The low ionic strength crystal structure of horse cytochrome c at 2.1 A resolution and comparison with its high ionic strength counterpart.

BACKGROUND: Cytochrome c is an integral part of the mitochondrial respiratory chain. It is confined to the intermembrane space of mitochondria, and has the function of transferring electrons between its redox partners. Solution studies of cytochrome c indicate that the conformation of the molecule is sensitive to the ionic strength of the medium. RESULTS: The crystal structures of cytochromes c from several species have been solved at extremely high ionic strengths of near-saturated solutions of ammonium sulfate. Here we present the first crystal structure of ferricytochrome c at low ionic strength refined at 2.1 A resolution. In general, the structure has the same features as those determined earlier. However, there are some differences in both backbone and side-chain conformations in several areas. These areas coincide with those observed by NMR and resonance Raman spectroscopy to be sensitive to ionic strength. CONCLUSIONS: Neither ionic strength nor crystal-packing interactions have much influence on the conformation of horse cytochrome c. Nevertheless, some differences in the side-chain conformations at high and low ionic strengths may be important for understanding how the protein functions. Close examination of the gamma-turn (residues 27-29) conserved in cytochromes c leads us to propose the 'negative classical' gamma-turn to describe this unusual feature.

Crystallization of wild-type and mutant ferricytochromes c at low ionic strength: seeding technique and X-ray diffraction analysis.

Ferricytochromes c were crystallized at low ionic strength by macroseeding techniques. Large crystals were grown by seed-induced self-nucleation which occurred anywhere in the drop, regardless of the location of the seed crystal. This unusual crystal-seeding method worked reproducibly in our hands, and X-ray quality crystals have been prepared of several ferricytochromes c: horse, rat (recombinant wild type), and two site-directed mutants of the latter, tyrosine 67 to phenylalanine (Y67F) and asparagine 52 to isoleucine (N52I). Crystals of any one of these four proteins could be used as seeds for the crystallization of any one of the others. All the crystals are of the same crystal form, with space group P2(1)2(1)2(1). There are two protein molecules per asymmetric unit. The crystals are stable in the X-ray beam and diffract to at least 2.0 A, resolution. Full crystallographic data sets have been collected from single crystals of all four proteins.

2.5 A structure of aspartate carbamoyltransferase complexed with the bisubstrate analog N-(phosphonacetyl)-L-aspartate.

In an X-ray diffraction study using the method of multiple isomorphous replacement, the structure of aspartate carbamoyltransferase (EC 2.1.3.2) complexed with the bisubstrate analog N-(phosphonacetyl)-L-aspartate (PALA) has been solved to 2.5 A. Ten rounds of model building and 123 cycles of restrained reciprocal space refinement have resulted in a model containing 94.4% of the theoretical atoms of the protein-inhibitor complex with an R-factor of 0.231. The fit of the model to the density is excellent, except for occasional side-chains and two sections of the regulatory chains that may be disordered. The electron density for the PALA molecule is readily identifiable for both catalytic (c) chains of the asymmetric unit and bonding interactions with several important residues including Ser52, Arg54, Thr55, Ser80, Lys84, Arg105, His134, Arg165, Arg229 and Gln231 are apparent. The carboxylate groups of the PALA molecule are in a nearly cis conformation. Gross quaternary changes between the T and R forms are noted and in agreement with earlier work from this laboratory. Namely, in the new structure the catalytic trimers move apart by 12 A along the 3-fold axis of the enzyme and relocate by 10 degrees relative to each other, adopting a more eclipsed position. The regulatory (r) chains in the new structure reorient about their 2-fold axis by 15 degrees. Large tertiary changes that include domain migration and rearrangement are also present between these two forms. In the R form both domains of the catalytic chain relocate closer to each other in order to bind to the inhibitor. The polar domain seems to bind primarily to the carbamoyl phosphate moiety of PALA, and the equatorial domain binds primarily to the L-aspartate moiety. Other changes in tertiary structure bring the 80s loop (from an adjacent catalytic chain) and the 240s loop into a position to interact with the PALA molecule. Changes have been searched for in all interface regions of the enzyme. While the C1-C4 and C1-R4 regions have been completely altered, most of the other interchain interfaces are similar in the T and R forms. The intrachain interfaces, between domains of the same catalytic chains, have undergone some reorganization as these domains move closer to each other when the inhibitor is bound. This new structure allows a reinterpretation of genetic and chemical modification studies done to date.(ABSTRACT TRUNCATED AT 400 WORDS)

The binding of N-(phosphonacetyl)-L-aspartate to aspartate carbamoyltransferase of Escherichia coli.

A more precise description of the binding of N-(phosphonacetyl)-L- aspartate to the catalytic chains of aspartate carbamoyltransferase clarifies aspects of the specificity of this enzyme toward its substrates, carbamoylphosphate and L-aspartate, and suggests a catalytic role for His-134.

Structure at 2.9-A resolution of aspartate carbamoyltransferase complexed with the bisubstrate analogue N-(phosphonacetyl)-L-aspartate.

In an x-ray diffraction study by the isomorphous replacement method, the structure of the complex of aspartate carbamoyltransferase (EC 2.1.3.2) bound to the bisubstrate analogue N-(phosphonacetyl)-L-aspartate has been solved to 2.9-A resolution (R = 0.24). The large quaternary structural changes previously deduced by molecular replacement methods have been confirmed: the two catalytic trimers (c3) move apart by 12 A and mutually reorient by 10 degrees, and the regulatory dimers (r2) reorient each about its twofold axis by about 15 degrees. In this, the T-to-R transition, new polar interactions develop between equatorial domains of c chains and the Zn domain of r chains. Within the c chain the two domains, one binding the phosphonate moiety (polar) and the other binding the aspartate moiety (equatorial) of the inhibitor N-(phosphonacetyl)-L-aspartate, move closer together. The Lys-84 loop makes a large relocation so that this residue and Ser-80 bind to the inhibitor of an adjacent catalytic chain within c3. A very large change in tertiary structure brings the 230-245 loop nearer the active site, allowing Arg-229 and Gln-231 to bind to the inhibitor. His-134 is close to the carbonyl group of the inhibitor, and Ser-52 is adjacent to its phosphonate group. However, no evidence exists in the literature for phosphorylation of serine in the mechanism. Residues studied by other methods, including Cys-47, Tyr-165, Lys-232, and Tyr-240, are too far from the inhibitor to have a direct interaction.

Refined crystal structures of Escherichia coli and chicken liver dihydrofolate reductase containing bound trimethoprim.

Refined crystal structures are reported for complexes of Escherichia coli and chicken dihydrofolate reductase containing the antibiotic trimethoprim (TMP). Structural comparison of these two complexes reveals major geometrical differences in TMP binding that may be important in understanding the stereo-chemical basis of this inhibitor's selectivity for bacterial dihydrofolate reductases. For TMP bound to chicken dihydrofolate reductase we observe an altered binding geometry in which the 2,4-diaminopyrimidine occupies a position in closer proximity (by approximately 1 A) to helix alpha B compared to the pyrimidine position for TMP or methotrexate bound to E. coli dihydrofolate reductase. One important consequence of this deeper insertion of the pyrimidine into the active site of chicken dihydrofolate reductase is the loss of a potential hydrogen bond that would otherwise form between the carbonyl oxygen of Val-115 and the inhibitor's 4-amino group. In addition, for TMP bound to E. coli dihydrofolate reductase, the inhibitor's benzyl side chain is positioned low in the active-site pocket pointing down toward the nicotinamide-binding site, whereas, in chicken dihydrofolate reductase, the benzyl group is accommodated in a side channel running upward and away from the cofactor. As a result, the torsion angles about the C5-C7 and C7-C1' bonds for TMP bound to the bacterial reductase (177 degrees, 76 degrees) differ significantly from the corresponding angles for TMP bound to chicken dihydrofolate reductase (-85 degrees, 102 degrees). Finally, when TMP binds to the chicken holoenzyme, the Tyr-31 side chain undergoes a large conformational change (average movement is 5.4 A for all atoms beyond C beta), rotating down into a new position where it hydrogen bonds via an intervening water molecule to the backbone carbonyl oxygen of Trp-24.

Dihydrofolate reductase. The stereochemistry of inhibitor selectivity.

X-ray structural results are reported for 10 triazine and pyrimidine inhibitors of dihydrofolate reductase, each one studied as a ternary complex with NADPH and chicken dihydrofolate reductase. Analysis of these data and comparison with structural results from the preceding paper (Matthews, D.A., Bolin, J.T., Burridge, J.M., Filman, D.J., Volz, K.W., Kaufman, B. T., Beddell, C.R., Champness, J.N., Stammers, D.K., and Kraut, J. (1985) J. Biol. Chem. 260, 381-391) in which we contrasted binding of the antibiotic trimethoprim (TMP) to chicken dihydrofolate reductase on the one hand with its binding to Escherichia coli dihydrofolate reductase on the other, permit identification of differences that are important in accounting for TMP's selectivity. The crystallographic evidence strongly suggests that loss of a potential hydrogen bond between the 4-amino group of TMP and the backbone carbonyl of Val-115 when TMP binds to chicken dihydrofolate reductase but not when it binds to the E. coli reductase is the major factor responsible for this drug's more potent inhibition of bacterial dihydrofolate reductase. A key finding of the current study which is important in understanding why TMP binds differently to chicken and E. coli dihydrofolate reductases is that residues on opposite sides of the active-site cleft in chicken dihydrofolate reductase are about 1.5-2.0 A further apart than are structurally equivalent residues in the E. coli enzyme.

Leucine aminopeptidase from bovine lens and hog kidney. Comparison using immunological techniques, electron microscopy, and X-ray diffraction.

The crystallization of leucine aminopeptidase from hog kidney is reported for the first time. The crystals which diffract to 4-A resolution have the space group P2(1)2(1)2(1) (a = 186.3 A, b = 223.2 A, and c = 80.5 A) and contain four hexamers per unit cell, or one per asymmetric unit. Electron micrographic images of hog kidney leucine aminopeptidase are indistinguishable from micrographs of beef leucine aminopeptidase taken under the same conditions (10). These reveal an equilateral triangle of about 85 A per side, seemingly made of three 40-A diameter spheres. This triangle is circumscribed by another concentric, less-dense triangle of 120 A per side which is rotated 60 degrees with respect to the inner triangle. Immunodiffusion and microcomplement fixation assays indicate that the two enzymes share greater than 90% amino acid sequence homology. This similarity is corroborated by peptide maps of tryptic fragments of the radioiodinated enzymes. The model of the quaternary structure proposed to explain the appearance of electron micrographs of single molecule and crystalline bovine lens enzyme also describes the hog kidney enzyme equally well. That the model of leucine aminopeptidase originally proposed for the beef enzyme also can be used to describe hog kidney leucine aminopeptidase crystal packing in the highly anisometric unit cell provides further corroboration that leucine aminopeptidase in these two species is a hexamer based on two trimers each made of three bilobal promoters.

Crystallography, quantitative structure-activity relationships, and molecular graphics in a comparative analysis of the inhibition of dihydrofolate reductase from chicken liver and Lactobacillus casei by 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(substituted-phenyl)-s-triazine s.

The inhibition of dihydrofolate reductase from chicken liver and from Lactobacillus casei has been studied with 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(substituted-phenyl)-s-triazines. It was found that for the chicken enzyme, inhibitor potency for 101 triazines was correlated by the following equation: log 1/Kiapp = 0.85 sigma tau' - 1.04 log (beta X 10 sigma tau' + 1) + 0.57 sigma + 6.36. The parameter tau' indicates that for certain substituents, tau = 0. In the case of the L. casei DHFR results, meta and para derivatives could not be included in the same equation. For 38 meta-substituted compounds, it was found that log 1/Kiapp = 0.38 tau'3-0.91 log (beta X 10 tau'3 + 1) + 0.71I + 4.60 and for 32 para-substituted phenyltriazines log 1/Kiapp = 0.44 tau'4-0.65 log (beta tau'4 + 1') - 0.90 upsilon + 0.69I + 4.67. In the L. casei equation, I is an indicator variable for substituents of the type CH2ZC6H4-Y and ZCH2C6H4-Y, where Z = O, NH, S, or Se. The parameter upsilon is Charton's steric parameter, which is similar to Taft's Es. The mathematical models obtained from correlation analysis are compared with stereo color graphics models.

Gross quaternary changes in aspartate carbamoyltransferase are induced by the binding of N-(phosphonacetyl)-L-aspartate: A 3.5-A resolution study.

The three-dimensional structure of the complex of N-(phosphonacetyl)-L-aspartate with aspartate carbamoyltransferase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) has been determined to a nominal resolution of 3.5 A by single-crystal x-ray diffraction methods. Initial phases were obtained by the method of "molecular tectonics": beginning with the structure of the CTP-protein complex, the domains of the catalytic and regulatory chains were manipulated as separate rigid bodies. The resulting coordinates were used to calculate an electron density map, which was then back transformed to give a set of calculated amplitudes and phases. Using all observed data, we obtained a crystallographic R factor between observed and calculated amplitudes Fo and Fc of 0.46. An envelope was then applied to a 2Fo - Fc map and the density was averaged across the molecular twofold axis. Two cycles of averaging yielded an R factor of 0.25. In this complex, we find that the two catalytic trimers have separated from each other along the threefold axis by 11-12 A and have rotated in opposing directions around the threefold axis such that the total relative reorientation is 8-9 degrees. This rotation places the trimers in a more nearly eclipsed configuration. In addition, two domains in a single catalytic chain have changed slightly their spatial relationship to each other. Finally, the two chains of one regulatory dimer have rotated 14-15 degrees around the twofold axis, and the Zn domains have separated from each other by 4 A along the threefold axis. These movements enlarge the central cavity of the molecule and allow increased accessibility to this cavity through the six channels from the exterior surface of the enzyme.