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B W Matthews

Publications and source records attributed to B W Matthews.

At least 19 recordsLinked to original sources
PubMed2001

A structural view of the action of Escherichia coli (lacZ) beta-galactosidase.

The structures of a series of complexes designed to mimic intermediates along the reaction coordinate for beta-galactosidase are presented. These complexes clarify and enhance previous proposals regarding the catalytic mechanism. The nucleophile, Glu537, is seen to covalently bind to the galactosyl moiety. Of the two potential acids, Mg(2+) and Glu461, the latter is in better position to directly assist in leaving group departure, suggesting that the metal ion acts in a secondary role. A sodium ion plays a part in substrate binding by directly ligating the galactosyl 6-hydroxyl. The proposed reaction coordinate involves the movement of the galactosyl moiety deep into the active site pocket. For those ligands that do bind deeply there is an associated conformational change in which residues within loop 794-804 move up to 10 A closer to the site of binding. In some cases this can be inhibited by the binding of additional ligands. The resulting restricted access to the intermediate helps to explain why allolactose, the natural inducer for the lac operon, is the preferred product of transglycosylation.

Binding Sites↗
PubMed2001

The conformation of the epsilon- and gamma-subunits within the Escherichia coli F(1) ATPase.

F(1) is the water-soluble portion of the ubiquitous F(1)F(0) ATP synthase. Its structure includes three alpha- and three beta-subunits, arranged as a hexameric disc, plus a gamma-subunit that penetrates the center of the disc akin to an axle. Recently Hausrath et al. (Hausrath, A. C., Grüber, G., Matthews, B. W., and Capaldi, R. A. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 13697-13702) obtained an electron density map of E. coli F(1) at 4.4-A resolution in which the coiled-coil alpha-helices of the gamma-subunit could be seen to extend 45 A from the base of the alpha(3)beta(3) hexamer. Subsequently the structure of a truncated form of the E. coli gamma-subunit in complex with epsilon has been described (Rodgers, A. J. W., and Wilce, M. C. J. (2000) Nat. Struct. Biol. 7, 1051-1054). In the present study the 4.4-A resolution electron density map of E. coli F(1) is re-evaluated in light of the newly available data on the gamma- and epsilon-subunits. It is shown that the map of the F(1) complex is consistent with the structure of the isolated subunits. When E. coli F(1) is compared with that from beef heart, the structures of the E. coli gamma- and epsilon-subunits are seen to be generally similar to their counterparts in the bovine enzyme but to undergo major shifts in position. In particular, the two long, coiled-coil alpha-helices that lie along the axis of F(1) both unwind and rotate. Also the epsilon-subunit rotates around the axis by 81 degrees and undergoes a net translation of about 23 A. It is argued that these large-scale changes in conformation reflect distinct functional states that occur during the rotation of the gamma-subunit within the alpha(3)beta(3) hexamer.

Animals↗
PubMed2001

Reversible lattice repacking illustrates the temperature dependence of macromolecular interactions.

Flash-freezing, which has become routine in macromolecular X-ray crystallography, causes the crystal to contract substantially. In the case of Escherichia coli beta-galactosidase the changes are reversible and are shown to be due to lattice repacking. On cooling, the area of the protein surface involved in lattice contacts increases by 50 %. There are substantial alterations in intermolecular contacts, these changes being dominated by the long, polar side-chains. For entropic reasons such side-chains, as well as surface solvent molecules, tend to be somewhat disordered at room temperature but can form extensive hydrogen-bonded networks on cooling. Low-temperature density measurements suggest that, at least in some cases, the beneficial effect of cryosolvents may be due to a density increase on vitrification which reduces the volume of bulk solvent that needs to be expelled from the crystal. Analysis of beta-galactosidase and several other proteins suggests that both intramolecular and intermolecular contact interfaces can be perturbed by cryocooling but that the changes tend to be more dramatic in the latter case. The temperature-dependence of the intermolecular interactions suggests that caution may be necessary in interpreting protein-protein and protein-nucleic acid interactions based on low-temperature crystal structures.

Cold Temperature↗
PubMed2001

Crystal structure of the regulatory subunit H of the V-type ATPase of Saccharomyces cerevisiae.

In contrast to the F-type ATPases, which use a proton gradient to generate ATP, the V-type enzymes use ATP to actively transport protons into organelles and extracellular compartments. We describe here the structure of the H-subunit (also called Vma13p) of the yeast enzyme. This is the first structure of any component of a V-type ATPase. The H-subunit is not required for assembly but plays an essential regulatory role. Despite the lack of any apparent sequence homology the structure contains five motifs similar to the so-called HEAT or armadillo repeats seen in the importins. A groove, which is occupied in the importins by the peptide that targets proteins for import into the nucleus, is occupied here by the 10 amino-terminal residues of subunit H itself. The structural similarity suggests how subunit H may interact with the ATPase itself or with other proteins. A cleft between the amino- and carboxyl-terminal domains also suggests another possible site of interaction with other factors.

Amino Acid Sequence↗
PubMed2001

Corepressor-induced organization and assembly of the biotin repressor: a model for allosteric activation of a transcriptional regulator.

The Escherichia coli biotin repressor binds to the biotin operator to repress transcription of the biotin biosynthetic operon. In this work, a structure determined by x-ray crystallography of a complex of the repressor bound to biotin, which also functions as an activator of DNA binding by the biotin repressor (BirA), is described. In contrast to the monomeric aporepressor, the complex is dimeric with an interface composed in part of an extended beta-sheet. Model building, coupled with biochemical data, suggests that this is the dimeric form of BirA that binds DNA. Segments of three surface loops that are disordered in the aporepressor structure are located in the interface region of the dimer and exhibit greater order than was observed in the aporepressor structure. The results suggest that the corepressor of BirA causes a disorder-to-order transition that is a prerequisite to repressor dimerization and DNA binding.

Allosteric Regulation↗
PubMed2001

Docking molecules by families to increase the diversity of hits in database screens: computational strategy and experimental evaluation.

Molecular docking programs screen chemical databases for novel ligands that fit protein binding sites. When one compound fits the site well, close analogs typically do the same. Therefore, many of the compounds that are found in such screens resemble one another. This reduces the variety and novelty of the compounds suggested. In an attempt to increase the diversity of docking hit lists, the Available Chemicals Directory was grouped into families of related structures. All members of every family were docked and scored, but only the best scoring molecule of a high-ranking family was allowed in the hit list. The identity and scores of the other members of these families were recorded as annotations to the best family member, but they were not independently ranked. This family-based docking method was compared with molecule-by-molecule docking in screens against the structures of thymidylate synthase, dihydrofolate reductase (DHFR), and the cavity site of the mutant T4 lysozyme Leu99 --> Ala (L99A). In each case, the diversity of the hit list increased, and more families of known ligands were found. To investigate whether the newly identified hits were sensible, we tested representative examples experimentally for binding to L99A and DHFR. Of the six compounds tested against L99A, five bound to the internal cavity. Of the seven compounds tested against DHFR, six inhibited the enzyme with apparent K(i) values between 0.26 and 100 microM. The segregation of potential ligands into families of related molecules is a simple technique to increase the diversity of candidates suggested by database screens. The general approach should be applicable to most docking methods. Proteins 2001;42:279-293.

Algorithms↗
PubMed2001

Structural and thermodynamic analysis of the binding of solvent at internal sites in T4 lysozyme.

To investigate the structural and thermodynamic basis of the binding of solvent at internal sites within proteins a number of mutations were constructed in T4 lysozyme. Some of these were designed to introduce new solvent-binding sites. Others were intended to displace solvent from preexisting sites. In one case Val-149 was replaced with alanine, serine, cysteine, threonine, isoleucine, and glycine. Crystallographic analysis shows that, with the exception of isoleucine, each of these substitutions results in the binding of solvent at a polar site that is sterically blocked in the wild-type enzyme. Mutations designed to perturb or displace a solvent molecule present in the native enzyme included the replacement of Thr-152 with alanine, serine, cysteine, valine, and isoleucine. Although the solvent molecule was moved in some cases by up to 1.7 A, in no case was it completely removed from the folded protein. The results suggest that hydrogen bonds from the protein to bound solvent are energy neutral. The binding of solvent to internal sites within proteins also appears to be energy neutral except insofar as the bound solvent may prevent a loss of energy due to potential hydrogen bonding groups that would otherwise be unsatisfied. The introduction of a solvent-binding site appears to require not only a cavity to accommodate the water molecule but also the presence of polar groups to help satisfy its hydrogen-bonding potential. It may be easier to design a site to accommodate two or more water molecules rather than one as the solvent molecules can then hydrogen-bond to each other. For similar reasons it is often difficult to design a point mutation that will displace a single solvent molecule from the core of a protein.

Amino Acid Substitution↗
PubMed2001

A structural basis for processivity.

The structures of a number of processive enzymes have been determined recently. These proteins remain attached to their polymeric substrates and may perform thousands of rounds of catalysis before dissociating. Based on the degree of enclosure of the substrate, the structures fall into two broad categories. In one group, the substrate is partially enclosed, while in the other class, enclosure is complete. In the latter case, enclosure is achieved by way of an asymmetric structure for some enzymes while others use a symmetrical toroid. In those cases where the protein completely encloses its polymeric substrate, the two are topologically linked and an immediate explanation for processivity is provided. In cases where there is only partial enclosure, the structural basis for processivity is less obvious. There are, for example, pairs of proteins that have quite similar structures but differ substantially in their processivity. It does appear, however, that the enzymes that are processive tend to be those that more completely enclose their substrates. In general terms, proteins that do not use topological restraint appear to achieve processivity by using a large interaction surface. This allows the enzyme to bind with moderate affinity at a multitude of adjacent sites distributed along its polymeric substrate. At the same time, the use of a large interaction surface minimizes the possibility that the enzyme might bind at a small number of sites with much higher affinity, which would interfere with sliding. Proteins that can both slide along a polymeric substrate, and, as well, recognize highly specific sites (e.g., some site-specific DNA-binding proteins) appear to undergo a conformational change between the cognate and noncognate-binding modes.

Animals↗
PubMed2001

Competing protein:protein interactions are proposed to control the biological switch of the E coli biotin repressor.

A model is suggested for the complex between the biotin repressor of Escherichia coli, BirA, and BCCP, the biotin carboxyl carrier protein to which BirA transfers biotin. The model is consistent with prior physical and biochemical studies. Measurement of transfer rates for variants of BirA with single-site mutations in the proposed BirA:BCCP interface region also provides support. The unique feature of the proposed interaction between BirA and BCCP is that it uses the same beta-sheet region on the surface of BirA that the protein uses for homodimerization into a form competent to bind DNA. The resulting mutually exclusive protein:protein interfaces explain the novel feature of the BirA regulatory system, namely, that transcription of the genes involved in biotin synthesis is not determined by the level of biotin, per se, but by the level of unmodified BCCP. The model also provides a role for the C-terminal domain of BirA that is structurally similar to an SH3 domain.

Acetyl-CoA Carboxylase↗
PubMed2000

Structure and mechanism of peptide methionine sulfoxide reductase, an "anti-oxidation" enzyme.

Peptide methionine sulfoxide reductase (MsrA) reverses oxidative damage to both free methionine and methionine within proteins. As such, it helps protect the host organism against stochastic damage that can contribute to cell death. The structure of bovine MsrA has been determined in two different modifications, both of which provide different insights into the biology of the protein. There are three cysteine residues located in the vicinity of the active site. Conformational changes in a glycine-rich C-terminal tail appear to allow all three thiols to come together and to participate in catalysis. The structures support a unique, thiol-disulfide exchange mechanism that relies upon an essential cysteine as a nucleophile and additional conserved residues that interact with the oxygen atom of the sulfoxide moiety.

Animals↗
PubMed2000

Size versus polarizability in protein-ligand interactions: binding of noble gases within engineered cavities in phage T4 lysozyme.

To investigate the relative importance of size and polarizability in ligand binding within proteins, we have determined the crystal structures of pseudo wild-type and cavity-containing mutant phage T4 lysozymes in the presence of argon, krypton, and xenon. These proteins provide a representative sample of predominantly apolar cavities of varying size and shape. Even though the volumes of these cavities range up to the equivalent of five xenon atoms, the noble gases bind preferentially at highly localized sites that appear to be defined by constrictions in the walls of the cavities, coupled with the relatively large radii of the noble gases. The cavities within pseudo wild-type and L121A lysozymes each bind only a single atom of noble gas, while the cavities within mutants L133A and F153A have two independent binding sites, and the L99A cavity has three interacting sites. The binding of noble gases within two double mutants was studied to characterize the additivity of binding at such sites. In general, when a cavity in a protein is created by a "large-to-small" substitution, the surrounding residues relax somewhat to reduce the volume of the cavity. The binding of xenon and, to a lesser degree, krypton and argon, tend to expand the volume of the cavity and to return it closer to what it would have been had no relaxation occurred. In nearly all cases, the extent of binding of the noble gases follows the trend xenon>krypton>argon. Pressure titrations of the L99A mutant have confirmed that the crystallographic occupancies accurately reflect fractional saturation of the binding sites. The trend in noble gas affinity can be understood in terms of the effects of size and polarizability on the intermolecular potential. The plasticity of the protein matrix permits repulsion due to increased ligand size to be more than compensated for by attraction due to increased ligand polarizability. These results have implications for the mechanism of general anesthesia, the migration of small ligands within proteins, the detection of water molecules within apolar cavities and the determination of crystallographic phases.

Amino Acid Substitution↗
PubMed2000

Determination of the structure of Escherichia coli glyoxalase I suggests a structural basis for differential metal activation.

The metalloenzyme glyoxalase I (GlxI) converts the nonenzymatically produced hemimercaptal of cytotoxic methylglyoxal and glutathione to nontoxic S-D-lactoylglutathione. Human GlxI, for which the structure is known, is active in the presence of Zn(2+). Unexpectedly, the Escherichia coli enzyme is inactive in the presence of Zn(2+) and is maximally active with Ni(2+). To understand this difference in metal activation and also to obtain a representative of the bacterial enzymes, the structure of E. coli Ni(2+)-GlxI has been determined. Structures have also been determined for the apo enzyme as well as complexes with Co(2+), Cd(2+), and Zn(2+). It is found that each of the protein-metal complexes that is catalytically active has octahedral geometry. This includes the complexes of the E. coli enzyme with Ni(2+), Co(2+), and Cd(2+), as well as the structures reported for the human Zn(2+) enzyme. Conversely, the complex of the E. coli enzyme with Zn(2+) has trigonal bipyramidal coordination and is inactive. This mode of coordination includes four protein ligands plus a single water molecule. In contrast, the coordination in the active forms of the enzyme includes two water molecules bound to the metal ion, suggesting that this may be a key feature of the catalytic mechanism. A comparison of the human and E. coli enzymes suggests that there are differences between the active sites that might be exploited for therapeutic use.

Bacterial Proteins↗
PubMed2000

The structure of rhamnose isomerase from Escherichia coli and its relation with xylose isomerase illustrates a change between inter and intra-subunit complementation during evolution.

Using a new expression construct, rhamnose isomerase from Escherichia coli was purified and crystallized. The crystal structure was solved by multiple isomorphous replacement and refined to a crystallographic residual of 17.4 % at 1.6 A resolution. Rhamnose isomerase is a tight tetramer of four (beta/alpha)(8)-barrels. A comparison with other known structures reveals that rhamnose isomerase is most similar to xylose isomerase. Alignment of the sequences of the two enzymes based on their structures reveals a hitherto undetected sequence identity of 13 %, suggesting that the two enzymes evolved from a common precursor. The structure and arrangement of the (beta/alpha)(8)-barrels of rhamnose isomerase are very similar to xylose isomerase. Each enzyme does, however, have additional alpha-helical domains, which are involved in tetramer association, and largely differ in structure. The structures of complexes of rhamnose isomerase with the inhibitor l-rhamnitol and the natural substrate l-rhamnose were determined and suggest that an extended loop, which is disordered in the native enzyme, becomes ordered on substrate binding, and may exclude bulk solvent during catalysis. Unlike xylose isomerase, this loop does not extend across a subunit interface but contributes to the active site of its own subunit. It illustrates how an interconversion between inter and intra-subunit complementation can occur during evolution. In the crystal structure (although not necessarily in vivo) rhamnose isomerase appears to bind Zn(2+) at a "structural" site. In the presence of substrate the enzyme also binds Mn(2+) at a nearby "catalytic" site. An array of hydrophobic residues, not present in xylose isomerase, is likely to be responsible for the recognition of l-rhamnose as a substrate. The available structural data suggest that a metal-mediated hydride-shift mechanism, which is generally favored for xylose isomerase, is also feasible for rhamnose isomerase.

Aldose-Ketose Isomerases↗
PubMed2000

Thiol-disulfide exchange is involved in the catalytic mechanism of peptide methionine sulfoxide reductase.

Peptide methionine sulfoxide reductase (MsrA; EC ) reverses the inactivation of many proteins due to the oxidation of critical methionine residues by reducing methionine sulfoxide, Met(O), to methionine. MsrA activity is independent of bound metal and cofactors but does require reducing equivalents from either DTT or a thioredoxin-regenerating system. In an effort to understand these observations, the four cysteine residues of bovine MsrA were mutated to serine in a series of permutations. An analysis of the enzymatic activity of the variants and their free sulfhydryl states by mass spectrometry revealed that thiol-disulfide exchange occurs during catalysis. In particular, the strictly conserved Cys-72 was found to be essential for activity and could form disulfide bonds, only upon incubation with substrate, with either Cys-218 or Cys-227, located at the C terminus. The significantly decreased activity of the Cys-218 and Cys-227 variants in the presence of thioredoxin suggested that these residues shuttle reducing equivalents from thioredoxin to the active site. A reaction mechanism based on the known reactivities of thiols with sulfoxides and the available data for MsrA was formulated. In this scheme, Cys-72 acts as a nucleophile and attacks the sulfur atom of the sulfoxide moiety, leading to the formation of a covalent, tetracoordinate intermediate. Collapse of the intermediate is facilitated by proton transfer and the concomitant attack of Cys-218 on Cys-72, leading to the formation of a disulfide bond. The active site is returned to the reduced state for another round of catalysis by a series of thiol-disulfide exchange reactions via Cys-227, DTT, or thioredoxin.

Amino Acid Sequence↗
PubMed2000

Structure and function of the methionine aminopeptidases.

The removal of the N-terminal methionine from proteins and peptides is dependent upon a novel class of proteases typified by the dinuclear metalloenzyme methionine aminopeptidase from Escherichia coli (eMetAP). Substantial progress has recently been made in determining the structures of several members of this family. The identification of human MetAP as the target of putative anti-cancer drugs reiterates the importance of this family of enzymes. Determination of the modes of binding to E. coli MetAP of a substrate-like bestatin-based inhibitor, as well as phosphorus-containing transition-state analogs and reaction products has led to a rationalization of the substrate specificity and suggested the presumed catalytic mechanism. The conservation of key active site residues and ligand interactions between the MetAPs and other enzyme of the same fold suggest that avoidance of cross-reactivity may be an important consideration in the design of inhibitors directed toward a single member of the family.

Aminopeptidases↗
PubMed2000

The structural basis for enhanced stability and reduced DNA binding seen in engineered second-generation Cro monomers and dimers.

It was previously shown that the Cro repressor from phage lambda, which is a dimer, can be converted into a stable monomer by a five-amino acid insertion. Phe58 is the key residue involved in this transition, switching from interactions which stabilize the dimer to those which stabilize the monomer. Structural studies, however, suggested that Phe58 did not penetrate into the core of the monomer as well as it did into the native dimer. This was strongly supported by the finding that certain core-repacking mutations, including in particular, Phe58-->Trp, increased the stability of the monomer. Unexpectedly, the same substitution also increased the stability of the native dimer. At the same time it decreased the affinity of the dimer for operator DNA. Here we describe the crystal structures of the Cro F58W mutant, both as the monomer and as the dimer. The F58W monomer crystallized in a form different from that of the original monomer. In contrast to that structure, which resembled the DNA-bound form of Cro, the F58W monomer is closer in structure to wild-type (i.e. non-bound) Cro. The F58W dimer also crystallizes in a form different from the native dimer but has a remarkably similar overall structure which tends to confirm the large changes in conformation of Cro on binding DNA. Introduction of Trp58 perturbs the position occupied by the side-chain of Arg38, a DNA-contact residue, providing a structural explanation for the reduction in DNA-binding affinity. The improved thermal stability is seen to be due to the enhanced solvent transfer free energy of Trp58 relative to Phe58, supplemented in the dimer structure, although not the monomer, by a reduction in volume of internal cavities.

Bacteriophage lambda↗
PubMed2000

The introduction of strain and its effects on the structure and stability of T4 lysozyme.

In order to try to better understand the role played by strain in the structure and stability of a protein a series of "small-to-large" mutations was made within the core of T4 lysozyme. Three different alanine residues, one involved in backbone contacts, one in side-chain contacts, and the third adjacent to a small cavity, were each replaced with subsets of the larger residues, Val, Leu, Ile, Met, Phe and Trp. As expected, the protein is progressively destabilized as the size of the introduced side-chain becomes larger. There does, however, seem to be a limit to the destabilization, suggesting that a protein of a given size may be capable of maintaining only a certain amount of strain. The changes in stability vary greatly from site to site. Substitution of larger residues for both Ala42 and Ala98 substantially destabilize the protein, even though the primary contacts in one case are predominantly with side-chain atoms and in the other with backbone. The results suggest that it is neither practical nor meaningful to try to separate the effects of introduced strain on side-chains from the effects on the backbone. Substitutions at Ala129 are much less destabilizing than at sites 42 or 98. This is most easily understood in terms of the pre-existing cavity, which provides partial space to accommodate the introduced side-chains. Crystal structures were obtained for a number of the mutants. These show that the changes in structure to accommodate the introduced side-chains usually consist of essentially rigid-body displacements of groups of linked atoms, achieved through relatively small changes in torsion angles. On rare occasions, a side-chain close to the site of substitution may change to a different rotamer. When such rotomer changes occur, they permit the structure to dissipate strain by a response that is plastic rather than elastic. In one case, a surface loop moves 1.2 A, not in direct response to a mutation, but in an interaction mediated via an intermolecular contact. It illustrates how the structure of a protein can be modified by crystal contacts.

Alanine↗