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Biomedical subjects

F A Quiocho

Publications and source records attributed to F A Quiocho.

At least 55 records · Page 3Linked to original sources
PubMed1995

1.7 A structure of FR-1, a fibroblast growth factor-induced member of the aldo-keto reductase family, complexed with coenzyme and inhibitor.

Murine FR-1 is a protein that is induced by fibroblast growth factor-1 and, therefore, may play a role in the regulation of the cell cycle. Sequence comparison indicates that it is a member of the NADPH-dependent aldo-keto reductase family. It bears 70% identity to human aldose reductase, an enzyme implicated in diabetic complications and a target for drug design. We have determined the 1.7 A resolution structure of the FR-1 in a ternary complex with NADPH and zopolrestat, a potent aldose reductase inhibitor. FR-1 folds into a (beta/alpha)8 barrel with an active site characterized by a preponderance of hydrophobic residues residing in a deep oblong cavity at the C-terminal end of the beta-barrel. The nicotinamide moiety of the coenzyme sits in the base of the cavity. Zopolrestat occupies the active site cavity and makes numerous contacts with several hydrophobic residues. The FR-1 ternary complex structure indicates that it uses the same general catalytic mechanism as aldose reductase and other members of the family whose structures have been determined. The protein exhibits reductase activity with DL-glyceraldehyde as a substrate and is strongly inhibited by zopolrestat. When compared with the structure of a similar ternary complex of aldose reductase, the binding site retains many of the interactions with the coenzyme and inhibitor from the conserved residues. Some differences in sequence, however, create a larger binding site that contains six more water molecules than in the aldose reductase ternary complex structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Oxidoreductases↗
PubMed1995

Refined structures of two insertion/deletion mutants probe function of the maltodextrin binding protein.

The X-ray structures of the maltose bound forms of two insertion/deletion mutants of the Escherichia coli maltodextrin binding protein, MalE322 and MalE178, have been determined and refined. MalE322 involves a one residue deletion, two residue insertion in a hinge segment connecting the two (N and C) domains of the protein, an area already identified as being critical for the correct functioning of the protein. MalE178 involves a nine residue deletion and two residue insertion in a helix at the periphery of the C-domain. The function of both mutant proteins is similar to the wild-type, although MalE322 increases the ability to transport maltose and maltodextrin whilst inhibiting the ability of the cell to grow on dextrins. Both proteins exhibit very localized and conservative conformational changes due to their mutations. The structure of MalE322 shows some deformation of the third hinge strand, indicating the likely cause of change in its biochemistry. MalE178 is stable and its activity virtually unchanged from the wild-type. This is most likely due to the long distance of the mutation from the binding site and conservation of the number of interactions between the area around the deletion site and the main body of the protein.

Amino Acid Sequence↗
PubMed1995

Conservative and nonconservative mutations in proteins: anomalous mutations in a transport receptor analyzed by free energy and quantum chemical calculations.

Experimental studies on a bacterial sulfate receptor have indicated anomalous relative binding affinities for the mutations Ser130-->Cys,Ser130-->Gly, and Ser130-->Ala. The loss of affinity for sulfate in the former mutation was previously attributed to a greater steric effect on the part of the Cys side chain relative to the Ser side chain, whereas the relatively small loss of binding affinity for the latter two mutations was attributed to the loss of a single hydrogen bond. In this report we present quantum chemical and statistical thermodynamic studies of these mutations. Qualitative results from these studies indicate that for the Ser130-->Cys mutation the large decrease in binding affinity is in part caused by steric effects, but also significantly by the differential work required to polarize the Cys thiol group relative to the Ser hydroxyl group. The Gly mutant cobinds a water molecule in the same location as the Ser side chain resulting in a relatively small decrease in binding affinity. Results for the Ala mutant are in disagreement with experimental results but are likely to be limited by insufficient sampling of configuration space due to physical constraints applied during the simulation.

Bacterial Proteins↗
PubMed1995

Influence of divalent cations in protein crystallization.

We have tested the effect of several cations in attempts to crystallize the ligand-bound forms of the leucine/isoleucine/valine-binding protein (LIVBP) (M(r) = 36,700) and leucine-specific binding protein (LBP) (M(r) = 37,000), which act as initial periplasmic receptors for the high-affinity osmotic-shock-sensitive active transport system in bacterial cells. Success was achieved with Cd2+ promoting the most dramatic improvement in crystal size, morphology, and diffraction quality. This comes about 15 years after the ligand-free proteins were crystallized. Nine other different divalent cations were tried as additives in the crystallization of LIVBP with polyethylene glycol 8000 as precipitant, and each showed different effects on the crystal quality and morphology. Cd2+ produced large hexagonal prism crystals of LIVBP, whereas a majority of the cations resulted in less desirable needle-shaped crystals. Zn2+ gave crystals that are long rods with hexagonal cross sections, a shape intermediate between the hexagonal prism and needle forms. The concentration of Cd2+ is critical. The best crystals of the LIVBP were obtained in the presence of 1 mM CdCl2, whereas those of LBP, with trigonal prism morphology, were obtained at a much higher concentration of 100 mM. Both crystals diffract to at least 1.7 A resolution using a conventional X-ray source.

Bacterial Proteins↗
PubMed1994

Fine tuning the specificity of the periplasmic phosphate transport receptor. Site-directed mutagenesis, ligand binding, and crystallographic studies.

Phosphorous, primarily in the form of phosphate, is a critical nutrient for the life of a cell. We have previously determined the 1.7-A resolution structure of the phosphate-binding protein, an initial receptor for the high-affinity phosphate active transport system or permease in Escherichia coli (Luecke, H., and Quiocho, F.A. (1990) Nature 347, 402-406). This structure is the first to reveal the key role of hydrogen bonding interactions in conferring the high specificity of the permease, a specificity also shared by other phosphate transport systems. Both monobasic and dibasic phosphates are recognized by the phosphate-binding protein with Asp56 playing a key role. Here we report site-directed mutagenesis, ligand binding, and crystallographic studies of the binding protein which show that introduction of one additional Asp by mutagenesis of the Thr141 in the ligand-binding site restricts binding to only the monobasic phosphate.

Bacterial Proteins↗
PubMed1994

Crystallographic analysis of the epimeric and anomeric specificity of the periplasmic transport/chemosensory protein receptor for D-glucose and D-galactose.

The D-glucose/D-galactose-binding protein (M(r) = 33,000) found in the periplasm of bacterial cells serves as the primary high-affinity receptor of active transport for and chemotaxis toward both sugar epimers. This protein from Escherichia coli binds D-glucose with a Kd of 2 x 10(-7) M, which is about 2 times tighter than D-galactose. The 2.0-A resolution crystal structure of the binding protein complexed with D-galactose has been refined to a crystallographic R-factor of 0.167. This structure, combined with that previously refined for the complex with D-glucose [Vyas, N.K., Vyas., M. N., & Quiocho, F. A. (1988) Science 242, 1290-1295], provides understanding, in atomic detail, of recognition of sugar epimers and anomers. In the two complex structures, the sugar ring is positioned identically in the binding site, and each hydroxyl group common to both is involved in very similar cooperative hydrogen-bonding interactions with protein residues and ordered water molecules. Only the beta-anomer of both monosaccharides is bound, with Asp154 OD1 primarily responsible for accepting a hydrogen bond from the anomeric hydroxyl. Recognition of both sugar epimers is accomplished principally by hydrogen bonding of Asp14 OD1 with the equatorial OH4 of D-glucose and OD2 with the axial OH4 of D-galactose. These results are reconciled with equilibrium and fast kinetics data, which indicate binding of both anomers of the two sugars, and further compared with sugar recognition by other periplasmic sugar-binding proteins with specificities for arabinose/galactose/fucose, maltooligosaccharides, and ribose.

Bacterial Proteins↗
PubMed1994

Refined 1.89-A structure of the histidine-binding protein complexed with histidine and its relationship with many other active transport/chemosensory proteins.

The structure of the histidine-binding protein (HBP, M(r) = 26,100), involved solely in active transport, has been determined by the molecular replacement technique and refined to 1.89-A resolution and to an R-factor of 0.199. The structure is that of two protein molecules, each with a bound L-histidine, in the asymmetric unit. Replacement solution was achieved by using a model of the crystal structure of the ligand-free, open-cleft form of the lysine/arginine/ornithine-binding protein which was modified so that the two domains are close to each other by bending the hinge connecting the two domains. The bound histidine is held in place by 10 hydrogen bonds, 2 salt links, and about 60 van der Waals contacts. Elucidation of the HBP structure brings a total of eight different binding proteins structures determined in our laboratory, including those with specificities for monosaccharides, maltodextrins (linear and cyclic), aliphatic amino acids, and inorganic oxyanions. These structures comprise about a third of the entire family of periplasmic binding proteins which act as initial primary high-affinity receptors of active transport in Gram-negative bacteria. Two of the binding proteins with specificities for glucose/galactose and maltodextrins also serve in a similar capacity in chemotaxis. Though these proteins have different molecular weights (ranging from 26,000 to 40,000), amino acid sequences, and ligand specificities, their three-dimensional structures are similar overall. They are elongated (axial ratios of 2:1) and composed of two similar globular domains separated by a deep cleft wherein the ligand-binding site is located. These structures provide understanding of molecular recognition of a variety of ligands at the atomic level and functional roles of the binding proteins.

Bacterial Proteins↗
PubMed1994

The immunodominant 38-kDa lipoprotein antigen of Mycobacterium tuberculosis is a phosphate-binding protein.

Several antigens of Mycobacterium tuberculosis have been identified by monoclonal antibodies and are being exploited in the development of improved vaccines and diagnostic reagents, but none has been linked to a specific function. Herein we report that the 38-kDa extracellular lipoprotein antigen, the most potent immunogen of the mycobacteria, is a phosphate-binding protein with features very similar to those of the well characterized periplasmic phosphate-binding protein of Escherichia coli which serves as an initial receptor for active transport. This is also the first report definitively linking a function of a binding protein anchored to a membrane and found in other than Gram-negative bacteria.

Amino Acid Sequence↗
PubMed1994

Crystallization and preliminary X-ray crystallographic analysis of the 38-kDa immunodominant antigen of Mycobacterium tuberculosis.

The 38-kDa lipoprotein is one of the most potent cell surface immunogens of Mycobacterium tuberculosis in antibody-and T cell-mediated reactions. Using a pure recombinant form of the protein, we have recently shown that it binds phosphate much like that of the phosphate-binding protein (M(r) = 34.4 kDa) that is localized in the periplasm of Escherichia coli and is involved as an initial receptor for active transport of phosphate. The purified 38-kDa protein has been crystallized in 2 forms that are suitable for high-resolution structural analyses. One form belongs to the monoclinic space group P2(1) with unit cell dimensions of a = 67.42 A, b = 113.38 A, c = 42.68 A, and beta = 108.53 degrees. The other is of orthorhombic space group P2(1)2(1)2 with a = 125.46 A, b = 72.27 A, and c = 73.43 A. Both crystal forms diffract to about 2 A resolution on a fine focus rotating anode.

Antigens, Bacterial↗
PubMed1994

Crystallographic observation of a trapped tetrahedral intermediate in a metalloenzyme.

Formation of tetrahedral transition intermediates is a key step in many enzyme catalyzed reactions. Much of our understanding of these and other intermediates, at the atomic level, has come from crystallographic studies of very few enzymes with bound, synthetic, transition-state analogues. Here we present the structure of adenosine deaminase, a zinc-metalloenzyme critical in both purine metabolism and development of the lymphoid system, having performed a stereospecific hydroxide addition to the C6 of inosine. This addition causes the O6 oxygen of inosine to assume an orientation analogous to the position of the amino leaving group of the tetrahedral intermediate in the enzyme-catalyzed hydrolytic deamination of adenosine to inosine.

Adenosine Deaminase↗
PubMed1994

Aldose reductase catalysis and crystallography. Insights from recent advances in enzyme structure and function.

Enhanced metabolism of glucose via the polyol pathway may play an important role in the pathogenesis of diabetic retinopathy, neuropathy, and nephropathy. Aldose reductase catalyzes the NADPH-dependent conversion of glucose to sorbitol, the first step in the polyol pathway. Interruption of the polyol pathway by inhibition of aldose reductase holds considerable promise as a therapeutic measure to prevent or delay the onset and severity of these late complications of diabetes. Dramatic advances in our understanding of the molecular biology, enzymology, and three-dimensional structure of aldose reductase have occurred in recent years, providing new and challenging insights into the enzyme's catalytic mechanism. Recent developments in structure determination of aldose reductase and the implications for evaluation and development of aldose reductase inhibitors are summarized.

Aldehyde Reductase↗
PubMed1993

Modulation of calmodulin plasticity in molecular recognition on the basis of x-ray structures.

Calmodulin is the primary calcium-dependent signal transducer and regulator of a wide variety of essential cellular functions. The structure of calcium-calmodulin bound to the peptide corresponding to the calmodulin-binding domain of brain calmodulin-dependent protein kinase II alpha was determined to 2 angstrom resolution. A comparison to two other calcium-calmodulin structures reveals how the central helix unwinds in order to position the two domains optimally in the recognition of different target enzymes and clarifies the role of calcium in maintaining recognition-competent domain structures.

Amino Acid Sequence↗
PubMed1993

Probing the active site of human aldose reductase. Site-directed mutagenesis of Asp-43, Tyr-48, Lys-77, and His-110.

Structural models of human aldose reductase complexed with NADPH have revealed the apposition of C4 of the nicotinamide ring with tyrosine 48 and histidine 110, suggesting that either of these residues could function as the proton donor in the reaction mechanism. Tyrosine 48 is also part of a hydrogen-bonding network that includes lysine 77 and aspartate 43. In order to study the potential catalytic roles of these 4 residues, we evaluated the kinetic properties of mutants containing structurally conservative replacements at these sites. Enzymatic activity was undetectable when Tyr-48 was mutated to phenylalanine (Y48F) although affinity for NADPH was unchanged. In contrast, a mutant containing asparagine substituted for His-110 (H110N) was characterized by an almost 80,000-fold increase in Km, but only about a 14-fold reduction in kcat measured with D-glyceraldehyde. Modest changes in catalytic properties were observed in the mutant containing aspartate 43 substituted with asparagine (D43N): Km for aldehyde substrates was elevated up to 17-fold, and kcat decreased less than 16-fold. However, the Kd(NADP) values for D43N were about 5 times higher than those for wild type. Mutant enzyme containing methionine substituted for lysine 77 (K77M) was up to 1,460-fold less active than the wild type. These results are consistent with Tyr-48 acting as the acid-base catalyst in human aldose reductase and confirm the importance of Asp-43, Lys-77, and His-110 to the structure and function of the active site.

Aldehyde Reductase↗
PubMed1993

Refined 1.8 A structure of human aldose reductase complexed with the potent inhibitor zopolrestat.

As the action of aldose reductase (EC 1.1.1.21) is believed to be linked to the pathogenesis of diabetic complications affecting the nervous, renal, and visual systems, the development of therapeutic agents has attracted intense effort. We report the refined 1.8 A x-ray structure of the human holoenzyme complexed with zopolrestat, one of the most potent noncompetitive inhibitors. The zopolrestat fits snugly in the hydrophobic active site pocket and induces a hinge-flap motion of two peptide segments that closes the pocket. Excellent complementarity and affinity are achieved on inhibitor binding by the formation of 110 contacts (< or = 4 A) with 15 residues (10 hydrophobic), 13 with the NADPH coenzyme and 9 with four water molecules. The structure is key to understanding the mode of action of this class of inhibitors and for rational design of better therapeutics.

Aldehyde Reductase↗
PubMed1993

Refined 1.8-A structure reveals the mode of binding of beta-cyclodextrin to the maltodextrin binding protein.

The maltodextrin binding protein from Escherichia coli serves as the initial receptor for both the active transport of and chemotaxis toward a range of linear maltose sugars. The X-ray structures of both the maltose-bound and sugar-free forms of the protein have been previously described [Spurlino, J. C., Lu, G.-Y., & Quiocho, F. A. (1991) J. Biol. Chem. 266, 5202-5219; Sharff, A. J., Rodseth, L. E., Spurlino, J. C., & Quocho, F. A. (1992) Biochemistry 31, 10657-10663]. The X-ray crystal structure of the maltodextrin binding protein complexed with cyclomaltoheptaose (beta-cyclodextrin) has been determined from a single crystal. The structure has been refined to a final R-value of 21% at 1.8-A resolution. Although not a physiological ligand for the maltodextrin binding protein, beta-cyclodextrin has been shown to bind with a Kd of the same order as those of the linear maltodextrin substrates. The observed structure shows that the complexed protein remains in the fully open conformation and is almost identical to the structure of the unliganded protein. The sugar sits in the open cleft with three glucosyl units bound to the C-domain at the base of the cleft, in a similar position to maltotriose, the most tightly bound ligand. The top of the ring is loosely bound to the upper edge of the cleft on the N-domain. The sugar makes a total of 94 productive interactions (of less than 4.0-A length) with the protein and with bound water molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Model of lactose repressor core based on alignment with sugar-binding proteins is concordant with genetic and chemical data.

Using primary sequence similarity to arabinose-binding protein, D-glucose/D-galactose-binding protein, and ribose-binding protein (Vyas, N. K., Vyas, M. N., and Quiocho, F. A. (1991) J. Biol. Chem. 266, 5226-5237; Mowbray, S. L., and Cole, L. B. (1992) J. Mol. Biol. 225, 155-175), the core domain (residues 62-323) of the bacterial regulatory protein lac repressor has been aligned to these sugar-binding proteins of known structure. Although the sequence identity is not striking, there is strong overall homology based on two separate matrix scoring systems (minimum base change per codon (MBC/C) and amino acid homology per residue (AAH/R)) (mean score: MBC/C < 1.25, AAH/R > 5.50; random sequences: MBC/C = 1.45, AAH/R = 4.46). Similarly, the predicted secondary structure of the repressor exhibits excellent agreement with the known secondary structures of the sugar-binding proteins. Using this primary sequence alignment, the tertiary structure of the core domain of the lac repressor has been modeled based on the known structures of the sugar-binding proteins as templates. While the structure deduced for the repressor is hypothetical, the model generated allows a comparison between the predicted tertiary arrangement and the wealth of genetic and chemical data elucidated for the repressor. Important residues involved in operator and sugar binding and in protein assembly have been identified using genetic methods, and placement of these residues in the model is consistent with their known function. This approach, therefore, provides a means to visualize the core domain of the lac repressor that allows interpretation of genetic and chemical data for specific residues and rational design of future experiments.

Amino Acid Sequence↗
PubMed1993

Leucine/isoleucine/valine-binding protein contracts upon binding of ligand.

Small-angle x-ray scattering and computer modeling have been used to study the effects of ligand binding to the leucine/isoleucine/valine-binding protein, an initial component of the high-affinity active transport system for branched-chain aliphatic amino acids in Escherichia coli. Measurements were made with no ligand present and with either L-leucine or L-valine present. Upon binding of either leucine or valine, there is a decrease in the radius of gyration, from 23.2 +/- 0.2 to 22.2 +/- 0.2 A, and in the maximum particle dimension, from 82 +/- 3 to 73 +/- 3 A. The x-ray structure of the unbound form has been determined and gives a radius of gyration and a maximum dimension consistent with the values found for the solution structure in this study (Sack, J. S., Saper, M. A., and Quiocho, F. A. (1989) J. Mol. Biol. 206, 171-191). The reduction in the radius of gyration and maximum dimension upon ligand binding can be accounted for by a substrate-induced cleft closure in a combined "hinge-twist" motion. Modeling of the substrate-bound state was done by comparison of this protein with another periplasmic binding protein (L-arabinose-binding protein), which possesses a similar two-lobe structure and for which the x-ray structure is known in its ligand-bound form.

Bacterial Proteins↗