Biomedical subjects

S L Mowbray

Publications and source records attributed to S L Mowbray.

At least 19 recordsLinked to original sources

PubMed2000

Mutations that affect ligand binding to the Escherichia coli aspartate receptor: implications for transmembrane signaling.

Three arginine residues of the binding site of the Escherichia coli aspartate receptor contribute to its high affinity for aspartate (K(d) approximately 3 microm). Site-directed mutations at residue 64 had the greatest effect on aspartate binding. No residue could substitute for the native arginine; all changes resulted in an apparent K(d) of approximately 35 mm. These mutations had little impact on maltose responses. At residue Arg-69, a lysine substitution was least disruptive, conferring an apparent K(d) of 0.3 mm for aspartate. Results obtained for an alanine mutant were similar to those with cysteine and histidine mutants (K(d) approximately 5 mm) indicating that side chain size was not an important factor here. Proline and aspartate caused more severe defects, presumably for reasons related to conformation and charge. The impact of residue 69 mutations on the maltose response was small. Mutations at Arg-73 had similar effects on aspartate binding (K(d) 0.3-7 mm) but more severe consequences for maltose responses. Larger side chains resulted in the best aspartate binding, implying steric considerations are important here. Signaling in the mutant proteins was surprisingly robust. Given aspartate binding, signaling occurred with essentially wild-type efficiency. These results were evaluated in the context of available structural data.

Aspartic Acid↗

PubMed2000

Structure of Aspergillus niger epoxide hydrolase at 1.8 A resolution: implications for the structure and function of the mammalian microsomal class of epoxide hydrolases.

BACKGROUND: Epoxide hydrolases have important roles in the defense of cells against potentially harmful epoxides. Conversion of epoxides into less toxic and more easily excreted diols is a universally successful strategy. A number of microorganisms employ the same chemistry to process epoxides for use as carbon sources. RESULTS: The X-ray structure of the epoxide hydrolase from Aspergillus niger was determined at 3.5 A resolution using the multiwavelength anomalous dispersion (MAD) method, and then refined at 1.8 A resolution. There is a dimer consisting of two 44 kDa subunits in the asymmetric unit. Each subunit consists of an alpha/beta hydrolase fold, and a primarily helical lid over the active site. The dimer interface includes lid-lid interactions as well as contributions from an N-terminal meander. The active site contains a classical catalytic triad, and two tyrosines and a glutamic acid residue that are likely to assist in catalysis. CONCLUSIONS: The Aspergillus enzyme provides the first structure of an epoxide hydrolase with strong relationships to the most important enzyme of human epoxide metabolism, the microsomal epoxide hydrolase. Differences in active-site residues, especially in components that assist in epoxide ring opening and hydrolysis of the enzyme-substrate intermediate, might explain why the fungal enzyme attains the greater speeds necessary for an effective metabolic enzyme. The N-terminal domain that is characteristic of microsomal epoxide hydrolases corresponds to a meander that is critical for dimer formation in the Aspergillus enzyme.

Animals↗

PubMed1999

Conformational changes of ribose-binding protein and two related repressors are tailored to fit the functional need.

The structures and conformational changes of the periplasmic ribose-binding protein and two repressors, PurR and LacI, were compared. Although the closed, ligand-bound structures of the three proteins are very similar, they differ greatly in the degree and direction in which they open, as well as in the amount of internal rearrangement within the domains during that process. Water molecules and a relatively symmetrical inter-domain connection region assist in the large opening observed for the binding protein, while the design of the repressors appears to preclude such dramatic movements. The dimeric nature of the latter proteins, an important aspect in their binding of pseudo-symmetrical DNA sequences, also appears to be a determinant in the allowed motion. Slight differences in the structure of PurR and LacI explain how they can converge to a similar DNA-binding state in response to different binding states of their small molecule effector.

Amino Acid Sequence↗

PubMed1999

Induced fit on sugar binding activates ribokinase.

The enzyme ribokinase phosphorylates ribose at O5* as the first step in its metabolism. The original X-ray structure of Escherichia coli ribokinase represented the ternary complex including ribose and ADP. Structures are presented here for the apo enzyme, as well as the ribose-bound state and four new ternary complex forms. Combined, the structures suggest that large and small conformational changes play critical roles in the function of this kinase. An initially open apo form can allow entry of the ribose substrate. After ribose binding, the active site lid is observed in a closed conformation, with the sugar trapped underneath. This closure and associated changes in the protein appear to assist ribokinase in recognition of the co-substrate ATP as the next step. Binding of the nucleotide brings about further, less dramatic adjustments in the enzyme structure. Additional small movements are almost certainly required during the phosphoryltransfer reaction. Evidence is presented that some types of movements of the lid are allowed in the ternary complex, which may be critical to the creation and breakdown of the transition state. Similar events are likely to take place during catalysis by other related carbohydrate kinases, including adenosine kinase.

Adenosine Diphosphate↗

PubMed1999

Bacterial chemoreceptors: recent progress in structure and function.

The behavior of a bacterial cell is determined by the interplay between transmembrane receptor molecules and a cytoplasmic kinase that is linked to the flagellar apparatus. In the absence of external stimulus, a balance exists between stresses in the periplasmic region of receptor molecules, and compensating cytoplasmic forces. A response, positive or negative, is due to a temporary disturbance in this balance, with corresponding alterations in kinase activity, and ultimately, of swimming behavior. Methylation acts to restore the balance by changing the properties of the receptor. Because methylation is slow, a response will continue for a period of time following stimulation. The mechanisms by which these processes occur are now being elucidated at the molecular level, and should soon make bacterial chemotaxis the first available picture of a complete sensory system.

Bacterial Proteins↗

PubMed1999

Structure of D-allose binding protein from Escherichia coli bound to D-allose at 1.8 A resolution.

ABC transport systems for import or export of nutrients and other substances across the cell membrane are widely distributed in nature. In most bacterial systems, a periplasmic component is the primary determinant of specificity of the transport complex as a whole. We report here the crystal structure of the periplasmic binding protein for the allose system (ALBP) from Escherichia coli, solved at 1.8 A resolution using the molecular replacement method. As in the other members of the family (especially the ribose binding protein, RBP, with which it shares 35 % sequence homology), this structure consists of two similar domains joined by a three-stranded hinge region. The protein is believed to exist in a dynamic equilibrium of closed and open conformations in solution which is an important part of its function. In the closed ligand-bound form observed here, D-allose is buried at the domain interface. Only the beta-anomer of allopyranose is seen in the crystal structure, although the alpha-anomer can potentially bind with a similar affinity. Details of the ligand-binding cleft reveal the features that determine substrate specificity. Extensive hydrogen bonding as well as hydrophobic interactions are found to be important. Altogether ten residues from both the domains form 14 hydrogen bonds with the sugar. In addition, three aromatic rings, one from each domain with faces parallel to the plane of the sugar ring and a third perpendicular, make up a hydrophobic stacking surface for the ring hydrogen atoms. Our results indicate that the aromatic rings forming the sugar binding cleft can sterically block the binding of any hexose epimer except D-allose, 6-deoxy-allose or 3-deoxy-glucose; the latter two are expected to bind with reduced affinity, due to the loss of some hydrogen bonds. The pyranose form of the pentose, D-ribose, can also fit into the ALBP binding cleft, although with lower binding affinity. Thus, ALBP can function as a low affinity transporter for D-ribose. The significance of these results is discussed in the context of the function of allose and ribose transport systems.

ATP-Binding Cassette Transporters↗

PubMed1999

Errors and reproducibility in electron-density map interpretation.

Three investigators, with varying levels of experience, independently built and refined the structure of Escherichia coli ribokinase at 2.6 A resolution. At the end of the refinement/rebuilding processes the models had essentially converged, although each had its own particular pattern of remaining errors. The subsequent refinement of the same structure at 1.8 A resolution allowed an overall quality check of each of the lower resolution models, and an analysis of which graphics-based tools were generally most efficient in locating these errors. Criteria which are useful in the application of Ramachandran, main-chain and side-chain database and real-space fit analyses are presented.

Crystallography, X-Ray↗

PubMed1998

Chemotaxis receptors: a progress report on structure and function.

Recent biochemical and structural studies have provided many new insights into the structure and function of bacterial chemoreceptors. Aspects of their ligand binding, conformational changes, and interactions with other members of the signaling pathway are being defined at the structural level. It is anticipated that the combined effort will soon provide a detailed, unified view of an entire response system.

Bacterial Proteins↗

PubMed1998

Multiple open forms of ribose-binding protein trace the path of its conformational change.

Conformational changes are necessary for the function of bacterial periplasmic receptors in chemotaxis and transport. Such changes allow entry and exit of ligand, and enable the correct interaction of the ligand-bound proteins with the membrane components of each system. Three open, ligand-free forms of the Escherichia coli ribose-binding protein were observed here by X-ray crystallographic studies. They are opened by 43 degrees, 50 degrees and 64 degrees with respect to the ligand-bound protein reported previously. The three open forms are not distinct, but show a clear relationship to each other. All are the product of a similar opening motion, and are stabilized by a new, almost identical packing interface between the domains. The changes are generated by similar bond rotations, although some differences in the three hinge segments are needed to accommodate the various structural scenarios. Some local repacking also occurs as interdomain contacts are lost. The least open (43 degrees) form is probably the dominant one in solution under normal conditions, although a mixture of species seems likely. The open and closed forms have distinct surfaces in the regions known to be important in chemotaxis and transport, which will differentiate their interactions with the membrane components. It seems certain that the conformational path that links the forms described here is that followed during ligand retrieval, and in ligand release into the membrane-bound permease system.

Biological Transport↗

PubMed1998

Structure of Escherichia coli ribokinase in complex with ribose and dinucleotide determined to 1.8 A resolution: insights into a new family of kinase structures.

BACKGROUND: D-ribose must be phosphorylated at O5' before it can be used in either anabolism or catabolism. This reaction is catalysed by ribokinase and requires the presence of ATP and magnesium. Ribokinase is a member of a family of carbohydrate kinases of previously unknown structure. RESULTS: The crystal structure of ribokinase from Escherichia coli in complex with ribose and dinucleotide was determined at 1.84 A resolution by multiple isomorphous replacement. There is one 33 kDa monomer of ribokinase in the asymmetric unit but the protein forms a dimer around a crystallographic twofold axis. Each subunit consists of a central alpha/beta unit, with a new type of nucleotide-binding fold, and a distinct beta sheet that forms a lid over the ribose-binding site. Contact between subunits involves orthogonal packing of beta sheets, in a novel dimer interaction that we call a beta clasp. CONCLUSIONS: Inspection of the complex indicates that ribokinase utilises both a catalytic base for activation of the ribose in nucleophilic attack and an anion hole that stabilises the transition state during phosphoryl transfer. The structure suggests an ordered reaction mechanism, similar to those proposed for other carbohydrate kinases that probably involves conformational changes. We propose that the beta-clasp structure acts as a lid, closing and opening upon binding and release of ribose. From these observations, an understanding of the structure and catalytic mechanism of related sugar kinases can be obtained.

Adenylyl Imidodiphosphate↗

PubMed1997

Purification, characterization, and crystallization of Escherichia coli ribokinase.

Ribokinase phosphorylates ribose to form ribose-5-phosphate in the presence of ATP and magnesium. The phosphorylated sugar can enter the pentose phosphate pathway or be used for the synthesis of nucleotides, histidine, and tryptophan. Ribokinase belongs to the PfkB family of carbohydrate kinases, for which no three-dimensional structure is currently known. We describe an improved purification protocol for Escherichia coli ribokinase and give evidence from light-scattering and gel filtration studies that the protein forms a dimer in solution. Several types of crystals are also described that have been obtained of apo ribokinase, ribokinase in the presence of ATP, and in a ternary complex with an ATP-analogue and ribose. The latter crystals give the best X-ray diffraction. A complete data set has been collected at the synchrotron source in Hamburg, to 2.6 A resolution using a frozen crystal. The crystals belong to space group P6(1)22 or P6(5)22 with cell parameters a = b = 95 A and c = 155 A.

Crystallography, X-Ray↗

PubMed1996

Conformational changes of three periplasmic receptors for bacterial chemotaxis and transport: the maltose-, glucose/galactose- and ribose-binding proteins.

Small-angle X-ray scattering experiments were carried out for the maltose-, glucose/galactose- and ribose-binding proteins of Gram negative bacteria. All were shown to be monomers that decrease in radius of gyration on ligand binding. The results obtained for the maltose-binding protein agree well with crystal structures of the closed, ligand-bound, and open, ligand-free protein, suggesting that these are indeed the primary forms in solution. The closed form is stabilized by protein-sugar interactions, while the open conformation is stabilized by close contacts between the two domains. Since it is the proper special relationship of the domains in the closed form that is most important for interaction with chemotaxis and transport partners, the stabilization of the open form would help keep ligand-free molecules from interfering in function. The scattering results also provide evidence that a large conformational change takes place in association with ligand binding to the glucose/galactose- and ribose-binding proteins, and that the two changes are similar. Modeling suggests that the open forms resemble those found in the related leucine and leucine/isoleucine/valine-binding proteins, but are different from those observed for the maltose-binding protein and the related purine repressor.

Bacterial Proteins↗

PubMed1996

Crystal structures and solution conformations of a dominant-negative mutant of Escherichia coli maltose-binding protein.

A mutant of the periplasmic maltose-binding protein (MBP) with altered transport properties was studied. A change of residue 230 from tryptophan to arginine results in dominant-negative MBP: expression of this protein against a wild-type background causes inhibition of maltose transport. As part of an investigation of the mechanism of such inhibition, we have solved crystal structures of both unliganded and liganded mutant protein. In the closed, liganded conformation, the side-chain of R230 projects into a region of the surface of MBP that has been identified as important for transport while in the open form, the same side-chain takes on a different, and less ordered, conformation. The crystallographic work is supplemented with a small-angle X-ray scattering study that provides evidence that the solution conformation of unliganded mutant is similar to that of wild-type MBP. It is concluded that dominant-negative inhibition of maltose transport must result from the formation of a non-productive complex between liganded-bound mutant MBP and wild-type MalFGK2. A general kinetic framework for transport by either wild-type MalFGK2 or MBP-independent MalFGK2 is used to understand the effects of dominant-negative MBP molecules on both of these systems.

ATP-Binding Cassette Transporters↗

PubMed1995

Strange bedfellows: interactions between acidic side-chains in proteins.

The oxygen atoms of two acidic side-chains are frequently found within hydrogen-bonding distance of each other in proteins. Two distinct types of cases are common. In metal-binding sites, the oxygen atoms are brought near (average closest approach 3.0 A) by their common role as metal ligands. In a different location, either buried or on the protein surface, the two acidic groups can share a proton. The corresponding O-O distances in the latter case are shorter (usually 2.7 or less), and the geometry is typical of hydrogen-bonding interactions. The glucose/galactose-binding protein of Salmonella typhimurium provides an example of a well-ordered Asp-Glu pair on the surface of a protein with a very short O-O distance, at a pH of 7.0. Other instances have been found at pH values as high as 8.0, suggesting substantial alteration of the pKa involved. These observations have implications for the study of enzymes that use pairs of acidic residues in binding and catalysts.

Binding Sites↗

PubMed1995

Sugar recognition by a glucose/galactose receptor. Evaluation of binding energetics from molecular dynamics simulations.

A new theoretical method for free energy calculations is used to compute the absolute binding constants for beta-D-glucose and methyl-beta-D-galactoside to the periplasmic glucose/galactose receptor from Salmonella typhimurium. The computer simulation results agree well with available experimental data and make it possible to assess the sources of both the high affinity as well as the specificity for glucose. It was found that the major contribution to the binding energy comes from electrostatic interactions and particularly hydrogen bonds of the charge-dipole type. We also predict the structure of the complex with methyl-galactoside as this has not yet been experimentally determined.

Computer Simulation↗

PubMed1995

Simple models for the analysis of binding protein-dependent transport systems.

Mathematical modeling was used to evaluate experimental data for bacterial binding protein-dependent transport systems. Two simple models were considered in which ligand-free periplasmic binding protein interacts with the membrane-bound components of transport. In one, this interaction was viewed as a competition with the ligand-bound binding protein, whereas in the other, it was considered to be a consequence of the complexes formed during the transport process itself. Two sets of kinetic parameters were derived for each model that fit the available experimental results for the maltose system. By contrast, a model that omitted the interaction of ligand-free binding protein did not fit the experimental data. Some applications of the successful models for the interpretation of existing mutant data are illustrated, as well as the possibilities of using mutant data to test the original models and sets of kinetic parameters. Practical suggestions are given for further experimental design.

Adenosine Triphosphate↗

PubMed1995

C alpha-based torsion angles: a simple tool to analyze protein conformational changes.

A simple method is presented for the analysis of protein conformational changes based on the comparison of torsion angles defined by four consecutive C alpha atoms. The technique was applied successfully to proteins that undergo hinge motion and shear motion. In the case of both MBP and LAO, which represent examples of hinge motion, the plot of the differences in C alpha-torsion angles between the open and closed forms of the proteins helped us to formulate a more thorough description of the conformational change: a large displacement of one domain with respect to the other where one of the domains does not behave like a rigid body but exhibits some degree of flexibility. The analysis of citrate synthase, which is an example of shear motion, shows that the largest differences in C alpha-torsion angles between the open and closed conformations are clustered around residues that belong to segments connecting alpha-helices, whereas the helices themselves appear to be rigid; this is in agreement with previous results obtained by detailed least-squares superpositions (Lesk AM, Chothia C, 1984, J Mol Biol 174:175-191).

Calcium-Binding Proteins↗

PubMed1995

Crystal structure of the dipeptide binding protein from Escherichia coli involved in active transport and chemotaxis.

The Escherichia coli periplasmic dipeptide binding protein functions in both peptide transport and taxis toward peptides. The structure of the dipeptide binding protein in complex with Gly-Leu (glycyl-L-leucine) has been determined at 3.2 A resolution. The binding site for dipeptides is designed to recognize the ligand's backbone while providing space to accommodate a variety of side chains. Some repositioning of protein side chains lining the binding site must occur when the dipeptide's second residue is larger than leucine. The protein's fold is very similar to that of the Salmonella typhimurium oligopeptide binding protein, and a comparison of the structures reveals the structural basis for the dipeptide binding protein's preference for shorter peptides.

Bacterial Proteins↗