Identification of the high affinity Mn2+ binding site of bacteriophage lambda phosphoprotein phosphatase: effects of metal ligand mutations on electron paramagnetic resonance spectra and phosphatase activities

Bacteriophage lambda phosphoprotein phosphatase (lambdaPP) has structural similarity to the mammalian Ser/Thr phosphoprotein phosphatases (PPPs) including the immunosuppressant drug target calcineurin. PPPs possess a conserved active site containing a dinuclear metal cluster, with metal ligands provided by a phosphoesterase motif plus two additional histidine residues at the C-terminus. Multiple sequence alignment of lambdaPP with 28 eubacterial and archeal phosphoesterases identified active site residues from the phosphoesterase motif and in many cases 2 additional C-terminal His metal ligands. Most highly similar to lambdaPP are E. coli PrpA and PrpB. Using the crystal structure of lambdaPP [Voegtli, W. C., et al. (2000) Biochemistry 39, 15365-15374] as a structural and active site model for PPPs and related bacterial phosphoesterases, we have studied mutant forms of lambdaPP reconstituted with Mn(2+) by electron paramagnetic resonance (EPR) spectroscopy, Mn(2+) binding analysis, and phosphatase kinetics. Analysis of Mn(2+)-bound active site mutant lambdaPP proteins shows that H22N, N75H, and H186N mutations decrease phosphatase activity but still allow mononuclear Mn(2+) and [(Mn(2+))(2)] binding. The high affinity Mn(2+) binding site is shown to consist of M2 site ligands H186 and Asn75, but not H22 from the M1 site which is ascribed as the lower affinity site.     

Role of the ortholog and paralog amino acid invariants in the active site of the UDP-MurNAc-L-alanine:D-glutamate ligase (MurD).

To evaluate their role in the active site of the UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD) from Escherichia coli, 12 residues conserved either in the Mur superfamily [Eveland, S. S., Pompliano, D. L., and Anderson, M. S. (1997) Biochemistry 36, 6223-6229; Bouhss, A., Mengin-Lecreulx, D., Blanot, D., van Heijenoort, J., and Parquet, C. (1997) Biochemistry 36, 11556-11563] or in the sequences of 26 MurD orthologs were submitted to site-directed mutagenesis. All these residues lay within the cleft of the active site of MurD as defined by its 3D structure [Bertrand, J. A., Auger, D., Fanchon, E., Martin, L., Blanot, D., van Heijenoort, J., and Dideberg, O. (1997) EMBO J. 16, 3416-3425]. Fourteen mutant proteins (D35A, K115A, E157A/K, H183A, Y194F, K198A/F, N268A, N271A, H301A, R302A, D317A, and R425A) containing a C-terminal (His)(6) extension were prepared and their steady-state kinetic parameters determined. All had a reduced enzymatic activity, which in many cases was very low, but no mutation led to a total loss of activity. Examination of the specificity constants k(cat)/K(m) for the three MurD substrates indicated that most mutations affected both the binding of one substrate and the catalytic process. These kinetic results correlated with the assigned function of the residues based on the X-ray structures.     

Characterization and functional expression in Escherichia coli of the sodium/proton/glutamate symport proteins of Bacillus stearothermophilus and Bacillus caldotenax

The genes encoding the Na+/H+/L-glutamate symport proteins of the thermophilic organisms Bacillus stearothermophilus (gltTBs) and Bacillus caldotenax (gltTBc) were cloned by complementation of Escherichia coli JC5412 for growth on glutamate as sole source of carbon, energy and nitrogen. The nucleotide sequences of the gltTBs and gltTBc genes were determined. In both cases the translated sequences corresponded with proteins of 421 amino acid residues (96.7% amino acid identity between GltTBs and GltTBc). Putative promoter, terminator and ribosome-binding-site sequences were found in the flanking regions. These expression signals were functional in E. coli. The hydropathy profiles indicate that the proteins are hydrophobic and could form 12 membrane-spanning regions. The Na+/H+ coupled L-glutamate symport proteins GltTBs and GltTBc are homologous to the strictly H+ coupled L-glutamate transport protein of E. coli K-12 (overall 57.2% identity). Functional expression of glutamate transport activity was demonstrated by uptake of glutamate in whole cells and membrane vesicles. In accordance with previous observations (de Vrij et al., 1989; Heyne et al., 1991), glutamate uptake was driven by the electrochemical gradients of sodium ions and protons.     

Membrane interaction of the glycosyltransferase MurG: a special role for cardiolipin

MurG is a peripheral membrane protein that is one of the key enzymes in peptidoglycan biosynthesis. The crystal structure of Escherichia coli MurG (S. Ha, D. Walker, Y. Shi, and S. Walker, Protein Sci. 9:1045-1052, 2000) contains a hydrophobic patch surrounded by basic residues that may represent a membrane association site. To allow investigation of the membrane interaction of MurG on a molecular level, we expressed and purified MurG from E. coli in the absence of detergent. Surprisingly, we found that lipid vesicles copurify with MurG. Freeze fracture electron microscopy of whole cells and lysates suggested that these vesicles are derived from vesicular intracellular membranes that are formed during overexpression. This is the first study which shows that overexpression of a peripheral membrane protein results in formation of additional membranes within the cell. The cardiolipin content of cells overexpressing MurG was increased from 1 +/- 1 to 7 +/- 1 mol% compared to nonoverexpressing cells. The lipids that copurify with MurG were even further enriched in cardiolipin (13 +/- 4 mol%). MurG activity measurements of lipid I, its natural substrate, incorporated in pure lipid vesicles showed that the MurG activity is higher for vesicles containing cardiolipin than for vesicles with phosphatidylglycerol. These findings support the suggestion that MurG interacts with phospholipids of the bacterial membrane. In addition, the results show a special role for cardiolipin in the MurG-membrane interaction.     

An analysis of the structure of the product of the rbsA gene of Escherichia coli K12

The predicted amino acid sequence of rbsA, a gene from the high affinity ribose transport operon (rbs) of Escherichia coli K12, is homologous to the products of hisP, malK, and pstB, components of the histidine, maltose, and phosphate high affinity transport operons. The recent finding by Hobson et al. (Hobson, A. C., Weatherwax, R., and Ames, G.F.-L. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 7333-7337) that the hisP and malK products bind ATP suggests that these four gene products may be involved in coupling the energy from ATP to drive the active transport in their respective transport systems. Each gene product contains a sequence of glycine and basic residues which are characteristic of an ATP-binding site (Walker, J.E., Saraste, M., Runswick, M.J., and Gay, N.J. (1982) EMBO J. 1, 945-951). Interestingly the N- and C-terminal halves of rbsA are also homologous, suggesting that a primordial gene duplication and subsequent fusion of the products occurred.     

Mechanistic roles of Thr134, Tyr160, and Lys 164 in the reaction catalyzed by dTDP-glucose 4,6-dehydratase

Escherichia coli dTDP-glucose 4,6-dehydratase and UDP-galactose 4-epimerase are members of the short-chain dehydrogenase/reductase SDR family. A highly conserved triad consisting of Ser/Thr, Tyr, and Lys is present in the active sites of these enzymes as well in other SDR proteins. Ser124, Tyr149, and Lys153 in the active site of UDP-galactose 4-epimerase are located in similar positions as the corresponding Thr134, Tyr160, and Lys164, in the active site of dTDP-glucose 4,6-dehydratase. The role of these residues in the first hydride transfer step of the dTDP-glucose 4,6-dehydratase mechanism has been studied by mutagenesis and steady-state kinetic analysis. In all mutants except T134S, the k(cat) values are more than 2 orders of magnitude lower than of wild-type enzyme. The substrate analogue, dTDP-xylose, was used to investigate the effects of the mutations on rate of the first hydride transfer step. The first step becomes significantly rate limiting upon mutation of Tyr160 to Phe and only partly rate limiting in the reaction catalyzed by K164M and T134A dehydratases. The pH dependence of k(cat), the steady-state NADH level, and the fraction of NADH formed with saturating dTDP-xylose show shifts in the pK(a) assigned to Tyr160 to more basic values by mutation of Lys164 and Thr134. The pK(a) of Tyr160, as determined by the pH dependence of NADH formation by dTDP-xylose, is 6.41. Lys164 and Thr134 are believed to play important roles in the stabilization of the anion of Tyr160 in a fashion similar to the roles of the corresponding residues in UDP-galactose 4-epimerase, which facilitate the ionization of Tyr149 in that enzyme [Liu, Y., et al. (1997) Biochemistry 35, 10675--10684]. Tyr160 is presumably the base for the first hydride transfer step, while Thr134 may relay a proton from the sugar to Tyr160.     

Insight into the roles of tyrosine 82 and glycine 253 in the Escherichia coli adenine glycosylase MutY

The oxidation product of 2'-deoxyguanosine, 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG), produces G:C to T:A transversion mutations. The Escherichia coli base excision repair glycosylase MutY plays an important role in preventing OG-associated mutations by removing adenines misincorporated opposite OG lesions during DNA replication. Recently, biallelic mutations in the human MutY homologue (hMYH) have been correlated with the development of colorectal cancer. The two most common mutations correspond to two single amino acid substitutions in the hMYH protein: Y165C and G382D [Al-Tassan, N., et al. (2002) Nat. Genet. 30, 227-232]. Previously, our laboratory analyzed the adenine glycosylase activity of the homologous variant E. coli MutY enzymes, Y82C and G253D [Chmiel, N. H., et al. (2003) J. Mol. Biol. 327, 431-443]. This work demonstrated that both variants have a reduced adenine glycosylase activity and affinity for substrate analogues compared to wild-type MutY. Recent structural work on Bacillus stearothermophilus MutY bound to an OG:A mismatch-containing duplex indicates that both residues aid in recognition of OG [Fromme, J. C., et al. (2004) Nature 427, 652-656]. To determine the extent with which Tyr 82 and Gly 253 contribute to catalysis of adenine removal by E. coli MutY, we made a series of additional modifications in these residues, namely, Y82F, Y82L, and G253A. When the substrate analogue 2'-deoxy-2'-fluoroadenosine (FA) in duplex paired with G or OG is used, both Y82F and G253A showed reduced binding affinity, and G253A was unable to discriminate between OG and G when paired with FA. Additionally, compromised glycosylase activity of Y82F, Y82C, and G253A MutY was observed using the nonoptimal G:A substrate, or at low reaction temperatures. Interestingly, adenine removal from an OG:A-containing DNA substrate by Y82C MutY was also shown to be extremely sensitive to the NaCl concentration. The most surprising result was the remarkably similar activity of Y82L MutY to the WT enzyme under all conditions examined, indicating that a leucine residue may effectively replace tyrosine for intercalation at the OG:A mismatch. The results contained herein provide further insight regarding the intricate roles of Tyr 82 and Gly 253 in the OG recognition and adenine excision functions of MutY.     

Nucleotide sequence of the Escherichia coli polA gene and primary structure of DNA polymerase I

We report the nucleotide sequence of 3.2 kilobase pair region of the Escherichia coli polA gene, comprising the coding region for DNA polymerase I with about 400 base pairs of flanking sequence. The amino acid sequence for DNA polymerase I derived from our DNA sequence is largely consistent with previous protein chemical data. In the following paper, Brown et al. (Brown, W. E., Stump, K. H., and Kelley, W. S. (1982) J. Biol. Chem. 257, 1965-1972) present additional protein chemistry experiments that further confirm our sequence. Mild proteolysis of DNA polymerase I is known to produce two enzymatically active fragments (Brutlag, D., Atkinson, M. R., Setlow, P., and Kornberg, A. (1969) Biochem. Biophys. Res. Commun. 37, 982-989; Klenow, H., and Henningsen, I. (1970) Proc. Natl. Acad. Sci. U. S. A. 74, 5632-5636). We have located the site of this cleavage between residues 323 and 324 of the 928 amino acid polymerase molecule. By sequence comparison of the polA1 and wild type alleles, we have identified the polA1 mutation as a change from Trp (TGG) to amber (TAG) at residue 342.     

Site-directed alteration of the active-site residues of histidine decarboxylase from Clostridium perfringens

To clarify the mechanism of biogenesis and catalysis by the pyruvoyl-dependent histidine decarboxylase (HisDCase) from Clostridium perfringens, 12 mutant genes encoding amino acid substitutions at the active site of this enzyme were constructed and expressed in Escherichia coli. The resulting mutant proteins were purified to homogeneity, characterized, and subjected to kinetic analysis. The results (a) exclude all polar amino acid residues in the active site except Glu-214 as donor of the proton that replaces the carboxyl group of histidine during decarboxylation and, since E214I and E214H are nearly inactive, indicate that Glu-214 is the essential proton donor; (b) demonstrate the importance to substrate binding of hydrophobic interactions between Phe-98, Ile-74, and the imidazole ring of histidine, and of hydrogen bonding between Asp-78 and N2 of the substrate; and (c) demonstrate a significant unidentified role for Glu-81 in the maintenance of the active-site structure. The proposed roles of these amino acid residues are consistent with those assigned on the basis of crystallographic evidence to the corresponding residues at the active site of the related HisDCase from Lactobacillus 30a [Gallagher, T., Snell, E. E., & Hackert, M. L. (1989) J. Biol. Chem. 264, 12737-12743]. Of the residues altered, only Ser-97 was essential for the autocatalytic serinolysis reaction by which this HisDCase, (alpha beta)6, is derived from its inactive, pyruvate-free precursor, proHisDCase, pi 6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure and function of the Escherichia coli RecE protein, a member of the RecB nuclease domain family

The RecB subunit of the Escherichia coli RecBCD enzyme has both helicase and nuclease activities. The helicase function was localized to an N-terminal domain, whereas the nuclease activity was found in a C-terminal domain. Recent analysis has uncovered a group of proteins that have weak amino acid sequence similarity to the RecB nuclease domain and that are proposed to constitute a family of related proteins (Aravind, L., Walker, D. R., and Koonin, E. V. (1999) Nucleic Acids Res. 27, 1223-1242). One is the E. coli RecE protein (exonuclease VIII), an ATP-independent exonuclease that degrades the 5'-terminated strand of double-stranded DNA. We have made mutations in several residues of RecE that align with the critical residues of RecB, and we find that the mutations reduce or abolish the nuclease activity of RecE but do not affect the enzyme binding to linear double-stranded DNA. Proteolysis experiments with subtilisin show that a stable 34-kilodalton C-terminal domain that contains these critical residues has nuclease activity, whereas no stable proteolytic fragments accumulate from the N-terminal portion of RecE. These results show that RecE has a nuclease domain and active site that are similar to RecB, despite the very weak sequence similarity between the two proteins. These similarities support the hypothesis that the nuclease domains of the two proteins are evolutionarily related.     

Strategies for structural proteomics of prokaryotes: Quantifying the advantages of studying orthologous proteins and of using both NMR and X-ray crystallography approaches

Only about half of non-membrane-bound proteins encoded by either bacterial or archaeal genomes are soluble when expressed in Escherichia coli (Yee et al., Proc Natl Acad Sci USA 2002;99:1825-1830; Christendat et al., Prog Biophys Mol Biol 200;73:339-345). This property limits genome-scale functional and structural proteomics studies, which depend on having a recombinant, soluble version of each protein. An emerging strategy to increase the probability of deriving a soluble derivative of a protein is to study different sequence homologues of the same protein, including representatives from thermophilic organisms, based on the assumption that the stability of these proteins will facilitate structural analysis. To estimate the relative merits of this strategy, we compared the recombinant expression, solubility, and suitability for structural analysis by NMR and/or X-ray crystallography for 68 pairs of homologous proteins from E. coli and Thermotoga maritima. A sample suitable for structural studies was obtained for 62 of the 68 pairs of homologs under standardized growth and purification procedures. Fourteen (eight E. coli and six T. maritima proteins) samples generated NMR spectra of a quality suitable for structure determination and 30 (14 E. coli and 16 T. maritima proteins) samples formed crystals. Only three (one E. coli and two T. maritima proteins) samples both crystallized and had excellent NMR properties. The conclusions from this work are: (1) The inclusion of even a single ortholog of a target protein increases the number of samples for structural studies almost twofold; (2) there was no clear advantage to the use of thermophilic proteins to generate samples for structural studies; and (3) for the small proteins analyzed here, the use of both NMR and crystallography approaches almost doubled the number of samples for structural studies.     

Essential catalytic role of Glu134 in endo-beta-1,3-1,4-D-glucan 4-glucanohydrolase from B. licheniformis as determined by site-directed mutagenesis.

Site-directed mutagenesis experiments designed to identify the active site of Bacillus licheniformis endo-beta-1,3-1,4-D-glucan 4-glucanohydrolase (beta-glucanase) have been performed. Putative catalytic residues were chosen on the basis of sequence similarity analysis to viral and eukaryotic lysozymes. Four mutant enzymes were expressed and purified from recombinant E. coli and their kinetics analysed with barley beta-glucan. Replacement of Glu134 by Gln produced a mutant (E134Q) that retains less than 0.3% of the wild-type activity. The other mutants, D133N, E160Q and D179N, are active but show different kinetic parameters relative to wild-type indicative of their participation in substrate binding and transition-state complex stabilization. Glu134 is essential for activity; it is comprised in a region of high sequence similarity to the active site of T4 lysozyme and matches the position of the general acid catalyst. These results strongly support a lysozyme-like mechanism for this family of Bacillus beta-glucan hydrolases with Glu134 being the essential acid catalyst.     

In vitro mutagenesis studies at the arginine residues of adenylate kinase. A revised binding site for AMP in the X-ray-deduced model

Although X-ray crystallographic and NMR studies have been made on the adenylate kinases, the substrate-binding sites are not unequivocally established. In an attempt to shed light on the binding sites for MgATP2- and for AMP2- in human cytosolic adenylate kinase (EC 2.7.4.3, hAK1), we have investigated the enzymic effects of replacement of the arginine residues (R44, R132, R138, and R149), which had been assumed by Pai et al. [Pai, E. F., Sachsenheimer, W., Schirmer, R. H., & Schulz, G. E. (1977) J. Mol. Biol. 114, 37-45] to interact with the phosphoryl groups of AMP2- and MgATP2-. With use of the site-directed mutagenesis method, point mutations were made in the artificial gene for hAK1 [Kim, H. J., Nishikawa, S., Tanaka, T., Uesugi, S., Takenaka, H., Hamada, M., & Kuby, S. A. (1989) Protein Eng. 2, 379-386] to replace these arginine residues with alanyl residues and yield the mutants R44A hAK1, R132A hAK1, R138A hAK1, and R149A hAK1. The resulting large increases in the Km,app values for AMP2- of the mutant enzymes, the relatively small increases in the Km,app values for MgATP2-, and the fact that the R132A, R138A, and R149A mutant enzymes proved to be very poor catalysts are consistent with the idea that the assigned substrate binding sites of Pai et al. (1977) have been reversed and that their ATP-binding site may be assigned as the AMP site.     

Half-of-the-sites binding of reactive intermediates and their analogues to 4-oxalocrotonate tautomerase and induced structural asymmetry of the enzyme

4-Oxalocrotonate tautomerase (4-OT), a homohexameric enzyme, converts the unconjugated enone, 2-oxo-4-hexenedioate (1), to the conjugated enone, 2-oxo-3-hexenedioate (3), via a dienolic intermediate, 2-hydroxymuconate (2). Pro-1 serves as the general base, and both Arg-11 and Arg-39 function in substrate binding and catalysis in an otherwise hydrophobic active site. Although 4-OT exhibits hyperbolic kinetics and no structural asymmetry either by X-ray or by NMR, inactivation by two affinity labels showed half-site stoichiometry [Stivers, J. T., et al. (1996) Biochemistry 35, 803-813; Johnson, W. H., Jr., et al. (1997) Biochemistry 36, 15724-15732], and titration of the R39Q mutant with cis,cis-muconate showed negative cooperativity [Harris, T. K., et al. (1999) Biochemistry 38, 12343-12357]. To test for anticooperativity during catalysis, 4-OT was titrated with equilibrium mixtures (> or = 81% product) of the reactive dicarboxylate or monocarboxylate intermediates, 2 or 2-hydroxy-2,4-pentadienoate (4), respectively, in three types of NMR experiments: two-dimensional 1H-15N HSQC titrations of backbone NH and of Arg N epsilonH resonances and one-dimensional 15N NMR titrations of Arg N epsilon resonances. All titrations showed substoichiometric binding of the equilibrium mixtures to 3 +/- 1 sites per hexamer with apparent dissociation constants comparable to the Km values of the intermediates. Compound 4 also bound 1 order of magnitude less tightly at another site, suggesting negative cooperativity. Consistent with negative cooperativity, asymmetry of the resulting complexes at saturating levels of 2 and 4 is indicated by splitting of the backbone NH resonances of 11 residues and 10 residues of 4-OT, respectively. The dicarboxylate competitive inhibitor, (2E)-fluoromuconate (5), with a KI of 45 +/- 7 microM, also exhibited substoichiometric binding to 3 +/- 1 sites per hexamer, with a KD of 25 +/- 18 microM, and splitting of the backbone NH resonance of L8. The monocarboxylate inhibitors (2E)- (6) and (2Z)-2-fluoro-2,4-pentadienoate (7) showed much weaker binding (KD = 3.1 +/- 1.3 mM), as well as splitting of two and five backbone NH resonances, respectively, indicating asymmetry of the complexes. The N epsilon resonances of both Arg-11 and Arg-39 were shifted downfield, and that of Pro-1N was broadened by all ligands, consistent with the major catalytic roles of these residues. Structural pathways for the site-site interactions which result in negative cooperativity are proposed on the basis of the X-ray structures of free and affinity-labeled 4-OT. Selective resonance broadenings induced by the binding of inactive analogues and active intermediates indicate residues which may be mobilized during reversible ligand binding and during catalysis, respectively.     

An assay for exogenous sources of purified MurG, enabled by the complementation of Escherichia coli murG(Ts) by the Mycobacterium tuberculosis homologue

The Mycobacterium tuberculosis murG gene, Rv2153, was expressed in Escherichia coli murG(Ts) strain OV58 on a plasmid under the control of the arabinose-inducible araBAD promoter. Mycobacterium tuberculosis murG rescued the growth of E.?coli murG(Ts) at the nonpermissive temperature: transformants were only obtained in the presence of 0.2% arabinose at 42?°C, and their growth rate was dependent on arabinose concentrations. However, no MurG activity was detected in membranes from the transformant grown in arabinose at 42?°C, while MraY activity was normal. This observation led to the development of a membrane-based scintillation proximity assay for exogenous sources of MurG. Addition of purified E.?coli MurG resulted in the reconstitution of MurG and peptidoglycan synthesis in these membranes. MurG is an attractive target for drug discovery, but assays to measure the activity of purified MurG are challenging. This presents an easy method to measure the activity of exogenous sources of MurG for structure-activity studies of mutant MurG proteins. It can also be used to compare the activity of, or effect of inhibitors on, MurG from other bacterial species.     

Effects of mutations and truncations on the kinetic behavior of IIAGlc, a phosphocarrier and regulatory protein of the phosphoenolpyruvate phosphotransferase system of Escherichia coli

IIAGlc, a component of the glucose-specific phosphoenolpyruvate:phosphotransferase system (PTS) of Escherichia coli, is important in regulating carbohydrate metabolism. In Glc uptake, the phosphotransfer sequence is: phosphoenolpyruvate --> Enzyme I --> HPr --> IIAGlc --> IICBGlc --> Glc. (HPr is the first phosphocarrier protein of the PTS.) We previously reported two classes of IIAGlc mutations that substantially decrease the P-transfer rate constants to/from IIAGlc. A mutant of His75 which adjoins the active site (His90), (H75Q), was 0.5% as active as wild-type IIAGlc in the reversible P-transfer to HPr. Two possible explanations were offered for this result: (a) the imidazole ring of His75 is required for charge delocalization and (b) H75Q disrupts the hydrogen bond network: Thr73, His75, phospho-His90. The present studies directly test the H-bond network hypothesis. Thr73 was replaced by Ser, Ala, or Val to eliminate the network. Because the rate constants for phosphotransfer to/from HPr were largely unaffected, we conclude that the H-bond network hypothesis is not correct. In the second class of mutants, proteolytic truncation of seven residues of the IIAGlc N terminus caused a 20-fold reduction in phosphotransfer to membrane-bound IICBGlc from Salmonella typhimurium. Here, we report the phosphotransfer rates between two genetically constructed N-terminal truncations of IIAGlc (Delta7 and Delta16) and the proteins IICBGlc and IIBGlc (the soluble cytoplasmic domain of IICBGlc). The truncations did not significantly affect reversible P-transfer to IIBGlc but substantially decreased the rate constants to IICBGlc in E. coli and S. typhimurium membranes. The results support the hypothesis (Wang, G., Peterkofsky, A., and Clore, G. M. (2000) J. Biol. Chem. 275, 39811-39814) that the N-terminal 18-residue domain "docks" IIAGlc to the lipid bilayer of membranes containing IICBGlc.     

Mapping the glycoprotein Ib-binding site in the von willebrand factor A1 domain

The von Willebrand factor (vWF) mediates platelet adhesion to exposed subendothelium at sites of vascular injury. It does this by forming a bridge between subendothelial collagen and the platelet glycoprotein Ib-IX-V complex (GPIb). The GPIb-binding site within vWF has been localized to the vWF-A1 domain. Based on the crystal structure of the vWF-A1 domain (Emsley, J., Cruz, M., Handin, R., and Liddington, R. (1998) J. Biol. Chem. 273, 10396-10401), we introduced point mutations into 16 candidate residues that might form all or part of the GPIb interaction site. We also introduced two mutations previously reported to impair vWF function yielding a total of 18 mutations. The recombinant vWF-A1 mutant proteins were then expressed in Escherichia coli, and the activity of the purified proteins was assessed by their ability to support flow-dependent platelet adhesion and their ability to inhibit ristocetin-induced platelet agglutination. Six mutations located on the front and upper anterior face of the folded vWF-A1 domain, R524S, G561S, H563T, T594S/E596A, Q604R, and S607R, showed reduced activity in all the assays, and we suggest that these residues form part of the GPIb interaction site. One mutation, G561S, with impaired activity occurs in the naturally occurring variant form of von Willebrand's disease-type 2M underscoring the physiologic relevance of the mutations described here.     

Fluorescent properties of the Escherichia coli D-xylose isomerase active site.

The consequences of active site mutations of the Escherichia coli D-xylose isomerase (E.C. 5.3.1.5) on substrate binding were examined by fluorescence spectroscopy. Site-directed mutagenesis of conserved tryptophan residues in the E. coli enzyme (Trp49 and Trp188) reveals that fluorescence quenching of these residues occurs during the binding of xylose by the wild-type enzyme. The fluorescent properties of additional active site substitutions at His101 were also examined. Substitutions of His101 which inactivate the enzyme were shown to have altered spectral characteristics, which preclude detection of substrate binding. In the case of H101S, a mutant protein with measurable isomerizing activity, substrate binding with novel fluorescent properties was observed, possibly the bound pyranose form of xylose under steady-state conditions.