A C-reactive protein mutant that does not bind to phosphocholine and pneumococcal C-polysaccharide

C-reactive protein (CRP), the major human acute-phase plasma protein, binds to phosphocholine (PCh) residues present in pneumococcal C-polysaccharide (PnC) of Streptococcus pneumoniae and to PCh exposed on damaged and apoptotic cells. CRP also binds, in a PCh-inhibitable manner, to ligands that do not contain PCh, such as fibronectin (Fn). Crystallographic data on CRP-PCh complexes indicate that Phe(66) and Glu(81) contribute to the formation of the PCh binding site of CRP. We used site-directed mutagenesis to analyze the contribution of Phe(66) and Glu(81) to the binding of CRP to PCh, and to generate a CRP mutant that does not bind to PCh-containing ligands. Five CRP mutants, F66A, F66Y, E81A, E81K, and F66A/E81A, were constructed, expressed in COS cells, purified, and characterized for their binding to PnC, PCh-BSA, and Fn. Wild-type and F66Y CRP bound to PnC with similar avidities, while binding of E81A and E81K mutants to PnC was substantially reduced. The F66A and F66A/E81A mutants did not bind to PnC. Identical results were obtained with PCh-BSA. In contrast, all five CRP mutants bound to Fn as well as did wild-type CRP. We conclude that Phe(66) is the major determinant of CRP-PCh interaction and is critical for binding of CRP to PnC. The data also suggest that the binding sites for PCh and Fn on CRP are distinct. A CRP mutant incapable of binding to PCh provides a tool to assess PCh-inhibitable interactions of CRP with its other biologically significant ligands, and to further investigate the functions of CRP in host defense and inflammation.     

Mutational analysis of Gbetagamma and phospholipid interaction with G protein-coupled receptor kinase 2

Agonist-dependent regulation of G protein-coupled receptors is dependent on their phosphorylation by G protein-coupled receptor kinases (GRKs). GRK2 and GRK3 are selectively regulated in vitro by free Gbetagamma subunits and negatively charged membrane phospholipids through their pleckstrin homology (PH) domains. However, the molecular binding determinants and physiological role for these ligands remain unclear. To address these issues, we generated an array of site-directed mutants within the GRK2 PH domain and characterized their interaction with Gbetagamma and phospholipids in vitro. Mutation of several residues in the loop 1 region of the PH domain, including Lys-567, Trp-576, Arg-578, and Arg-579, resulted in a loss of receptor phosphorylation, likely via disruption of phospholipid binding, that was reversed by Gbetagamma. Alternatively, mutation of residues distal to the C-terminal amphipathic alpha-helix, including Lys-663, Lys-665, Lys-667, and Arg-669, resulted in decreased responsiveness to Gbetagamma. Interestingly, mutation of Arg-587 in beta-sheet 3, a region not previously thought to interact with Gbetagamma, resulted in a specific and profound loss of Gbetagamma responsiveness. To further characterize these effects, two mutants (GRK2(K567E/R578E) and GRK2(R587Q)) were expressed in Sf9 cells and purified. Analysis of these mutants revealed that GRK2(K567E/R578E) was refractory to stimulation by negatively charged phospholipids but bound Gbetagamma similar to wild-type GRK2. In contrast, GRK2(R587Q) was stimulated by acidic phospholipids but failed to bind Gbetagamma. In order to examine the role of phospholipid and Gbetagamma interaction in cells, wild-type and mutant GRK2s were expressed with a beta(2)-adrenergic receptor (beta(2)AR) mutant that is responsive to GRK2 phosphorylation (beta(2)AR(Y326A)). In these cells, GRK2(K567E/R578E) and GRK2(R587Q) were largely defective in promoting agonist-dependent phosphorylation and internalization of beta(2)AR(Y326A). Similarly, wild-type GRK2 but not GRK2(K567E/R578E) or GRK2(R587Q) promoted morphinedependent phosphorylation of the mu-opioid receptor in cells. Thus, we have (i) identified several specific GRK2 binding determinants for Gbetagamma and phospholipids, and (ii) demonstrated that Gbetagamma binding is the limiting step for GRK2-dependent receptor phosphorylation in cells.     

Time-resolved fluorescence and computational studies of adenylylated glutamine synthetase: analysis of intersubunit interactions.

Adenylylation of Tyr-397 of each subunit of Escherichia coli glutamine synthetase (GS) down-regulates enzymatic activity in vivo. The overall structure of the enzyme consists of 12 subunits arranged as two hexamers, face to face. Research reported in this paper addresses the question of whether the covalently attached adenylyl group interacts with neighboring amino acid residues to produce the regulatory phenomenon. Wild-type GS has two Trp residues (positions 57 and 158) and the adenylylation site lies within 7-8 A of the Trp-57 loop in the adjacent subunit of the same hexameric ring; Trp-158 is about 35 A from the site of adenylylation. Fluorescence lifetimes and quantum yields have been determined for two fluorophores with wild-type and mutant GS. One fluorophore is epsilon-AMP adenylylated GS (at Tyr-397), and the other fluorophore is the intrinsic protein residue Trp-57. These experiments were conducted in order to detect possible intersubunit interactions between adenylyl groups and the neighboring Trp-57 to search for a role for the Trp-57 loop in the regulation of GS. The fluorescence due to epsilon-AMP of two adenylylated enzymes, wild-type GS and the W158F mutant, exhibits heterogeneous decay kinetics; the data adequately fit to a double exponential decay model with recovered average lifetime values of 18.2 and 2.1 ns, respectively. The pre-exponential factors range from 0.66 to 0.73 for the long lifetime component, at five emission wavelengths. The W57L-epsilon-AMP enzyme yields longer average lifetime values of 19.5 and 2.4 ns, and the pre-exponential factors range from 0.82 to 0.85 for the long lifetime component. An additional residue in the Trp-57 loop, Lys-58, has been altered and the K58C mutant enzyme has been adenylylated with epsilon-AMP on Tyr-397. Lys-58 is near the ATP binding site and may represent a link by which the adenylyl group controls the activity of GS. The fluorescence of epsilon-AMP-adenylylated K58C mutant GS is best described by a triple exponential decay with average recovered lifetime values of 19.9, 4.6, and 0.58 ns, with the largest fraction being the median lifetime component. Relative quantum yields of epsilon-AMP-Tyr-397 were measured in order to determine if static quenching occurs from adenine-indole stacking in the wild-type GS. The relative quantum yield of the epsilon-AMP-adenylylated W57L mutant is larger than the wild-type protein by the amount predicted from the difference in lifetime values: thus, no static quenching is evident.(ABSTRACT TRUNCATED AT 400 WORDS)     

Variability among the sites by which curaremimetic toxins bind to torpedo acetylcholine receptor, as revealed by identification of the functional residues of alpha-cobratoxin

alpha-Cobratoxin, a long chain curaremimetic toxin from Naja kaouthia venom, was produced recombinantly (ralpha-Cbtx) from Escherichia coli. It was indistinguishable from the snake toxin. Mutations at 8 of the 29 explored toxin positions resulted in affinity decreases for Torpedo receptor with DeltaDeltaG higher than 1.1 kcal/mol. These are R33E > K49E > D27R > K23E > F29A >/= W25A > R36A >/= F65A. These positions cover a homogeneous surface of approximately 880 A(2) and mostly belong to the second toxin loop, except Lys-49 and Phe-65 which are, respectively, on the third loop and C-terminal tail. The mutations K23E and K49E, and perhaps R33E, induced discriminative interactions at the two toxin-binding sites. When compared with the short toxin erabutoxin a (Ea), a number of structurally equivalent residues are commonly implicated in binding to muscular-type nicotinic acetylcholine receptor. These are Lys-23/Lys-27, Asp-27/Asp-31, Arg-33/Arg-33, Lys-49/Lys-47, and to a lesser and variable extent Trp-25/Trp-29 and Phe-29/Phe-32. In addition, however, the short and long toxins display three major differences. First, Asp-38 is important in Ea in contrast to the homologous Glu-38 in alpha-Cbtx. Second, all of the first loop is insensitive to mutation in alpha-Cbtx, whereas its tip is functionally critical in Ea. Third, the C-terminal tail may be specifically critical in alpha-Cbtx. Therefore, the functional sites of long and short curaremimetic toxins are not identical, but they share common features and marked differences that might reflect an evolutionary pressure associated with a great diversity of prey receptors.     

Arg-52 in the Melibiose Carrier of Escherichia coli Is Important for Cation-Coupled Sugar Transport and Participates in an Intrahelical Salt Bridge

Arg-52 of the Escherichia coli melibiose carrier was replaced by Ser (R52S), Gln (R52Q), or Val (R52V). While the level of carrier in the membrane for each mutant remained similar to that for the wild type, analysis of melibiose transport showed an uncoupling of proton cotransport and a drastic reduction in Na(+)-coupled transport. Second-site revertants were selected on MacConkey plates containing melibiose, and substitutions were found at nine distinct locations in the carrier. Eight revertant substitutions were isolated from the R52S strain: Asp-19-->Gly, Asp-55-->Asn, Pro-60-->Gln, Trp-116-->Arg, Asn-244-->Ser, Ser-247-->Arg, Asn-248-->Lys, and Ile-352-->Val. Two revertants were also isolated from the R52V strain: Trp-116-->Arg and Thr-338-->Arg revertants. The R52Q strain yielded an Asp-55-->Asn substitution and a first-site revertant, Lys-52 (R52K). The R52K strain had transport properties similar to those of the wild type. Analysis of melibiose accumulation showed that proton-driven accumulation was still defective in the second-site revertant strains, and only the Trp-116-->Arg, Ser-247-->Arg, and Asn-248-->Lys revertants regained significant Na(+)-coupled accumulation. In general, downhill melibiose transport in the presence of Na(+) was better in the revertant strains than in the parental mutants. Three revertant strains, Asp-19-->Gly, Asp-55-->Asn, and Thr-338-->Arg strains, required a high Na(+) concentration (100 mM) for maximal activity. Kinetic measurements showed that the N248K and W116R revertants lowered the K(m) for melibiose, while other revertants restored transport velocity. We suggest that the insertion of positive charges on membrane helices is compensating for the loss of Arg-52 and that helix II is close to helix IV and VII. We also suggest that Arg-52 is salt bridged to Asp-55 (helix II) and Asp-19 (helix I).     

3'-Phosphoadenosine 5'-phosphosulfate binding site of flavonol 3-sulfotransferase studied by affinity chromatography and 31P NMR

The function of Lys-59, Arg-141, and Arg-277 in PAPS binding and catalysis of the flavonol 3-sulfotransferase was investigated. Affinity chromatography of conservative mutants with PAPS analogues allowed us to determine that Lys-59 interacts with the 5' portion of the nucleotide, while Arg-141 interacts with the 3' portion, confirming assignments deduced from the crystal structure of mouse estrogen sulfotransferase [Kakuta, Y., Pedersen, L. G., Carter, C. W. , Negishi, M., and Pedersen, L. C. (1997) Nat. Struct. Biol. 4, 904-908]. The affinity chromatography method could be used to characterize site-directed mutants for other types of enzymes that bind nucleoside 3',5'- or 2',5'-diphosphates. 31P NMR spectra of enzyme-PAP complexes were recorded for the wild-type enzyme and K59R and K59A mutants. The results of these experiments suggest that Lys-59 is involved in the determination of the proper orientation of the phosphosulfate group for catalysis.     

Cytoplasmic loop connecting helices IV and V of the melibiose permease from Escherichia coli is involved in the process of Na+-coupled sugar translocation

Previous photolabeling and limited proteolysis studies suggested that one of the four basic residues (Arg-141) of the N-terminal cytoplasmic loop connecting helices IV and V (loop 4-5) of the melibiose permease (MelB) from Escherichia coli has a potential role in its symport function (Ambroise, Y., Leblanc, G., and Rousseau, B. (2000) Biochemistry 39, 1338-1345). A mutagenesis study of Arg-141 and of the other three basic residues of loop 4-5 was undertaken to further examine this hypothesis. Cys replacement analysis indicated that Arg-141 and Arg-149, but not Lys-138 and Arg-139, are essential for MelB transport activity. Replacement of Arg-141 by neutral residues (Cys or Gln) inactivated transport and energy-independent carrier-mediated flows of substrates (counterflow, efflux), whereas it had a limited effect on co-substrate binding. R141C sugar transport was partially rescued on reintroducing a positive charge with a charged and permeant thiol reagent. Whereas R149C was completely inactive, R149K and R149Q remained functional. Strikingly, introduction of an additional mutation in the C-terminal helix X (Gly for Val-343) of R149C restored sugar transport. Impermeant thiol reagents inhibited R149C/V343G transport activity in right-side-out membrane vesicles and prevented sugar binding in a sugar-protected manner. All these data suggest that MelB loop 4-5 is close to the sugar binding site and that the charged residue Arg-141 is involved in the reaction of co-substrate translocation or substrate release in the inner compartment.     

Arg-158 is critical in both binding the substrate and stabilizing the transition-state oxyanion for the enzymatic reaction of malonamidase E2

Malonamidase E2 (MAE2) from Bradyrhizobium japonicum is an enzyme that hydrolyzes malonamate to malonate and has a Ser-cis-Ser-Lys catalytic triad at the active site. The crystal structures of wild type and mutant MAE2 exhibited that the guanido group of Arg-158 could be involved in the binding of malonamate in which the negative charge of the carboxyl group could destabilize a negatively charged transition-state oxyanion in the enzymatic reaction. In an attempt to elucidate the specific roles of Arg-158, site-directed mutants, R158Q, R158E, and R158K, were prepared (see Table 1). The crystal structure of R158Q determined at 2.2 Angstrom resolution showed that the guanido group of Arg-158 was important for the substrate binding with the marginal structural change upon the mutation. The k(cat) value of R158Q significantly decreased by over 1500-fold and the catalytic activity of R158E could not be detected. The k(cat) value of R158K was similar to that of the wild type with the K(m) value drastically increased by 100-fold, suggesting that Lys-158 of R158K can stabilize the negative charge of the carboxylate in the substrate to some extent and contribute to the stabilization of the transition-state oxyanion, but a single amine group of Lys-158 in R158K could not precisely anchor the carboxyl group of malonamate compared with the guanido group of Arg-158. Our kinetic and structural evidences demonstrate that Arg-158 in MAE2 should be critical to both binding the substrate and stabilizing the transition-state oxyanion for the catalytic reaction of MAE2.     

The contribution of adjacent subunits to the active sites of D-3-phosphoglycerate dehydrogenase

D-3-Phosphoglycerate dehydrogenase (PGDH) from Escherichia coli is allosterically inhibited by L-serine, the end product of its metabolic pathway. Previous results have shown that inhibition by serine has a large effect on Vmax and only a small or negligible effect on Km. PGDH is thus classified as a V-type allosteric enzyme. In this study, the active site of PGDH has been studied by site-directed mutagenesis to assess the role of certain residues in substrate binding and catalysis. These consist of a group of cationic residues (Arg-240, Arg-60, Arg-62, Lys-39, and Lys-141') that potentially form an electrostatic environment for the binding of the negatively charged substrate, as well as the only tryptophan residue found in PGDH and which fits into a hydrophobic pocket immediately adjacent to the active site histidine residue. Interestingly, Trp-139' and Lys-141' are part of the polypeptide chain of the subunit that is adjacent to the active site. The results of mutating these residues show that Arg-240, Arg-60, Arg-62, and Lys-141' play distinct roles in the binding of the substrate to the active site. Mutants of Trp-139' show that this residue may play a role in stabilizing the catalytic center of the enzyme. Furthermore, these mutants appear to have a significant effect on the cooperativity of serine inhibition and suggest a possible role for Trp-139' in the cooperative interactions between subunits.     

Delineation of the roles of amino acids involved in the catalytic functions of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase

The roles of particular amino acids in substrate and coenzyme binding and catalysis of glucose-6-phosphate dehydrogenase of Leuconostoc mesenteroides have been investigated by site-directed mutagenesis, kinetic analysis, and determination of binding constants. The enzyme from this species has functional dual NADP(+)/NAD(+) specificity. Previous investigations in our laboratories determined the three-dimensional structure. Kinetic studies showed an ordered mechanism for the NADP-linked reaction while the NAD-linked reaction is random. His-240 was identified as the catalytic base, and Arg-46 was identified as important for NADP(+) but not NAD(+) binding. Mutations have been selected on the basis of the three-dimensional structure. Kinetic studies of 14 mutant enzymes are reported and kinetic mechanisms are reported for 5 mutant enzymes. Fourteen substrate or coenzyme dissociation constants have been measured for 11 mutant enzymes. Roles of particular residues are inferred from k(cat), K(m), k(cat)/K(m), K(d), and changes in kinetic mechanism. Results for enzymes K182R, K182Q, K343R, and K343Q establish Lys-182 and Lys-343 as important in binding substrate both to free enzyme and during catalysis. Studies of mutant enzymes Y415F and Y179F showed no significant contribution for Tyr-415 to substrate binding and only a small contribution for Tyr-179. Changes in kinetics for T14A, Q47E, and R46A enzymes implicate these residues, to differing extents, in coenzyme binding and discrimination between NADP(+) and NAD(+). By the same measure, Lys-343 is also involved in defining coenzyme specificity. Decrease in k(cat) and k(cat)/K(m) for the D374Q mutant enzyme defines the way Asp-374, unique to L. mesenteroides G6PD, modulates stabilization of the enzyme during catalysis by its interaction with Lys-182. The greatly reduced k(cat) values of enzymes P149V and P149G indicate the importance of the cis conformation of Pro-149 in accessing the correct transition state.     

The Carbohydrate-linked Phosphorylcholine of the Parasitic Nematode Product ES-62 Modulates Complement Activation

Parasitic nematodes manufacture various carbohydrate-linked phosphorylcholine (PCh)-containing molecules, including ES-62, a protein with an N-linked glycan terminally substituted with PCh. The PCh component is biologically important because it is required for immunomodulatory effects. We showed that most ES-62 was bound to a single protein, C-reactive protein (CRP), in normal human serum, displaying a calcium-dependent, high-avidity interaction and ability to form large complexes. Unexpectedly, CRP binding to ES-62 failed to efficiently activate complement as far as the C3 convertase stage in comparison with PCh-BSA and PCh-containing Streptococcus pneumoniae cell wall polysaccharide. C1q capture assays demonstrated an ES-62-CRP-C1q interaction in serum. The three ligands all activated C1 and generated C4b to similar extents. However, a C2a active site was not generated following ES-62 binding to CRP, demonstrating that C2 cleavage was far less efficient for ES-62-containing complexes. We proposed that failure of C2 cleavage was due to the flexible nature of carbohydrate-bound PCh and that reduced proximity of the C1 complex was the reason that C2 was poorly cleaved. This was confirmed using synthetic analogues that were similar to ES-62 only in respect of having a flexible PCh. Furthermore, ES-62 was shown to deplete early complement components, such as the rate-limiting C4, following CRP interaction and thereby inhibit classical pathway activation. Thus, flexible PCh-glycan represents a novel mechanism for subversion of complement activation. These data illustrate the importance of the rate-limiting C4/C2 stage of complement activation and reveal a new addition to the repertoire of ES-62 immunomodulatory mechanisms with possible therapeutic applications.     

Molecular basis of the interaction between complement receptor type 2 (CR2/CD21) and Epstein-Barr virus glycoprotein gp350

The binding of the Epstein-Barr virus glycoprotein gp350 by complement receptor type 2 (CR2) is critical for viral attachment to B lymphocytes. We set out to test hypotheses regarding the molecular nature of this interaction by developing an enzyme-linked immunosorbent assay (ELISA) for the efficient analysis of the gp350-CR2 interaction by utilizing wild-type and mutant forms of recombinant gp350 and also of the CR2 N-terminal domains SCR1 and SCR2 (designated CR2 SCR1-2). To delineate the CR2-binding site on gp350, we generated 17 gp350 single-site substitutions targeting an area of gp350 that has been broadly implicated in the binding of both CR2 and the major inhibitory anti-gp350 monoclonal antibody (MAb) 72A1. These site-directed mutations identified a novel negatively charged CR2-binding surface described by residues Glu-21, Asp-22, Glu-155, Asp-208, Glu-210, and Asp-296. We also identified gp350 amino acid residues involved in non-charge-dependent interactions with CR2, including Tyr-151, Ile-160, and Trp-162. These data were supported by experiments in which phycoerythrin-conjugated wild-type and mutant forms of gp350 were incubated with CR2-expressing K562 cells and binding was assessed by flow cytometry. The ELISA was further utilized to identify several positively charged residues (Arg-13, Arg-28, Arg-36, Lys-41, Lys-57, Lys-67, Arg-83, and Arg-89) within SCR1-2 of CR2 that are involved in the binding interaction with gp350. These experiments allowed a comparison of those CR2 residues that are important for binding gp350 to those that define the epitope for an effective inhibitory anti-CR2 MAb, 171 (Asn-11, Arg-13, Ser-32, Thr-34, Arg-36, and Tyr-64). The mutagenesis data were used to calculate a model of the CR2-gp350 complex using the soft-docking program HADDOCK.     

Multiple activation loop conformations and their regulatory properties in the insulin receptor's kinase domain

Low catalytic efficiency of protein kinases often results from intrasteric inhibition caused by the activation loop blocking the active site. In the insulin receptor's kinase domain, Asp-1161 and Tyr-1162 in the peptide substrate-like sequence of the unphosphorylated activation loop can interact with four invariant residues in the active site: Lys-1085, Asp-1132, Arg-1136, and Gln-1208. Contributions of these six residues to intrasteric inhibition were tested by mutagenesis, and the unphosphorylated kinase domains were characterized. The mutations Q1208S, K1085N, and Y1162F each relieved intrasteric inhibition, increasing catalytic efficiency but without changing the rate-limiting step of the reaction. The mutants R1136Q and D1132N were virtually inactive. Steric accessibility of the active site was ranked by relative changes in iodide quenching of intrinsic fluorescence, and A-loop conformation was ranked by limited tryptic cleavage. Together these ranked the openness of the active site cleft as R1136Q approximately D1132N > or = D1161A > Y1162F approximately K1085N > Q1208S > or = wild-type. These findings demonstrate the importance of specific invariant residues for intrasteric inhibition and show that diverse activation loop conformations can produce similar steady-state kinetic properties. This suggests a broader range of regulatory properties for the activation loop than expected from a simple off-versus-on switch for kinase activation.     

Primary structure of glucagon from the gut of the common dogfish (Scyliorhinus canicula)

The primary structure of glucagon isolated from the intestine of the common dogfish, Scyliorhinus canicula, was established as H S E G T F T S D Y S K Y M D N R R A K D F V Q W L M N T. The peptide shows four substitutions compared with human glucagon: Glu-3 for Gln, Met-14 for Leu, Asn-16 for Ser and Lys-20 for Gln. Glucagon represented the predominant molecular form of the glucagon-like immunoreactivity in the dogfish gut extracts demonstrating that the pathway of posttranslational processing of proglucagon in the gut of this fish differs markedly from the pathway in the mammalian gut.     

The complete amino acid sequence of the low molecular weight cytosolic acid phosphatase

This paper presents the complete amino acid sequence of the low molecular weight acid phosphatase from bovine liver. This isoenzyme of the acid phosphatase family is located in the cytosol, is not inhibited by L-(+)-tartrate and fluoride ions, but is inhibited by sulfhydryl reagents. The enzyme consists of 157 amino acid residues, has an acetylated NH2 terminus, and has arginine as the COOH-terminal residue. All 8 half-cystine residues are in the free thiol form. The molecular weight calculated from the sequence is 17,953. The sequence was determined by characterizing the peptides purified by reverse-phase high performance liquid chromatography from tryptic, thermolytic, peptic, Staphylococcus aureus protease, and chymotryptic digests of the carboxymethylated protein. No sequence homologies were found with the two known acylphosphatase isoenzymes or the metalloproteins porcine uteroferrin and purple acid phosphatase from bovine spleen (both of which have acid phosphatase activity). Two half-cystines at or near the active site were identified through the reaction of the enzyme with [14C] iodoacetate in the presence or in the absence of a competitive inhibitor (i.e. inorganic phosphate). Ac-A E Q V T K S V L F V C L G N I C R S P I A E A V F R K L V T D Q N I S D N W V I D S G A V S D W N V G R S P N P R A V S C L R N H G I N T A H K A R Q V T K E D F V T F D Y I L C M D E S N L R D L N R K S N Q V K N C R A K I E L L G S Y D P Q K Q L I I E D P Y Y G N D A D F E T V Y Q Q C V R C C R A F L E K V R-OH.     

Characterization of herpes simplex virus type 1 thymidine kinase mutants selected under a single round of high-dose brivudin

A broad variety of herpes simplex virus type 1 clones was selected under a single round of high-dose selection with brivudin. Mutations in the thymidine kinase (TK) genes consisted of 42% frameshift mutations within homopolymer repeats of G's and C's and single nucleotide substitutions (58%) that produced stop codons (Q261 and R281) or a new codon at the site of the substitution (A168T, R51W, G59W, G206R, R220H, Y239S, and T287 M). The A168T change, associated with an altered TK phenotype, proved to be the most commonly selected substitution. For the different mutants, a correlation between phenotype, genotype, and in vivo neurovirulence was observed.     

The alpha-glucuronidase,GlcA67A,of Cellvibrio japonicus utilizes the carboxylate and methyl groups of aldobiouronic acid as important substrate recognition determinants

alpha-Glucuronidases are key components of the ensemble of enzymes that degrade the plant cell wall. They hydrolyze the alpha1,2-glycosidic bond between 4-O-methyl-d-glucuronic acid (4-O-MeGlcA) and the xylan or xylooligosaccharide backbone. Here we report the crystal structure of an inactive mutant (E292A) of the alpha-glucuronidase, GlcA67A, from Cellvibrio japonicus in complex with its substrate. The data show that the 4-O-methyl group of the substrate is accommodated within a hydrophobic sheath flanked by Val-210 and Trp-160, whereas the carboxylate moiety is located within a positively charged region of the substrate-binding pocket. The carboxylate side chains of Glu-393 and Asp-365, on the "beta-face" of 4-O-MeGlcA, form hydrogen bonds with a water molecule that is perfectly positioned to mount a nucleophilic attack at the anomeric carbon of the target glycosidic bond, providing further support for the view that, singly or together, these amino acids function as the catalytic base. The capacity of reaction products and product analogues to inhibit GlcA67A shows that the 4-O-methyl group, the carboxylate, and the xylose sugar of aldobiouronic acid all play an important role in substrate binding. Site-directed mutagenesis informed by the crystal structure of enzyme-ligand complexes was used to probe the importance of highly conserved residues at the active site of GlcA67A. The biochemical properties of K288A, R325A, and K360A show that a constellation of three basic amino acids (Lys-288, Arg-325, and Lys-360) plays a critical role in binding the carboxylate moiety of 4-O-MeGlcA. Disruption of the apolar nature of the pocket created by Val-210 (V210N and V210S) has a detrimental effect on substrate binding, although the reduction in affinity is not reflected by an inability to accommodate the 4-O-methyl group. Replacing the two tryptophan residues that stack against the sugar rings of the substrate with alanine (W160A and W543A) greatly reduced activity.     

C-reactive protein-bound enzymatically modified low-density lipoprotein does not transform macrophages into foam cells

The formation of low-density lipoprotein (LDL) cholesterol-loaded macrophage foam cells contributes to the development of atherosclerosis. C-reactive protein (CRP) binds to atherogenic forms of LDL, but the role of CRP in foam cell formation is unclear. In this study, we first explored the binding site on CRP for enzymatically modified LDL (E-LDL), a model of atherogenic LDL to which CRP binds. As reported previously, phosphocholine (PCh) inhibited CRP-E-LDL interaction, indicating the involvement of the PCh-binding site of CRP in binding to E-LDL. However, the amino acids Phe66 and Glu81 in CRP that participate in CRP-PCh interaction were not required for CRP-E-LDL interaction. Surprisingly, blocking of the PCh-binding site with phosphoethanolamine (PEt) dramatically increased the binding of CRP to E-LDL. The PEt-mediated enhancement in the binding of CRP to E-LDL was selective for E-LDL because PEt inhibited the binding of CRP to another PCh-binding site-ligand pneumococcal C-polysaccharide. Next, we investigated foam cell formation by CRP-bound E-LDL. We found that, unlike free E-LDL, CRP-bound E-LDL was inactive because it did not transform macrophages into foam cells. The function of CRP in eliminating the activity of E-LDL to form foam cells was not impaired by the presence of PEt. Combined data lead us to two conclusions. First, PEt is a useful compound because it potentiates the binding of CRP to E-LDL and, therefore, increases the efficiency of CRP to prevent transformation of macrophages into E-LDL-loaded foam cells. Second, the function of CRP to prevent formation of foam cells may influence the process of atherogenesis.     

Maize phosphoenolpyruvate carboxylase. Mutations at the putative binding site for glucose 6-phosphate caused desensitization and abolished responsiveness to regulatory phosphorylation

Phosphoenolpyruvate carboxylases (PEPC, EC 4.1.1.31) from higher plants are regulated by both allosteric effects and reversible phosphorylation. Previous x-ray crystallographic analysis of Zea mays PEPC has revealed a binding site for sulfate ion, speculated to be the site for an allosteric activator, glucose 6-phosphate (Glc-6-P) (Matsumura, H., Xie, Y., Shirakata, S., Inoue, T., Yoshinaga, T., Ueno, Y., Izui, K., and Kai, Y. (2002) Structure (Lond.) 10, 1721-1730). Because kinetic experiments have also supported this notion, each of the four basic residues (Arg-183, -184, -231, and -372' on the adjacent subunit) located at or near the binding site was replaced by Gln, and the kinetic properties of recombinant mutant enzymes were investigated. Complete desensitization to Glc-6-P was observed for R183Q, R184Q, R183Q/R184Q (double mutant), and R372Q, as was a marked decrease in the sensitivity for R231Q. The heterotropic effect of Glc-6-P on an allosteric inhibitor, l-malate, was also abolished, but sensitivity to Gly, another allosteric activator of monocot PEPC, was essentially not affected, suggesting the distinctness of their binding sites. Considering the kinetic and structural data, Arg-183 and Arg-231 were suggested to be involved directly in the binding with phosphate group of Glc-6-P, and the residues Arg-184 and Arg-372 were thought to be involved in making up the site for Glc-6-P and/or in the transmission of an allosteric regulatory signal. Most unexpectedly, the mutant enzymes had almost lost responsiveness to regulatory phosphorylation at Ser-15. An apparent lack of kinetic competition between the phosphate groups of Glc-6-P and of phospho-Ser at 15 suggested the distinctness of their binding sites. The possible roles of these Arg residues are discussed.