cys198. Since bound dUMP forms the binding surface against which the pterin ring of cofactor binds, misorientation of dUMP results in higher Km values for cofactor. At the same time, binding of the cofactor aids in ordering and positioning dUMP for catalysis. Hydrophobic mutants, such as N229I, favor an arrangement of solvent molecules and side-chains around the ligands similar to that in a proposed transition state for ternary complex formation in wild-type TS, and kcat values are similar to the wild-type value. Smaller, more hydrophilic mutants favor arrangements of the solvent and side-chains surrounding the ligands that do not resemble the proposed transition state. These changes correspond to decreases in kcat of up to 2000-fold, with only modest increases in Km or Kd. These results are consistent with the proposal that the hydrogen-bonding network between water, dUMP and side-chains in the active-site cavity contributes to catalysis in TS. Asn229 has the unique ability to maintain this critical network, without sterically interfering with dUMP binding.

Design of potent selective zinc-mediated serine protease inhibitors.

Many serine proteases are targets for therapeutic intervention because they often play key roles in disease. Small molecule inhibitors of serine proteases with high affinity are especially interesting as they could be used as scaffolds from which to develop drugs selective for protease targets. One such inhibitor is bis(5-amidino-2-benzimidazolyl)methane (BABIM), standing out as the best inhibitor of trypsin (by a factor of over 100) in a series of over 60 relatively closely related analogues. By probing the structural basis of inhibition, we discovered, using crystallographic methods, a new mode of high-affinity binding in which a Zn2+ ion is tetrahedrally coordinated between two chelating nitrogens of BABIM and two active site residues, His57 and Ser 195. Zn2+, at subphysiological levels, enhances inhibition by over 10(3)-fold. The distinct Zn2+ coordination geometry implies a strong dependence of affinity on substituents. This unique structural paradigm has enabled development of potent, highly selective, Zn2+-dependent inhibitors of several therapeutically important serine proteases, using a physiologically ubiquitous metal ion.

Asparagine 229 mutants of thymidylate synthase catalyze the methylation of 3-methyl-2'-deoxyuridine 5'-monophosphate.

The conserved Asn 229 of thymidylate synthase (TS) forms a cyclic hydrogen bond network with the 3-NH and 4-O of the nucleotide substrate 2'-deoxyuridine 5'-monophosphate (dUMP). Asn 229 is not essential for substrate binding or catalysis [Liu, l., & Santi, D. B. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8604-8608] but is a major determinant in substrate specificity [Liu, l., & Santi, D. V. (1993) Biochemistry 32, 9263-9267]. 3-Methyl-dUMP (3-MedUMP) is neither a substrate nor an inhibitor of wild type TS but is converted to 3-methyl 2'-deoxythymidine 5'-monophosphate by many TS Asn 229 mutants. Some of the Asn 229 mutants (N229C, -I, -M, -A, and -V) have kcat values for 3-MedUMP methylation which are up to about 20% of that for wild type TS-catalyzed methylation of dUMP, and some mutants (N229C and -A) catalyze methylation of 3-MedUMP more efficiently than that of dUMP. Mutants with hydrophobic side chains tended to be more active in catalysis of methylation of 3-MedUMP than those with hydrophilic side chains. The ability of 3-MedUMP to serve as a substrate for Asn 229 mutants shows that the active form of dUMP involves the neutral pyrimidine base and that ionization of the 3-NH group does not occur in the course of catalysis. In contrast to the negligible binding of 3-MedUMP to wild type TS, both 3-MedUMP and dUMP showed similar Km values with the Asn 229 mutants, suggesting similar binding affinities to the mutants. The X-ray crystal structure of the TS N229C--3-MedUMP complex showed that the side chain of Cys 229 was rotated away from the pyrimidine ring to allow placement of a water molecule and the 3-methyl group of 3-MedUMP in the active site. Our results suggest that the inability of 3-MedUMP to undergo methylation by wild type TS is due to its inability to bind to the enzyme, which in turn is simply a result of steric interference of the 3-methyl group with the side chain of Asn 229.

Partitioning roles of side chains in affinity, orientation, and catalysis with structures for mutant complexes: asparagine-229 in thymidylate synthase.

Thymidylate synthase (TS) methylates only dUMP, not dCMP. The crystal structure of TS.dCMP shows sCMP 4-NH2 excluded from the space between Asn-229 and His-199 by the hydrogen bonding and steric properties and Asn-229. Consequently, 6-C of dCMP is over 4 A from the active site sulfhydryl. The Asn-229 side chain is prevented from flipping 180 degrees to and orientation the could hydrogen bond to dCMP by a hydrogen bond network between conserved residues. Thus, the specific binding of dUMP by TS results from occlusion of competing substrates by steric and electronic effects of residues in the active site cavity. When Asn-229 is replaced by a cysteine, the Cys-229 S gamma rotates out of the active site, and the mutant enzyme binds both dCMP and dUMP tightly but does not methylate dCMP. Thus simply admitting dCMP into the dUMP binding site of TS is not sufficient for methylation of dCMP. Structures of nucleotide complexes of TS N229D provide a reasonable explanation for the preferential methylation of dCMP instead of dUMP by this mutant. In TS N229D.dCMP, Asp-229 forms hydrogen bonds to 3-N and 40NH2 of dCMP. Neither the Asp-229 carboxyl moiety nor ordered water appears to hydrogen bond to 4-O of dUMP. Hydrogen bonds to 4-O (or 4-NH2) have been proposed to stabilize reaction intermediates. If their absence in TS N229D.dUMP persists in the ternary complex, it could explain the 10(4)-fold decrease in kcat/Km for dUMP.

Contribution of a salt bridge to binding affinity and dUMP orientation to catalytic rate: mutation of a substrate-binding arginine in thymidylate synthase.

Invariant arginine 179, one of four arginines that are conserved in all thymidylate synthases (TS) and that bind the phosphate moiety of the substrate 2'-deoxyuridine-5'-monophosphate (dUMP), can be altered even to a negatively charged glutamic acid with little effect on kcat. In the mutant structures, ordered water or the other phosphate-binding arginines compensate for the hydrogen bonds made by Arg179 in the wild-type enzyme and there is almost no change in the conformation or binding site of dUMP. Correlation of dUMP Kds for TS R179A and TS R179K with the structures of their binary complexes shows, that the positive charge on Arg179 contributes significantly to dUMP binding affinity. kcat/K(m) for dUMP measures the rate of dUMP binding to TS during the ordered bi-substrate reaction, and in the ternary complex dUMP provides a binding surface for the cofactor. kcat/K(m) reflects the ability of the enzyme to accept a properly oriented dUMP for catalysis and is less sensitive than is Kd to the changes in electrostatics at the phosphate binding site.

Crystal structure of thymidylate synthase from T4 phage: component of a deoxynucleoside triphosphate-synthesizing complex.

Thymidylate synthase from phage T4 (T4TS) is part of a complex of several enzymes required for coordinate DNA synthesis in infected Escherichia coli cells. It has been proposed that similar complexes of enzymes related to DNA synthesis are also functional in eukaryotes [Pardee, A. B. (1989) Science 246, 603-608]. To delineate the role of structure in the function of this complex, we have solved the structure of T4TS as a basis for mapping the complex by mutagenesis. The 3.1 A structure of the unliganded enzyme was determined by molecular replacement and refined to 19.9% for all data. Three inserts and one deletion in the coding region are unique to T4TS, and all sites lie on one side of the enzyme surface, possibly encoding unique T4 specific intermolecular interactions during the infective cycle. The crystal structure is generally in the open, unliganded conformation seen in unliganded E. coli TS, as opposed to the closed, ternary complex conformation, except that the critically important C-terminus is inserted into the active site hydrogen bonded to residue Asn85, as seen in functional ternary complex structures. Other differences between E. coli TS and T4TS appear to explain the enhanced binding of folyl polyglutamate to the latter.

Stereochemistry of a multistep/bipartite methyl transfer reaction: thymidylate synthase.

Atomic structures of thymidylate synthase (TS) reveal key steps in a multi-step reaction and show quantitatively how conformation change is involved in mediating the methyl transfer reaction catalyzed by TS. Numerous alterations in TS produced by mutation, screened by complementation, and further characterized can be understood in terms of the structure and profound structure change required during the TS reaction.

Cofactor triggers the conformational change in thymidylate synthase: implications for an ordered binding mechanism.

We have solved crystal structures of two complexes with Escherichia coli thymidylate synthase (TS) bound either to the cofactor analog N10-propargyl-5,8-dideazafolate (CB3717) or to a tighter binding polygutamyl derivative of CB3717. These structures suggest that cofactor binding alone is sufficient to induce the conformational change in TS; dUMP binding is not required. Because polyglutamyl folates are the primary cofactor form in vivo, and because they can bind more tightly than dUMP to TS, these structures may represent a key intermediate along the TS reaction pathway. These structures further suggest that the dUMP binding site is accessible in the TS-cofactor analog binary complexes. Conformational flexibility of the binary complex may permit dUMP to enter the active site of TS while the cofactor is bound. Alternatively, dUMP may enter the active site from the opposite side that the cofactor appears to enter; that is, through a portal flanked by arginines that also coordinate the phosphate group in the active site. Entry of dUMP through this portal may allow dUMP to bind to a TS-cofactor binary complex in which the complex has completed its conformational transition to the catalytically competent structure.

Solvent structure in crystals of trypsin determined by X-ray and neutron diffraction.

The solvent structure in orthorhombic crystals of bovine trypsin has been independently determined by X-ray diffraction to 1.35 A resolution and by neutron diffraction to 2.1 A resolution. A consensus model of the water molecule positions was obtained using oxygen positions identified in the electron density map determined by X-ray diffraction, which were verified by comparison to D2O-H2O difference neutron scattering density. Six of 184 water molecules in the X-ray structure, all with B-factors greater than 50 A2, were found to be spurious after comparison with neutron results. Roughly two-thirds of the water of hydration expected from thermodynamic data for proteins was localized by neutron diffraction; approximately one-half of the water of hydration was located by X-ray diffraction. Polar regions of the protein are well hydrated, and significant D2O-H2O difference density is seen for a small number of water molecules in a second shell of hydration. Hydrogen bond lengths and angles calculated from unconstrained refinement of water positions are distributed about values typically seen in small molecule structures. Solvent models found in seven other bovine trypsin and trypsinogen and rat trypsin structures determined by X-ray diffraction were compared. Internal water molecules are well conserved in all trypsin structures including anionic rat trypsin, which is 65% homologous to bovine trypsin. Of the 22 conserved waters in trypsin, 19 were also found in trypsinogen, suggesting that they are located in regions of the apoprotein that are structurally conserved in the transition to the mature protein. Seven waters were displaced upon activation of trypsinogen. Water structure at crystal contacts is not generally conserved in different crystal forms. Three groups of integral structural water molecules are highly conserved in all solvent structures, including a spline of water molecules inserted between two beta-strands, which may resemble an intermediate in the formation of beta sheets during the folding of a protein.

Structure, multiple site binding, and segmental accommodation in thymidylate synthase on binding dUMP and an anti-folate.

The structure of Escherichia coli thymidylate synthase (TS) complexed with the substrate dUMP and an analogue of the cofactor methylenetetrahydrofolate was solved by multiple isomorphous replacement and refined at 1.97-A resolution to a residual of 18% for all data (16% for data greater than 2 sigma) for a highly constrained structure. All residues in the structure are clearly resolved and give a very high confidence in total correctness of the structure. The ternary complex directly suggests how methylation of dUMP takes place. C-6 of dUMP is covalently bound to gamma S of Cys-198(146) during catalysis, and the reactants are surrounded by specific hydrogen bonds and hydrophobic interactions from conserved residues. Comparison with the independently solved structure of unliganded TS reveals a large conformation change in the enzyme, which closes down to sequester the reactants and several highly ordered water molecules within a cavernous active center, away from bulk solvent. A second binding site for the quinazoline ring of the cofactor analogue was discovered by withholding addition of reducing agent during crystal storage. The chemical change in the protein is slight, and from difference density maps modification of sulfhydryls is not directly responsible for blockade of the primary site. The site, only partially overlapping with the primary site, is also surrounded by conserved residues and thus may play a functional role. The ligand-induced conformational change is not a domain shift but involves the segmental accommodation of several helices, beta-strands, and loops that move as units against the beta-sheet interface between monomers.

Pairwise specificity and sequential binding in enzyme catalysis: thymidylate synthase.

The structures of thymidylate synthase (TS) from Escherichia coli, in ternary complexes with substrate and an analogue of the cofactor, are the basis of a stereochemical model for a key reaction intermediate in the catalyzed reaction. This model is used to compare the reaction chemistry and chirality of the transferred methyl group with structures of the components, to identify those residues that participate, and to propose a stereochemical mechanism for catalysis by TS. Effects of chemical modification of specific amino acid residues and site-directed mutations of residues are correlated with structure and effects on enzyme mechanism. The ordered binding sequence of substrate deoxyuridine monophosphate and methylenetetrahydrofolate can be understood from the structure, where each forms a large part of the binding site for the other. The catalytic site serves to orient the reactants, which are sequestered along with many water molecules within a cavernous active center. Conformational changes during the reaction could involve nearby residues in ways that are not obvious in this complex.

Plastic adaptation toward mutations in proteins: structural comparison of thymidylate synthases.

The structure of thymidylate synthase (TS) from Escherichia coli was solved from cubic crystals with a = 133 A grown under reducing conditions at pH 7.0, and refined to R = 22% at 2.1 A resolution. The structure is compared with that from Lactobacillus casei solved to R = 21% at 2.3 A resolution. The structures are compared using a difference distance matrix, which identifies a common core of residues that retains the same relationship to one another in both species. After subtraction of the effects of a 50 amino acid insert present in Lactobacillus casei, differences in position of atoms correlate with temperature factors and with distance from the nearest substituted residue. The dependence of structural difference on thermal factor is parameterized and reflects both errors in coordinates that correlate with thermal factor, and the increased width of the energy well in which atoms of high thermal factor lie. The dependence of structural difference on distance from the nearest substitution also depends on thermal factors and shows an exponential dependence with half maximal effect at 3.0 A from the substitution. This represents the plastic accommodation of the protein which is parameterized in terms of thermal B factor and distance from a mutational change.

Atomic structure of thymidylate synthase: target for rational drug design.

The atomic structure of thymidylate synthase from Lactobacillus casei was determined at 3 angstrom resolution. The native enzyme is a dimer of identical subunits. The dimer interface is formed by an unusual association between five-stranded beta sheets present in each monomer. Comparison of known sequences with the Lactobacillus casei structure suggests that they all have a common core structure around which loops are inserted or deleted in different sequences. Residues from both subunits contribute to each active site. Two arginine side chains can contribute to binding phosphate on the substrate. The side chains of several conserved amino acids can account for other determinants of substrate binding.