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F A Quiocho

Publications and source records attributed to F A Quiocho.

At least 73 records · Page 4Linked to original sources
PubMed1993

Inversion of receptor binding preferences by mutagenesis: free energy thermodynamic integration studies on sugar binding to L-arabinose binding proteins.

The Escherichia coli L-arabinose-binding protein (ABP) participates as a specific receptor in the transport of L-arabinose, D-fucose, and D-galactose through the periplasmic space. The wild-type protein binds L-arabinose about 40 times more strongly than D-fucose. A mutation of the protein at position 108 (Met-->Leu) causes a specificity change. The Met108Leu ABP slightly prefers binding of D-fucose over L-arabinose. Molecular dynamics (MD) and thermodynamic integration (TI) computer simulations were performed to study the mechanism of sugar discrimination and specificity change based on the known high-resolution X-ray structures. The specificity change was evaluated by calculating the difference in free energy of L-arabinose versus D-fucose bound to wild-type and Met108Leu ABP. The calculated free energy differences are consistent with the experimentally observed specificity of wild-type and Met108Leu ABP. The simulations indicate that the specificity change of Met108Leu ABP is accomplished mainly by reduced Lennard-Jones interactions of residue 108 with L-arabinose and improved interactions with D-fucose. In addition to MD/TI calculations on sugar binding, finite difference Poisson-Boltzmann calculations were performed to identify the most stable ionization state of buried ionizable residues in ABP.

Arabinose↗
PubMed1993

Preliminary crystallographic analysis of a Fab specific for the O-antigen of Shigella flexneri cell surface lipopolysaccharide with and without bound saccharides.

The Fab of a monoclonal anti-carbohydrate antibody, SYA/J6 (IgG3, kappa, murine), raised against the O-polysaccharide antigen of the cell surface lipopolysaccharide of variant Y Shigella flexneri, a Gram negative bacterium, has been crystallized in the unliganded form and in complex with tri- and pentasaccharide antigens. The three crystal forms belong to the tetragonal space group P4(3)2(1)2, or P4(1)2(1)2, with very similar unit cell dimensions and an asymmetric unit that contains one molecule of about 50,000 Daltons, and a fourth crystal form belongs to monoclinic space group P2(1) that contains four molecules of Fab in an asymmetric unit. Whereas diffractions of these crystals on an area detector-rotating anode system extend to only about 3.5 A resolution, those measured using an imaging plate and synchrotron radiation at the Photon Factory facility extend to 2.5 A.

Antibodies, Monoclonal↗
PubMed1993

Crystallization of the maltodextrin-binding protein for active transport and chemotaxis in several different liganded and mutant forms.

We have crystallized the maltodextrin-binding protein complexed with two cyclic maltodextrins, and with no ligand at all. We have also grown crystals of two complexes of a mutant of maltodextrin-binding protein whose transport specificity has been altered. Studies of these crystals should provide considerable information about protein-oligosaccharide interactions, the effects of these interactions on oligosaccharide conformation, and particularly about the function of the periplasmic receptors in transport and chemotaxis.

Bacterial Proteins↗
PubMed1993

A pre-transition-state mimic of an enzyme: X-ray structure of adenosine deaminase with bound 1-deazaadenosine and zinc-activated water.

The refined 2.4-A structure of adenosine deaminase, recently discovered to be a zinc metalloenzyme [Wilson, D. K., Rudolph, F. B., & Quiocho, F. A. (1991) Science 252, 1278-1284], complexed with the ground-state analog 1-deazaadenosine shows the mode of binding of the analog and, unexpectedly, a zinc-activated water (hydroxide). This structure of a pre-transition-state mimic, combined with that previously determined for the complex with 6(R)-hydroxy-1,6-dihydropurine ribonucleoside, a nearly ideal transition-state analog, sheds new understanding of the precise stereospecificity and hydrolytic catalysis of an important and well-characterized member of a large group of zinc metalloenzymes. As both of these excellent mimics were generated in the active site, they demonstrate a powerful means of dissecting the course of an enzymatic reaction by direct crystallographic analysis.

Adenosine Deaminase↗
PubMed1993

Dominant role of local dipoles in stabilizing uncompensated charges on a sulfate sequestered in a periplasmic active transport protein.

Electrostatic interactions are among the key factors determining the structure and function of proteins. Here we report experimental results that illuminate the functional importance of local dipoles to these interactions. The refined 1.7-A X-ray structure of the liganded form of the sulfate-binding protein, a primary sulfate active transport receptor of Salmonella typhimurium, shows that the sulfate dianion is completely buried and bound by hydrogen bonds (mostly main-chain peptide NH groups) and van der Waals forces. The sulfate is also closely linked, via one of these peptide units, to a His residue. It is also adjacent to the N-termini of three alpha-helices, of which the two shortest have their C-termini "capped" by Arg residues. Site-directed mutagenesis of the recombinant Escherichia coli sulfate receptor had no effect on sulfate-binding activity when an Asn residue was substituted for the positively charged His and the two Arg (changed singly and together) residues. These results, combined with other observations, further solidify the idea that stabilization of uncompensated charges in a protein is a highly localized process that involves a collection of local dipoles, including those of peptide units confined to the first turns of helices. The contribution of helix macrodipoles appears insignificant.

Bacterial Proteins↗
PubMed1992

Calmodulin structure refined at 1.7 A resolution.

We have determined and refined the crystal structure of a recombinant calmodulin at 1.7 A resolution. The structure was determined by molecular replacement, using the 2.2 A published native bovine brain structure as the starting model. The final crystallographic R-factor, using 14,469 reflections in the 10.0 to 1.7 A range with structure factors exceeding 0.5 sigma, is 0.216. Bond lengths and bond angle distances have root-mean-square deviations from ideal values of 0.009 A and 0.032 A, respectively. The final model consists of 1279 non-hydrogen atoms, including four calcium ions, 1130 protein atoms, including three Asp118 side-chain atoms in double conformation, 139 water molecules and one ethanol molecule. The electron densities for residues 1 to 4 and 148 of calmodulin are poorly defined, and not included in our model, except for main-chain atoms of residue 4. The calmodulin structure from our crystals is very similar to the earlier 2.2 A structure described by Babu and coworkers with a root-mean-square deviation of 0.36 A. Calmodulin remains a dumb-bell-shaped molecule, with similar lobes and connected by a central alpha-helix. Each lobe contains three alpha-helices and two Ca2+ binding EF hand loops, with a short antiparallel beta-sheet between adjacent EF hand loops and one non-EF hand loop. There are some differences in the structure of the central helix. The crystal packing is extensively studied, and facile crystal growth along the z-axis of the triclinic crystals is explained. Herein, we describe hydrogen bonding in the various secondary structure elements and hydration of calmodulin.

Calcium↗
PubMed1992

Crystallographic evidence of a large ligand-induced hinge-twist motion between the two domains of the maltodextrin binding protein involved in active transport and chemotaxis.

The periplasmic maltodextrin binding protein of Escherichia coli serves as an initial receptor for the active transport of and chemotaxis toward maltooligosaccharides. The three-dimensional structure of the binding protein complexed with maltose has been previously reported [Spurlino, J. C., Lu, G.-Y., & Quiocho, F. A. (1991) J. Biol. Chem. 266, 5202-5219]. Here we report the structure of the unliganded form of the binding protein refined to 1.8-A resolution. This structure, combined with that for the liganded form, provides the first crystallographic evidence that a major ligand-induced conformational change occurs in a periplasmic binding protein. The unliganded structure shows a rigid-body "hinge-bending" between the two globular domains by approximately 35 degrees, relative to the maltose-bound structure, opening the sugar binding site groove located between the two domains. In addition, there is an 8 degrees twist of one domain relative to the other domain. The conformational changes observed between this structure and the maltose-bound structure are consistent with current models of maltose/maltodextrin transport and maltose chemotaxis and solidify a mechanism for receptor differentiation between the ligand-free and ligand-bound forms in signal transduction.

Amino Acid Sequence↗
PubMed1992

Target enzyme recognition by calmodulin: 2.4 A structure of a calmodulin-peptide complex.

The crystal structure of calcium-bound calmodulin (Ca(2+)-CaM) bound to a peptide analog of the CaM-binding region of chicken smooth muscle myosin light chain kinase has been determined and refined to a resolution of 2.4 angstroms (A). The structure is compact and has the shape of an ellipsoid (axial ratio approximately 2:1). The bound CaM forms a tunnel diagonal to its long axis that engulfs the helical peptide, with the hydrophobic regions of CaM melded into a single area that closely covers the hydrophobic side of the peptide. There is a remarkably high pseudo-twofold symmetry between the closely associated domains. The central helix of the native CaM is unwound and expanded into a bend between residues 73 and 77. About 185 contacts (less than 4 A) are formed between CaM and the peptide, with van der Waals contacts comprising approximately 80% of this total.

Amino Acid Sequence↗
PubMed1992

Refined 2.5 A structure of murine adenosine deaminase at pH 6.0.

The X-ray structure of murine adenosine deaminase complexed with the transition-state analogue 6-hydroxyl-1,6-dihydropurine ribonucleoside has been determined from a single crystal grown at pH 4.2 and transferred to mother liquor of increasing pH up to a final pH of 6.0 prior to data collection. The structure has been refined to 2.5 A to a final crystallographic R-factor of 20% using phases from the previously refined 2.4 A structure at pH 4.2. Kinetic measurements show that the enzyme is only 20% active at pH 4.2 whereas it is fully active between pH 6.0 and pH 8.5. The refined structures at either pH are essentially the same. Consideration of the pKa values of the key catalytic residues and the mechanism proposed on the basis of the structure suggests that the ionization state of these residues is largely responsible for the pH dependence on activity.

Adenosine Deaminase↗
PubMed1992

Protein-ligand energetics assessed using deoxy and fluorodeoxy sugars in equilibrium binding and high resolution crystallographic studies.

Hydrogen bonding interactions are one of the most important single factors in protein-ligand interactions and molecular recognition. To probe the energetics of the interactions, we have analyzed the binding of 1-deoxy-, 2-deoxy- and 6-fluoro-6-deoxy- analogues of D-galactose (Gal) to a primary high-affinity periplasmic receptor for monosaccharide active transport. Kd values and atomic structures refined at 1.81 to 1.45 A resolution of the complexes have been determined and compared with those of Gal binding. With binding site residues and the bound modified sugars in nearly identical positions as found in the complex with Gal, the binding of 1-deoxy-Gal or 2-deoxy-Gal reflects the overall contribution of 1.8 kcal mol-1 per hydrogen bond (neutral-charge type) to the affinity of Gal. Neglected in these estimates is the contribution of van der Waals' forces that accompany the formation of hydrogen bonds with each sugar hydroxyl. Contrary to expectations, the 6-fluoro-6-deoxy analogue proved to be an inadequate probe of Gal OH6 as a hydrogen bond donor due to the binding of a new water molecule and structural changes arising from the electronegative fluoro group. This study sheds new light on the energetics of protein-ligand interactions and the use of engineered ligands in assessing these interactions.

Biological Transport, Active↗
PubMed1992

Atomic interactions in protein-carbohydrate complexes. Tryptophan residues in the periplasmic maltodextrin receptor for active transport and chemotaxis.

We have refined the 1.9 A resolution crystal structures of two maltodextrin receptor mutants in which tryptophan residues 230 and 232 have been changed to alanine and compared these structures with the refined 1.7 A structure of the wild-type protein. In the wild-type structure, Trp230, which is located in the maltodextrin-binding groove, stacks against the B-face of the reducing sugar of the bound maltose. Trp232, which is located near the protein surface, does not participate directly in sugar binding. Relative to the wild-type structure, neither mutation caused a significant rearrangement in the overall protein structure or in the mode of binding maltose. Although the position once occupied by Trp230 remains empty, a new water molecule has moved near the void. In contrast, a new water molecule has entered into the space once occupied by Trp232. Whereas one hydrogen bond is formed with the water molecule near the Trp230 void, no hydrogen bond is associated with the water molecule occupying the space vacated by Trp232. The three van der Waals' contacts between Trp230 and maltose in the wild-type structure that are lost in the W230A mutation could contribute to the 12-fold decrease in ligand-binding activity of the mutant protein. The W232A mutation causes little change in binding activity. The structures of these mutant proteins also provide some insight into the complicated tryptophan fluorescence spectra of the maltodextrin binding-protein. The change in fluorescence due to the deletion of Trp230 can readily be explained as resulting directly from loss of Trp230 in the sugar-binding site. The change in fluorescence due to deletion of Trp232, however, is ascribed to the modification of local interactions mediated by the binding of maltodextrin since the tryptophan is not directly involved in any sugar-binding interaction.

Bacterial Proteins↗
PubMed1992

An unlikely sugar substrate site in the 1.65 A structure of the human aldose reductase holoenzyme implicated in diabetic complications.

Aldose reductase, which catalyzes the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of a wide variety of aromatic and aliphatic carbonyl compounds, is implicated in the development of diabetic and galactosemic complications involving the lens, retina, nerves, and kidney. A 1.65 angstrom refined structure of a recombinant human placenta aldose reductase reveals that the enzyme contains a parallel beta 8/alpha 8-barrel motif and establishes a new motif for NADP-binding oxidoreductases. The substrate-binding site is located in a large, deep elliptical pocket at the COOH-terminal end of the beta barrel with a bound NADPH in an extended conformation. The highly hydrophobic nature of the active site pocket greatly favors aromatic and apolar substrates over highly polar monosaccharides. The structure should allow for the rational design of specific inhibitors that might provide molecular understanding of the catalytic mechanism, as well as possible therapeutic agents.

Aldehyde Reductase↗
PubMed1992

Interdomain salt bridges modulate ligand-induced domain motion of the sulfate receptor protein for active transport.

The refined crystal structure of the liganded form of the Salmonella typhimurium sulfate-binding protein, a periplasmic receptor of active transport, is made up of two globular domains bisected by a deep cleft wherein the dehydrated sulfate is completely engulfed and bound by hydrogen bonds and van der Waals' forces. Two salt bridges (between Glu15 and Arg174 and between Asp68 and Arg134) span the cleft opening. To elucidate the role of the inter-domain salt bridges in the ligand-induced domain motion, the acidic residues were changed (singly and together) to their corresponding amide side-chains by site-directed mutagenesis of the recombinant Escherichia coli sulfate-binding protein. Rapid kinetics and equilibrium measurements of sulfate binding to the purified mutant proteins demonstrate that these salt bridges stabilize the closed liganded form of the receptor and modulate the rate of cleft opening. Our results have new implications in understanding the dynamics of many other multidomain proteins that undergo similar large-scale domain motions.

Bacterial Proteins↗
PubMed1991

Structure of a recombinant calmodulin from Drosophila melanogaster refined at 2.2-A resolution.

The crystal structure of calmodulin (Mr 16,700, 148 residues) from Drosophila melanogaster as expressed in a bacterial system has been determined and refined at 2.2-A resolution. Starting with the structure of mammalian calmodulin, we produced an extensively refitted and refined model with a conventional crystallographic R value of 0.197 for the 5,239 reflections (F greater than or equal to 2 sigma (F)) within the 10.0-2.2-A resolution range. The model includes 1,164 protein atoms, 4 calcium ions, and 78 water molecules and has root mean square deviations from standard values of 0.018 A for bond lengths and 0.043 A for angle distances. The overall structure is similar to mammalian calmodulin, with a seven-turn central helix connecting the two calcium-binding domains. The "dumb-bell" shaped molecule contains seven alpha-helices and four "EF hand" calcium-binding sites. Although the amino acid sequences of mammalian and Drosophila calmodulins differ by only three conservative amino acid changes, the refined model reveals a number of significant differences between the two structures. Superimposition of the structures yields a root mean square deviation of 1.22 A for the 1,120 equivalent atoms. The calcium-binding domains have a root mean square deviation of 0.85 A for the 353 equivalent atoms. There are also differences in the amino terminus, the bend of the central alpha-helix, and the orientations of some of the side chains.

Amino Acid Sequence↗
PubMed1991

Sugar-binding and crystallographic studies of an arabinose-binding protein mutant (Met108Leu) that exhibits enhanced affinity and altered specificity.

In addition to hydrogen bonds, van der Waals forces contribute to the affinity of protein-carbohydrate interactions. Nonpolar van der Waals contacts in the complexes of the L-arabinose-binding protein (ABP) with monosaccharides have been studied by means of site-directed mutagenesis, equilibrium and rapid kinetic binding techniques, and X-ray crystallography. ABP, a periplasmic transport receptor of Escherichia coli, binds L-arabinose, D-galactose, and D-fucose with preferential affinity in the order of Ara greater than Gal much greater than Fuc. Well-refined, high-resolution structures of ABP complexed with the three sugars revealed that the structural differences in the ABP-sugar complexes are localized around C5 of the sugars, where the equatorial H of Ara has been substituted for CH3 (Fuc) or CH2OH (Gal). The side chain of Met108 undergoes a sterically dictated, ligand-specific, conformational change to optimize nonpolar interactions between its methyl group and the sugar. We found that the Met108Leu ABP binds Gal tighter than wild-type ABP binds Ara and exhibits a preference for ligand in the order of Gal much greater than Fuc greater than Ara. The differences in affinity can be attributed to differences in the dissociation rates of the ABP-sugar complexes. We have refined at better than 1.7-A resolution the crystal structures of the Met108Leu ABP complexed with each of the sugars and offer a molecular explanation for the altered binding properties.

Amino Acid Sequence↗
PubMed1991

Atomic structure of adenosine deaminase complexed with a transition-state analog: understanding catalysis and immunodeficiency mutations.

The crystal structure of a murine adenosine deaminase complexed with 6-hydroxyl-1,6-dihydropurine ribonucleoside, a nearly ideal transition-state analog, has been determined and refined at 2.4 angstrom resolution. The structure is folded as an eight-stranded parallel alpha/beta barrel with a deep pocket at the beta-barrel COOH-terminal end wherein the inhibitor and a zinc are bound and completely sequestered. The presence of the zinc cofactor and the precise structure of the bound analog were not previously known. The 6R isomer of the analog is very tightly held in place by the coordination of the 6-hydroxyl to the zinc and the formation of nine hydrogen bonds. On the basis of the structure of the complex a stereoselective addition-elimination or SN2 mechanism of the enzyme is proposed with the zinc atom and the Glu and Asp residues playing key roles. A molecular explanation of a hereditary disease caused by several point mutations of an enzyme is also presented.

Adenosine Deaminase↗