Joseph Kraut

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

In the crystal structure of a substrate complex, the side chains of residues Asn279, Tyr271, and Arg283 of DNA polymerase β 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 β 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.

Atomic coordinates for subtilisin BPN' (or Novo).

Abstract Atomic coordinates and backbone torsion angles are tabulated for the active form of subtilisin BPN′ (or Novo). Coordinates are also given for well defined solvent molecules inside the structure and in the neighborhood of the catalytic site.

Low-resolution electron-density and anomalous-scattering-density maps of Chromatium high-potential iron protein.

Electron-density and Bijvoet-difference Fourier (anomalous-scattering-density) maps, at a resolution of 4 A, have been calculated for the Chromatium high-potential iron protein. Phases were obtained from two heavy-atom isomorphous derivatives, PtCl2−6 and UO2F3−5. The most prominent peak in the electron density map coincides with the most prominent peak in the Bijvoet-difference Fourier map, and there are no other coincidences of minor peaks in the two maps. It is concluded that the four iron atoms of Chromatium high-potential iron protein reside in a single unresolved cluster.

pH-Dependent Conformational Changes in Escherichia coli Dihydrofolate Reductase Revealed by Raman Difference Spectroscopy

The catalytic site of all dihydrofolate reductases contains an invariant carboxylic acid, equivalent to Asp-27 in Escherichia coli dihydrofolate reductase (ecDHFR). It has been found that various kinetic and ligand binding properties of ecDHFR show a pH profile with a pKa of about 6.5. The group responsible for this pKa is often assumed to be carboxyl group of Asp-27. To determine the ionization state of this carboxyl and its pKa, we have employed a novel method, based on Raman difference spectroscopy, to obtain its vibrational spectrum in situ. The method is general for the study of protein carboxyl groups, which are often significantly implicated in protein function and structure; this study establishes the methods limits and problems. The Raman difference spectrum between wild-type ecDHFR and the Asp-27 to serine mutant (D27S) in the pH range 5.6-9.0 has been taken. No protonation of the carboxyl group was detected, implying that its pKa is probably less than 5.0. We did, however, detect a pH dependence in the intensity of Raman bands in the difference spectrum with a pKa of 6.3, indicating that the apo enzyme undergoes a pH-dependent conformational change. Because the carboxyl group of Asp-27 at the active site is the only ionizable group in the binding site, other groups, away from the catalytic site, must be responsible for the pH behavior of ecDHFR.

Exploring the molecular mechanism of dihydrofolate reductase

A description of the transition-state complex of the enzyme dihydrofolate reductase is presented based upon extensive crystallographic studies of substrate/cofactor complexes from various sources. Structural elements of DHFR have been identified which contribute in different ways to effect the chemical step involving protonation and hydride transfer. Emphasis is placed upon residues, structures and solvent which create the appropriate environment for stabilization of the positively charged carbenium ions which are thought to be developed in the transition state of the enzyme-catalysed reaction. Changes in the positions of the substrate and cofactor in the active site which must occur to achieve the correct geometry for hydride transfer are also described. Finally, the structures of several site-directed mutants of DHFR are presented and the results are discussed in the context of the proposed structure for the transition state.

Crystal structure of a subtilisin BPN′ complex with N-benzoyl-l-arginine☆

The structure of a complex between subtilisin BPN′ and the product-inhibitor N-benzoyl-l-arginine has been determined from a 2.5-A electron-density difference map. The inhibitor is bound adjacent to the catalytic site of the enzyme with its carboxylate group 3 A from the reactive Ser 221 side chain, and its amido NH forming a hydrogen bond with the backbone CO of Asn 218. The guanidinium group of the inhibitor lies parallel to and in van der Waals contact with the exposed side chain of Tyr 217. This binding geometry is different from that observed in peptide chloromethyl ketone derivatives of subtilisin. Possible interpretations are, either that the new geometry represents an alternative productive binding mode, or that it represents an important non-productive binding mode for N-benzoyl-l-arginine esters and amides.

Atomic coordinates for ferricytochrome c2 of Rhodospirillum rubrum

Abstract Atomic coordinates and backbone torsion angles are tabulated for ferricytochrome c 2 of Rhodospirillum rubrum .

A structure-function study of dihydrofolate reductase by protein engineering

Several mutants of the enzyme dihydrofolate reductase (DHFR) have been engineered by oligonucleotide-directed mutagenesis of the cloned E. coli gene. The mutations were designed to address specific questions about DHFR structure-function relations that arose from the analysis of the high-resolution structure. Mutations at the active site have revealed that the invariant residue aspartate-27 is involved in substrate protonation, and not in transition-state stabilization as previously thought. The 2.0 Å (1 Å = 10-1 nm = 10-10 m) refined structures of the Asn-27 and Ser-27 mutant enzymes reveal that the enhanced binding observed for the 2,4-diamino pteridine and pyrimidine inhibitors is probably not attributable to the charge interaction between Asp-27 and a protonated N-1 of the inhibitor. Substitution of a cysteine for a proline at position 39 places two sulphydryls within bonding distance, and under certain oxidation conditions they will quantitatively form a disulphide bond. The refined 2.0 Å structures of both reduced and oxidized forms of this mutant show that only minor conformational changes occur for disulphide bond formation. The crosslinked enzyme is significantly more conformationally stable to denaturants such as guanidine hydrochloride and urea.

Crystallization and purification of the enzyme anthranilate phosphoribosyl transferase

Anthranilate phosphoribosyl transferase from the bacterium Hafnia alvei has been crystallized. This enzyme is one of a small number that constitute the biosynthetic pathway for tryptophan. Large cubic crystals were grown at 4 degrees C by dialyzing away the glycerol from a protein solution that included ammonium sulfate, polyethylene glycol and glycerol. The crystals were much more temperature stable and resistant to X-ray deterioration than a previous, similar crystal form that had included glycerol. The crystals belong to the space group I432, a = b = c = 189 A (1 A = 0.1 nm). The ratio of the monomer molecular weight, 37,000, to the volume of the unit cell suggests that there is one homodimer per asymmetric unit. The crystals diffracted to a resolution of 3.0 A at the Stanford Synchotron Radiation Laboratory X-ray source.