Structure-based inhibitors of tau aggregation.

Aggregated tau protein is associated with over 20 neurological disorders, which include Alzheimer's disease. Previous work has shown that tau's sequence segments VQIINK and VQIVYK drive its aggregation, but inhibitors based on the structure of the VQIVYK segment only partially inhibit full-length tau aggregation and are ineffective at inhibiting seeding by full-length fibrils. Here we show that the VQIINK segment is the more powerful driver of tau aggregation. Two structures of this segment determined by the cryo-electron microscopy method micro-electron diffraction explain its dominant influence on tau aggregation. Of practical significance, the structures lead to the design of inhibitors that not only inhibit tau aggregation but also inhibit the ability of exogenous full-length tau fibrils to seed intracellular tau in HEK293 biosensor cells into amyloid. We also raise the possibility that the two VQIINK structures represent amyloid polymorphs of tau that may account for a subset of prion-like strains of tau.

The formation, function and regulation of amyloids: insights from structural biology.

Amyloid diseases are characterized by the accumulation of insoluble, ß-strand-rich aggregates. The underlying structural conversions are closely associated with cellular toxicity, but can also drive the formation of functional protein assemblies. In recent years, studies in the field of structural studies have revealed astonishing insights into the origins, mechanisms and implications of amyloid formation. Notably, high-resolution crystal structures of peptides in amyloid-like fibrils and prefibrillar oligomers have become available despite their challenging chemical nature. Nuclear magnetic resonance spectroscopy has revealed that dynamic local polymorphisms in the benign form of the prion protein affect the transformation into amyloid fibrils and the transmissibility of prion diseases. Studies of the structures and interactions of chaperone proteins help us to understand how the cellular proteostasis network is able to recognize different stages of aberrant protein folding and prevent aggregation. In this review, we will focus on recent developments that connect the different aspects of amyloid biology and discuss how understanding the process of amyloid formation and the associated defence mechanisms can reveal targets for pharmacological intervention that may become the first steps towards clinically viable treatment strategies.

Self-assembly in the carboxysome: a viral capsid-like protein shell in bacterial cells.

Many proteins self-assemble to form large supramolecular complexes. Numerous examples of these structures have been characterized, ranging from spherical viruses to tubular protein assemblies. Some new kinds of supramolecular structures are just coming to light, while it is likely there are others that have not yet been discovered. The carboxysome is a subcellular structure that has been known for more than 40 years, but whose structural and functional details are just now emerging. This giant polyhedral body is constructed as a closed shell assembled from several thousand protein subunits. Within this protein shell, the carboxysome encapsulates the CO(2)-fixing enzymes, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) and carbonic anhydrase; this arrangement enhances the efficiency of cellular CO(2) fixation. The carboxysome is present in many photosynthetic and chemoautotrophic bacteria, and so plays an important role in the global carbon cycle. It also serves as the prototypical member of what appears to be a large class of primitive protein-based organelles in bacteria. A series of crystal structures is beginning to reveal the secrets of how the carboxysome is assembled and how it enhances the efficiency of CO(2) fixation. Some of the assembly principles revealed in the carboxysome are reminiscent of those seen in icosahedral viral capsids. In addition, the shell appears to be perforated by pores for metabolite transport into and out of the carboxysome, suggesting comparisons to the pores through oligomeric transmembrane proteins, which serve to transport small molecules across the membrane bilayers of cells and eukaryotic organelles.

Crystal structure of a protein repair methyltransferase from Pyrococcus furiosus with its L-isoaspartyl peptide substrate.

Protein L-isoaspartyl (D-aspartyl) methyltransferases (EC 2.1.1.77) are found in almost all organisms. These enzymes catalyze the S-adenosylmethionine (AdoMet)-dependent methylation of isomerized and racemized aspartyl residues in age-damaged proteins as part of an essential protein repair process. Here, we report crystal structures of the repair methyltransferase at resolutions up to 1.2 A from the hyperthermophilic archaeon Pyrococcus furiosus. Refined structures include binary complexes with the active cofactor AdoMet, its reaction product S-adenosylhomocysteine (AdoHcy), and adenosine. The enzyme places the methyl-donating cofactor in a deep, electrostatically negative pocket that is shielded from solvent. Across the multiple crystal structures visualized, the presence or absence of the methyl group on the cofactor correlates with a significant conformational change in the enzyme in a loop bordering the active site, suggesting a role for motion in catalysis or cofactor exchange. We also report the structure of a ternary complex of the enzyme with adenosine and the methyl-accepting polypeptide substrate VYP(L-isoAsp)HA at 2.1 A. The substrate binds in a narrow active site cleft with three of its residues in an extended conformation, suggesting that damaged proteins may be locally denatured during the repair process in cells. Manual and computer-based docking studies on different isomers help explain how the enzyme uses steric effects to make the critical distinction between normal L-aspartyl and age-damaged L-isoaspartyl and D-aspartyl residues.

Structures of cytochrome c-549 and cytochrome c6 from the cyanobacterium Arthrospira maxima.

Cytochrome c(6) and cytochrome c-549 are small (89 and 130 amino acids, respectively) monoheme cytochromes that function in photosynthesis. They appear to have descended relatively recently from the same ancestral gene but have diverged to carry out very different functional roles, underscored by the large difference between their midpoint potentials of nearly 600 mV. We have determined the X-ray crystal structures of both proteins isolated from the cyanobacterium Arthrospira maxima. The two structures are remarkably similar, superimposing on backbone atoms with an rmsd of 0.7 A. Comparison of the two structures suggests that differences in solvent exposure of the heme and the electrostatic environment of the heme propionates, as well as in heme iron ligation, are the main determinants of midpoint potential in the two proteins. In addition, the crystal packing of both A. maxima cytochrome c-549 and cytochrome c(6) suggests that the proteins oligomerize. Finally, the cytochrome c-549 dimer we observe can be readily fit into the recently described model of cyanobacterial photosystem II.

The crystal structure of a heptameric archaeal Sm protein: Implications for the eukaryotic snRNP core.

Sm proteins form the core of small nuclear ribonucleoprotein particles (snRNPs), making them key components of several mRNA-processing assemblies, including the spliceosome. We report the 1.75-A crystal structure of SmAP, an Sm-like archaeal protein that forms a heptameric ring perforated by a cationic pore. In addition to providing direct evidence for such an assembly in eukaryotic snRNPs, this structure (i) shows that SmAP homodimers are structurally similar to human Sm heterodimers, (ii) supports a gene duplication model of Sm protein evolution, and (iii) offers a model of SmAP bound to single-stranded RNA (ssRNA) that explains Sm binding-site specificity. The pronounced electrostatic asymmetry of the SmAP surface imparts directionality to putative SmAP-RNA interactions.

Structural basis of non-specific lipid binding in maize lipid-transfer protein complexes revealed by high-resolution X-ray crystallography.

Non-specific lipid-transfer proteins (nsLTPs) are involved in the movement of phospholipids, glycolipids, fatty acids, and steroids between membranes. Several structures of plant nsLTPs have been determined both by X-ray crystallography and nuclear magnetic resonance. However, the detailed structural basis of the non-specific binding of hydrophobic ligands by nsLTPs is still poorly understood. In order to gain a better understanding of the structural basis of the non-specific binding of hydrophobic ligands by nsLTPs and to investigate the plasticity of the fatty acid binding cavity in nsLTPs, seven high-resolution (between 1.3 A and 1.9 A) crystal structures have been determined. These depict the nsLTP from maize seedlings in complex with an array of fatty acids.A detailed comparison of the structures of maize nsLTP in complex with various ligands reveals a new binding mode in an nsLTP-oleate complex which has not been seen before. Furthermore, in the caprate complex, the ligand binds to the protein cavity in two orientations with equal occupancy. The volume of the hydrophobic cavity in the nsLTP from maize shows some variation depending on the size of the bound ligands. The structural plasticity of the ligand binding cavity and the predominant involvement of non-specific van der Waals interactions with the hydrophobic tail of the ligands provide a structural explanation for the non-specificity of maize nsLTP. The hydrophobic cavity accommodates various ligands from C10 to C18. The C18:1 ricinoleate with its hydroxyl group hydrogen bonding to Ala68 possibly mimics cutin monomer binding which is of biological importance. Some of the myristate binding sites in human serum albumin resemble the maize nsLTP, implying the importance of a helical bundle in accommodating the non-specific binding of fatty acids.

Crystal structure of T7 gene 4 ring helicase indicates a mechanism for sequential hydrolysis of nucleotides.

We have determined the crystal structure of an active, hexameric fragment of the gene 4 helicase from bacteriophage T7. The structure reveals how subunit contacts stabilize the hexamer. Deviation from expected six-fold symmetry of the hexamer indicates that the structure is of an intermediate on the catalytic pathway. The structural consequences of the asymmetry suggest a "binding change" mechanism to explain how cooperative binding and hydrolysis of nucleotides are coupled to conformational changes in the ring that most likely accompany duplex unwinding. The structure of a complex with a nonhydrolyzable ATP analog provides additional evidence for this hypothesis, with only four of the six possible nucleotide binding sites being occupied in this conformation of the hexamer. This model suggests a mechanism for DNA translocation.

Crystal structure of the helicase domain from the replicative helicase-primase of bacteriophage T7.

Helicases that unwind DNA at the replication fork are ring-shaped oligomeric enzymes that move along one strand of a DNA duplex and catalyze the displacement of the complementary strand in a reaction that is coupled to nucleotide hydrolysis. The helicase domain of the replicative helicase-primase protein from bacteriophage T7 crystallized as a helical filament that resembles the Escherichia coli RecA protein, an ATP-dependent DNA strand exchange factor. When viewed in projection along the helical axis of the crystals, six protomers of the T7 helicase domain resemble the hexameric rings seen in electron microscopic images of the intact T7 helicase-primase. Nucleotides bind at the interface between pairs of adjacent subunits where an arginine is near the gamma-phosphate of the nucleotide in trans. The bound nucleotide stabilizes the folded conformation of a DNA-binding motif located near the center of the ring. These and other observations suggest how conformational changes are coupled to DNA unwinding activity.

An open and closed case for all polymerases.

The recently determined structures of HIV-1 reverse transcriptase and Taq DNA polymerase in complex with DNA primer-template and an incoming nucleotide have shown that a large conformational change configures the polymerase active site for nucleotidyl transfer. The structure of reverse transcriptase in the catalytic complex will open the path to the rational design of novel nucleoside analog inhibitors of viral replication.

Crystal structures of human DNA polymerase beta complexed with gapped and nicked DNA: evidence for an induced fit mechanism.

DNA polymerase beta (pol beta) fills single nucleotide (nt) gaps in DNA produced by the base excision repair pathway of mammalian cells. Crystal structures have been determined representing intermediates in the 1 nt gap-filling reaction of pol beta: the binary complex with a gapped DNA substrate (2.4 A resolution), the ternary complex including ddCTP (2.2 A), and the binary product complex containing only nicked DNA (2.6 A). Upon binding ddCTP to the binary gap complex, the thumb subdomain rotates into the closed conformation to contact the otherwise solvent-exposed ddCTP-template base pair. Thumb movement triggers further conformational changes which poise catalytic residue Asp192, dNTP, and template for nucleotidyl transfer, effectively assembling the active site. In the product nicked DNA complex, the thumb returns to the open conformation as in the gapped binary DNA complex, facilitating dissociation of the product. These findings suggest that pol beta may enhance fidelity by an induced fit mechanism in which correct base pairing between template and incoming dNTP induces alignment of catalytic groups for catalysis (via thumb closure), but incorrect base pairing will not. The structures also reveal that pol beta binds both gapped and nicked DNA with a 90 degrees kink occurring precisely at the 5'-phosphodiester linkage of the templating residue. If the DNA were not kinked in this way, contact between the thumb and dNTP-template base pair, presumably important for the checking mechanism, would be impossible, especially when the gap is but a single nucleotide. Such a 90 degrees kink may be a mechanistic feature employed by any polymerase involved in filling gaps to completion.

Loop and subdomain movements in the mechanism of Escherichia coli dihydrofolate reductase: crystallographic evidence.

The reaction catalyzed by Escherichia coli dihydrofolate reductase (ecDHFR) cycles through five detectable kinetic intermediates: holoenzyme, Michaelis complex, ternary product complex, tetrahydrofolate (THF) binary complex, and THF.NADPH complex. Isomorphous crystal structures analogous to these five intermediates and to the transition state (as represented by the methotrexate-NADPH complex) have been used to assemble a 2.1 A resolution movie depicting loop and subdomain movements during the catalytic cycle (see Supporting Information). The structures suggest that the M20 loop is predominantly closed over the reactants in the holoenzyme, Michaelis, and transition state complexes. But, during the remainder of the cycle, when nicotinamide is not bound, the loop occludes (protrudes into) the nicotinamide-ribose binding pocket. Upon changing from the closed to the occluded conformation, the central portion of the loop rearranges from beta-sheet to 3(10) helix. The change may occur by way of an irregularly structured open loop conformation, which could transiently admit a water molecule into position to protonate N5 of dihydrofolate. From the Michaelis to the transition state analogue complex, rotation between two halves of ecDHFR, the adenosine binding subdomain and loop subdomain, closes the (p-aminobenzoyl)glutamate (pABG) binding crevice by approximately 0.5 A. Resulting enhancement of contacts with the pABG moiety may stabilize puckering at C6 of the pteridine ring in the transition state. The subdomain rotation is further adjusted by cofactor-induced movements (approximately 0.5 A) of helices B and C, producing a larger pABG cleft in the THF.NADPH analogue complex than in the THF analogue complex. Such movements may explain how THF release is assisted by NADPH binding. Subdomain rotation is not observed in vertebrate DHFR structures, but an analogous loop movement (residues 59-70) appears to similarly adjust the pABG cleft width, suggesting that these movements are important for catalysis. Loop movement, also unobserved in vertebrate DHFR structures, may preferentially weaken NADP+ vs NADPH binding in ecDHFR, an evolutionary adaptation to reduce product inhibition in the NADP+ rich environment of prokaryotes.

A structural basis for metal ion mutagenicity and nucleotide selectivity in human DNA polymerase beta.

When crystals of human DNA polymerase beta (pol beta) complexed with DNA [Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., & Kraut, J. (1996) Biochemistry 35, 12742-12761] are soaked in the presence of dATP and Mn2+, X-ray structural analysis shows that nucleotidyl transfer to the primer 3'-OH takes place directly in the crystals, even though the DNA is blunt-ended at the active site. Under similar crystal-soaking conditions, there is no evidence for a reaction when Mn2+ is replaced by Mg2+, which is thought to be the divalent metal ion utilized by most polymerases in vivo. These results suggest that one way Mn2+ may manifest its mutagenic effect on polymerases is by promoting greater reactivity than Mg2+ at the catalytic site, thereby allowing the nucleotidyl transfer reaction to take place with little or no regard to instructions from a template. Non-template-directed nucleotidyl transfer is also observed when pol beta-DNA cocrystals are soaked in the presence of dATP and Zn2+, but the reaction products differ in that the sugar moiety of the incorporated nucleotide appears distorted or otherwise cleaved, in agreement with reports that Zn2+ may act as a polymerase inhibitor rather than as a mutagen [Sirover, M. A., & Loeb, L. A. (1976) Science 194, 1434-1436]. Although no reaction is observed when crystals are soaked in the presence of dATP and other metal ions such as Ca2+, Co2+, Cr3+, or Ni2+, X-ray structural analyses show that these metal ions coordinate the triphosphate moiety of the nucleotide in a manner that differs from that observed with Mg2+. In addition, all metal ions tested, with the exception of Mg2+, promote a change in the side-chain position of aspartic acid 192, which is one of three highly conserved active-site carboxylate residues. Soaking experiments with nucleotides other than dATP (namely, dCTP, dGTP, dTTP, ATP, ddATP, ddCTP, AZT-TP, and dATP alpha S) reveal a non-base-specific binding site on pol beta for the triphosphate and sugar moieties of a nucleotide, suggesting a possible mechanism for nucleotide selectivity whereby triphosphate-sugar binding precedes a check for correct base pairing with the template.

Crystal structures of human DNA polymerase beta complexed with DNA: implications for catalytic mechanism, processivity, and fidelity.

Mammalian DNA polymerase beta (pol beta) is a small (39 kDa) DNA gap-filling enzyme that comprises an amino-terminal 8-kDa domain and a carboxy-terminal 31-kDa domain. In the work reported here, crystal structures of human pol beta complexed with blunt-ended segments of DNA show that, although the crystals belong to a different space group, the DNA is nevertheless bound in the pol beta binding channel in the same way as the DNA in previously reported structures of rat pol beta complexed with a template-primer and ddCTP [Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H., & Kraut, J. (1994) Science 264, 1891-1903]. The 8-kDa domain is in one of three previously observed positions relative to the 31-kDa domain, suggesting that the 8-kDa domain may assume only a small number of stable conformations. The thumb subdomain is in a more open position in the human pol beta-DNA binary complex than it is in the rat pol beta-DNA-ddCTP ternary complex, and a closing thumb upon nucleotide binding could represent the rate-limiting conformational change that has been observed in pre-steady-state kinetic studies. Intermolecular contacts between the DNA and the 8-kDa domain of a symmetry-related pol beta molecule reveal a plausible binding site on the 8-kDa domain for the downstream oligonucleotide of a gapped-DNA substrate; in addition to a lysine-rich binding pocket that accommodates a 5'-PO4 end group, the 8-kDa domain also contains a newly discovered helix-hairpin-helix (HhH) motif that binds to DNA in the same way as does a structurally and sequentially homologous HhH motif in the 31-kDa domain. DNA binding by both HhH motifs is facilitated by a metal ion. In that HhH motifs have been identified in other DNA repair enzymes and DNA polymerases, the HhH-DNA interactions observed in pol beta may be applicable to a broad range of DNA binding proteins. The sequence similarity between the HhH motif of endonuclease III from Escherichia coli and the HhH motif of the 8-kDa domain of pol beta is particularly striking in that all of the conserved residues are clustered in one short sequence segment, LPGVGXK, where LPGV corresponds to a type II beta-turn (the hairpin turn), and GXK corresponds to a part of the HhH motif that is proposed to be critical for DNA binding and catalysis for both enzymes. These results suggest that endonuclease III and the 8-kDa domain of pol beta may employ a similar mode of DNA binding and may have similar catalytic mechanisms for their respective DNA lyase activities. A model for productive binding of pol beta to a gapped-DNA substrate requires a 90 degrees bend in the single-stranded template, which could enhance nucleotide selectivity during DNA repair or replication.

Characterization of the metal ion binding helix-hairpin-helix motifs in human DNA polymerase beta by X-ray structural analysis.

X-ray crystallographic studies have shown that DNA binding by human polymerase beta (pol beta) occurs primarily through two structurally and sequentially homologous helix-hairpin-helix (HhH) motifs, one in the fingers subdomain and the other in the 8-kDa domain [Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., & Kraut, J. (1996a) Biochemistry 35, 12742-12761]. In that DNA binding by each HhH motif is facilitated by a metal ion, we set out to determine the identity of the metal ion that most likely binds to the HhH motif in vivo. Crystal soaking experiments were performed on human pol beta-DNA cocrystals with Mg2+, Ca2+, Na+, and K+, the four most prevalent metal ions in the cell, and in each case a data set was collected and the resulting structure was refined. Under the conditions tested, the HhH motifs of pol beta have an affinity for these biologically prevalent metal ions in the order Mg2+ < Ca2+ < Na+ < K+, with K+ displaying the strongest binding. Crystals soaked in the presence of Tl+, a commonly used spectroscopic probe for K+, were too X-ray-sensitive to establish the binding behavior of Tl+, but soaking experiments with Ba2+ and Cs+ resulted in relatively stable crystals that gave evidence of metal ion binding in both HhH motifs, confirming that larger monovalent and divalent metal ions are capable of binding to the HhH metal sites. Although Mn2+, which has been categorized as a potent polymerase mutagen, binds to the HhH motifs with a greater affinity than Mg2+, Mn2+ does not bind to the HhH motifs in the presence of equimolar concentrations of Na+. These results suggest that in vivo, where Mn2+ is present only in trace amounts, Mn2+ probably does not have a large effect on DNA binding and may instead manifest a mutagenic effect on pol beta primarily by distorting nucleotide binding or by directly affecting the catalytic step [Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., & Kraut, J. (1996b) Biochemistry 35, 12762-12777]. Crystal soaking experiments with 31-kDa apoenzyme crystals show that, in the absence of DNA, the HhH motif in the fingers subdomain binds metal ions with either much lower occupancy or not at all, indicating that metal ion binding is dependent on the presence of the DNA substrate.

Crystallization of the A alpha subunit of protein phosphatase 2A.

The A alpha subunit of human protein phosphatase 2A forms crystals in space group P2(1) with cell dimensions a = 104.0, b = 174.9, c = 168.2 A, and beta angle = 90.2 degrees. At cryogenic temperatures, the crystals diffracted to a resolution limit of approximately 3.0 A. Based on the unit cell dimensions and a calculated molecular mass of 65,277 Da, the Matthews coefficient suggests eight molecules per asymmetric unit. Two native data sets were collected to a nominal resolution of 3.0 A and merged to provide a set that is 93% complete, with Rsym of 9.9%.

Enzyme-DNA interactions required for efficient nucleotide incorporation and discrimination in human DNA polymerase beta.

In the crystal structure of a substrate complex, the side chains of residues Asn279, Tyr271, and Arg283 of DNA polymerase beta are within hydrogen bonding distance to the bases of the incoming deoxynucleoside 5'-triphosphate (dNTP), the terminal primer nucleotide, and the templating nucleotide, respectively (Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H., and Kraut, J. (1994) Science 264, 1891-1903). We have altered these side chains through individual site-directed mutagenesis. Each mutant protein was expressed in Escherichia coli and was soluble. The mutant enzymes were purified and characterized to probe their role in nucleotide discrimination and catalysis. A reversion assay was developed on a short (5 nucleotide) gapped DNA substrate containing an opal codon to assess the effect of the amino acid substitutions on fidelity. Substitution of the tyrosine at position 271 with phenylalanine or histidine did not influence catalytic efficiency (kcat/Km) or fidelity. The hydrogen bonding potential between the side chain of Asn279 and the incoming nucleotide was removed by replacing this residue with alanine or leucine. Although catalytic efficiency was reduced as much as 17-fold for these mutants, fidelity was not. In contrast, both catalytic efficiency and fidelity decreased dramatically for all mutants of Arg283 (Ala > Leu > Lys). The fidelity and catalytic efficiency of the alanine mutant of Arg283 decreased 160- and 5000-fold, respectively, relative to wild-type enzyme. Sequence analyses of the mutant DNA resulting from short gap-filling synthesis indicated that the types of base substitution errors produced by the wild-type and R283A mutant were similar and indicated misincorporations resulting in frequent T.dGTP and A.dGTP mispairing. With R283A, a dGMP was incorporated opposite a template thymidine as often as the correct nucleotide. The x-ray crystallographic structure of the alanine mutant of Arg283 verified the loss of the mutated side chain. Our results indicate that specific interactions between DNA polymerase beta and the template base, but not hydrogen bonding to the incoming dNTP or terminal primer nucleotide, are required for both high catalytic efficiency and nucleotide discrimination.

Isomorphous crystal structures of Escherichia coli dihydrofolate reductase complexed with folate, 5-deazafolate, and 5,10-dideazatetrahydrofolate: mechanistic implications.

Crystal structures of Escherichia coli dihydrofolate reductase (ecDHFR, EC 1.5.1.3) in binary complexes with folate, 5-deazafolate (5dfol), and 5,10-dideazatetrahydrofolate (ddTHF) have been refined to R-factors of 13.7%, 14.9%, and 14.5%, respectively, all at 1.9 A. All three are isomorphous with a previously reported binary complex of ecDHFR with methotrexate (MTX), in space group P6(1), two molecules per asymmetric unit [Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, R. C., & Kraut, J. (1982) J. Biol. Chem. 257, 13650-13662]. A hitherto unobserved water molecule is hydrogen bonded to the pteridine N5 and O4 in both molecules of the asymmetric unit of the folate complex (but not the 5dfol or ddTHF complexes), supporting the hypothesis that N5 protonation of bound substrate, an important step of the DHFR reaction, occurs by way of such a water molecule. There is no indication of a hydrogen bond between N8 of 5dfol and the backbone carbonyl of Ile-5, suggesting that the bacterial enzyme, unlike the human enzyme [Davies, J. F., II, Delcamp, T. J., Prendergast, N. J., Ashford, V. A., Freisheim, J. H., & Kraut, J. (1990) Biochemistry 29, 9467-9479], does not favor protonation at N8. Perhaps this explains why bacterial DHFR is much less effective than vertebrate DHFR in folate reduction. When the ecDHFR.NADPH complex (space group P3221; M. R. Sawaya, in preparation) is superimposed on the folate and 5dfol complexes, the distances from pteridine C6 to nicotinamide C4 were found to be 2.9 and 2.8 A, respectively, in close agreement with the theoretically calculated optimal distance in the transition state for hydride transfer [Wu, Y. D., & Houk, K. N. (1987) J. Am. Chem. Soc. 109, 906-908, 2226-2227]. In contrast to the planar ring system of folate or 5dfol, the reduced pteridine ring of ddTHF is severely puckered and bent toward the nicotinamide pocket, with the reduced pyridine ring assuming a half-chair type of conformation. This change in shape causes the pteridine ring to bind with O4 closer to Trp-22(N epsilon 1) by over 0.5 A, so that an invariant water molecule now bridges these two atoms with ideal hydrogen bonds. Furthermore, while the pABA rings of folate and 5dfol are nearly coincident and closer to the alpha C helix than to the alpha B helix, those of MTX and ddTHF are displaced along the binding crevice by approximately 1.1 and 0.6 A, respectively, and are equidistant from alpha B and alpha C.(ABSTRACT TRUNCATED AT 400 WORDS)

Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and ddCTP.

Two ternary complexes of rat DNA polymerase beta (pol beta), a DNA template-primer, and dideoxycytidine triphosphate (ddCTP) have been determined at 2.9 A and 3.6 A resolution, respectively. ddCTP is the triphosphate of dideoxycytidine (ddC), a nucleoside analog that targets the reverse transcriptase of human immunodeficiency virus (HIV) and is at present used to treat AIDS. Although crystals of the two complexes belong to different space groups, the structures are similar, suggesting that the polymerase-DNA-ddCTP interactions are not affected by crystal packing forces. In the pol beta active site, the attacking 3'-OH of the elongating primer, the ddCTP phosphates, and two Mg2+ ions are all clustered around Asp190, Asp192, and Asp256. Two of these residues, Asp190 and Asp256, are present in the amino acid sequences of all polymerases so far studied and are also spatially similar in the four polymerases--the Klenow fragment of Escherichia coli DNA polymerase I, HIV-1 reverse transcriptase, T7 RNA polymerase, and rat DNA pol beta--whose crystal structures are now known. A two-metal ion mechanism is described for the nucleotidyl transfer reaction and may apply to all polymerases. In the ternary complex structures analyzed, pol beta binds to the DNA template-primer in a different manner from that recently proposed for other polymerase-DNA models.

Crystal structure of rat DNA polymerase beta: evidence for a common polymerase mechanism.

Structures of the 31-kilodalton catalytic domain of rat DNA polymerase beta (pol beta) and the whole 39-kilodalton enzyme were determined at 2.3 and 3.6 angstrom resolution, respectively. The 31-kilodalton domain is composed of fingers, palm, and thumb subdomains arranged to form a DNA binding channel reminiscent of the polymerase domains of the Klenow fragment of Escherichia coli DNA polymerase I, HIV-1 reverse transcriptase, and bacteriophage T7 RNA polymerase. The amino-terminal 8-kilodalton domain is attached to the fingers subdomain by a flexible hinge. The two invariant aspartates found in all polymerase sequences and implicated in catalytic activity have the same geometric arrangement within structurally similar but topologically distinct palms, indicating that the polymerases have maintained, or possibly re-evolved, a common nucleotidyl transfer mechanism. The location of Mn2+ and deoxyadenosine triphosphate in pol beta confirms the role of the invariant aspartates in metal ion and deoxynucleoside triphosphate binding.