As part of our continuing efforts to accumulate information on the coding region of unidentified human genes, we newly determined the sequences of 40 cDNA clones of human cell line KG-1 which correspond to relatively long and nearly full-length transcripts, and predicted the coding sequences of the corresponding genes, named KIAA0161 to 0200. The average size of the cDNA clones analyzed was approximately 5.0 kb. A computer search of the sequences in public databases indicated that the sequences of 20 genes were unrelated to any reported genes, while the remaining 20 genes carried sequences which show some similarities to known genes. Among the genes in the latter category, KIAA0167 contained a Zn-finger motif with significant structural similarity to that of the yeast transcription factor GCS1, and KIAA0189 was classified into the RhoGAP gene family. Stretches of typical CAG (Gln) repeats, which were often correlated with genetic disorders, were found in KIAA0181 and KIAA0192. Another novel repeat composed of alternating Arg and Glu was identified in KIAA0182. Northern hybridization analysis demonstrated that 10 genes are expressed in a cell- or tissue-specific manner.

The epitope determinants of chimpanzee Fab antibody 1A5, which have been shown to be broadly reactive to flaviviruses and efficient for cross-neutralization of dengue virus type 1 and type 2 (DENV-1 and DENV-2), were studied by analysis of DENV-2 antigenic variants. Sequence analysis showed that one antigenic variant contained a Gly-to-Val substitution at position 106 within the flavivirus-conserved fusion peptide loop of the envelope protein (E), and another variant contained a His-to-Gln substitution at position 317 in E. Substitution of Gly(106)Val in DENV-2 E reduced the binding affinity of Fab 1A5 by approximately 80-fold, whereas substitution of His(317)Gln had little or no effect on antibody binding compared to the parental virus. Treatment of DENV-2 with beta-mercaptoethanol abolished binding of Fab 1A5, indicating that disulfide bridges were required for the structural integrity of the Fab 1A5 epitope. Binding of Fab 1A5 to DENV-2 was competed by an oligopeptide containing the fusion peptide sequence as shown by competition enzyme-linked immunosorbent assay. Both DENV-2 antigenic variants were shown to be attenuated, or at least similar to the parental virus, when evaluated for growth in cultured cells or for neurovirulence in mice. Fab 1A5 inhibited low pH-induced membrane fusion of mosquito C6/36 cells infected with DENV-1 or DENV-2, as detected by reduced syncytium formation. Both substitutions in DENV-2 E lowered the pH threshold for membrane fusion, as measured in a fusion-from-within assay. In the three-dimensional structure of E, Gly(106) in domain II and His(317) in domain III of the opposite E monomer were spatially close. From the locations of these amino acids, Fab 1A5 appears to recognize a novel epitope that has not been mapped before with a flavivirus monoclonal antibody.

All aspartic proteases, including retroviral proteases, share the triplet DTG critical for the active site geometry and catalytic function. These residues interact closely in the active, dimeric structure of HIV-1 protease (PR). We have systematically assessed the effect of the D25N mutation on the structure and stability of the mature PR monomer and dimer. The D25N mutation (PR(D25N)) increases the equilibrium dimer dissociation constant by a factor >100-fold (1.3 +/- 0.09 microm) relative to PR. In the absence of inhibitor, NMR studies reveal clear structural differences between PR and PR(D25N) in the relatively mobile P1 loop (residues 79-83) and flap regions, and differential scanning calorimetric analyses show that the mutation lowers the stabilities of both the monomer and dimer folds by 5 and 7.3 degrees C, respectively. Only minimal differences are observed in high resolution crystal structures of PR(D25N) complexed to darunavir (DRV), a potent clinical inhibitor, or a non-hydrolyzable substrate analogue, Ac-Thr-Ile-Nle-r-Nle-Gln-Arg-NH(2) (RPB), as compared with PR.DRV and PR.RPB complexes. Although complexation with RPB stabilizes both dimers, the effect on their T(m) is smaller for PR(D25N) (6.2 degrees C) than for PR (8.7 degrees C). The T(m) of PR(D25N).DRV increases by only 3 degrees C relative to free PR(D25N), as compared with a 22 degrees C increase for PR.DRV, and the mutation increases the ligand dissociation constant of PR(D25N).DRV by a factor of approximately 10(6) relative to PR.DRV. These results suggest that interactions mediated by the catalytic Asp residues make a major contribution to the tight binding of DRV to PR.

We have recently identified a new form of post-translational regulation of BACE1 (beta-site amyloid precursor protein (APP)-cleaving enzyme 1), a membrane protein that acts as the rate-limiting enzyme in the generation of the Alzheimer disease amyloid beta-peptide (Abeta). Specifically, BACE1 is transiently acetylated on seven lysine residues in the lumen of the endoplasmic reticulum/endoplasmic reticulum-Golgi intermediate compartment (ER/ERGIC). The acetylated intermediates of the nascent protein are able to reach the Golgi apparatus, whereas the non-acetylated ones are retained and degraded in a post-ER compartment. Here, we report that the serine protease PCSK9 (proprotein convertase subtilisin kexin type 9) contributes to the disposal of non-acetylated BACE1. Both overexpression and small interfering RNA-mediated downregulation of PCSK9 affected the levels of BACE1. The downregulation of PCSK9 affected the levels of the loss-of-acetylation mutants (BACE1(Ala) and BACE1(Arg)) but not those of the gain-of-acetylation mutant (BACE1(Gln)). In addition, Pcsk9(-/-) mice showed increased levels of BACE1 and Abeta in the brain. Finally, we found that nascent low-density lipoprotein receptor, a known substrate of PCSK9, is also acetylated.

sigmaE is an alternative sigma factor involved in a pathway of extracytoplasmic stress responses in Escherichia coli. Under normal growth conditions, sigmaE activity is down-regulated by the membrane-bound anti-sigmaE protein, RseA. Extracytoplasmic stress signals induce degradation of RseA by two successive proteolytic events: DegS-catalyzed first cleavage at a periplasmic site followed by YaeL-mediated second proteolysis at an intramembrane region. Normally, the second reaction (site-2 proteolysis) only occurs after the first cleavage (site-1 cleavage). Here, we show that YaeL variants with the periplasmic PDZ domain deleted or mutated allows unregulated cleavage of RseA and consequent sigmaE activation. It was also found that a glutamine-rich region in the periplasmic domain of RseA was required for the avoidance of the YaeL-mediated proteolysis in the absence of site-1 cleavage. These results indicate that multiple negative elements both in the enzyme (PDZ domain) and in the substrate (glutamine-rich region) determine the strict dependence of the site-2 proteolysis on the site-1 cleavage.

The crystal structure of the glutamine-binding protein (GlnBP) from Escherichia coli in a ligand-free "open" conformational state has been determined by isomorphous replacement methods and refined to an R-value of 21.4% at 2.3 A resolution. There are two molecules in the asymmetric unit, related by pseudo 4-fold screw symmetry. The refined model consists of 3587 non-hydrogen atoms from 440 residues (two monomers), and 159 water molecules. The structure has root-mean-square deviations of 0.013 A from "ideal" bond lengths and 1.5 degrees from "ideal" bond angles. The GlnBP molecule has overall dimensions of approximately 60 A x 40 A x 35 A and is made up of two domains (termed large and small), which exhibit a similar supersecondary structure, linked by two antiparallel beta-strands. The small domain contains three alpha-helices and four parallel and one antiparallel beta-strands. The large domain is similar to the small domain but contains two additional alpha-helices and three more short antiparallel beta-strands. A comparison of the secondary structural motifs of GlnBP with those of other periplasmic binding proteins is discussed. A model of the "closed form" GlnBP-Gln complex has been proposed based on the crystal structures of the histidine-binding protein-His complex and "open form" GlnBP. This model has been successfully used as a search model in the crystal structure determination of the "closed form" GlnBP-Gln complex by molecular replacement methods. The model agrees remarkably well with the crystal structure of the Gln-GlnBP complex with root-mean-square deviation of 1.29 A. Our study shows that, at least in our case, it is possible to predict one conformational state of a periplasmic binding protein from another conformational state of the protein. The glutamine-binding pockets of the model and the crystal structure are compared and the modeling technique is described.

Glutamine (Gln) protects gut mucosa against injury and promotes mucosal healing. Because the induction of heat shock proteins (HSP) protects cells under conditions of stress, we determined whether Gln conferred protection against stress in an intestinal epithelial cell line through HSP induction. Gln added to IEC-18 cells induces an increase in HSP70, a concentration-dependent effect also seen with mRNA. Two forms of injury, lethal heat (49 degrees C) and oxidant, were used, and viability was determined by 51Cr release. Gln-treated cells were significantly more resistant to injury. Treatment with 6-diazo-5-oxo-L-norleucine (DON), a nonmetabolizable analog of Gln, induced HSP70 and protected cells from injury, but less than Gln. These findings suggest that the effects of Gln on HSP70 induction and cellular protection are mediated by metabolic and nonmetabolic mechanisms. To determine whether HSP induction was central to the action of Gln and DON, quercetin, which blocks HSP induction, was used. Quercetin blocked HSP70 induction and the protective effect of Gln and DON. We conclude that the protective effects of Gln in intestinal epithelial cells are in part mediated by HSP70 induction.

The repressor encoded by bacteriophage 434 binds to its operators by inserting a 'recognition' alpha-helix into the major groove of the DNA. We have identified an amino acid-base pair contact that determines (in part) the DNA-binding specificity of 434 repressor. The identification is based on the properties of a 'new-specificity' mutant, named Repressor [Ala 28], which bears the substitution of Ala for Gln at the first residue of its recognition alpha-helix. Repressor [Ala 28] binds with high affinity to a particular doubly mutant operator bearing the same substitution at position 1 in each half-site, but does not bind to either the wild-type operator or to other mutant operators. We describe molecular models of residue 28-base pair 1 interactions that account for the binding specificities of both the mutant and wild-type proteins.

UvrB plays a major role in recognition and processing of DNA lesions during nucleotide excision repair. The crystal structure of UvrB revealed a similar fold as found in monomeric DNA helicases. Homology modeling suggested that the beta-hairpin motif of UvrB might be involved in DNA binding (Theis, K., Chen, P. J., Skorvaga, M., Van Houten, B., and Kisker, C. (1999) EMBO J. 18, 6899-6907). To determine a role of the beta-hairpin of Bacillus caldotenax UvrB, we have constructed a deletion mutant, Deltabetah UvrB, which lacks residues Gln-97-Asp-112 of the beta-hairpin. Deltabetah UvrB does not form a stable UvrB-DNA pre-incision complex and is inactive in UvrABC-mediated incision. However, Deltabetah UvrB is able to bind to UvrA and form a complex with UvrA and damaged DNA, competing with wild type UvrB. In addition, Deltabetah UvrB shows wild type-like ATPase activity in complex with UvrA that is stimulated by damaged DNA. In contrast to wild type UvrB, the ATPase activity of mutant UvrB does not lead to a destabilization of the damaged duplex. These results indicate that the conserved beta-hairpin motif is a major factor in DNA binding.

Strain SF22, a glutamine-requiring (Gln-) mutant of Bacillus subtilis SMY, is likely to have a mutation in the structural gene for glutamine synthetase, since this strain synthesized 22 to 55% as much glutamine synthetase antigen as did wild-type cells in a 10-min period but had less than 3% of wild-type glutamine synthetase enzymatic activity. The expression of several genes subject to glucose catabolite repression was altered in the Gln- mutant. The induced levels of alpha-glucosidase, histidase, and aconitase were 3.5- to 4-fold higher in SF22 cells than in wild-type cells grown in glucose-glutamine medium, and citrate synthase levels were 8-fold higher in the Gln- mutant than in wild-type cells. The relief of glucose catabolite repression in the Gln- mutant may result from poor utilization of glucose. Examination of the intracellular metabolite pools of cells grown in glucose-glutamine medium showed that the glucose-6-phosphate pool was 2.5-fold lower, the pyruvate pool was 4-fold lower, and the 2-ketoglutarate pool was 2.5-fold lower in the Gln- cells than they were in wild-type cells. Intracellular levels of glutamine were sixfold higher in the Gln- mutant than in wild-type cells. Measurements of enzymes involved in glutamine transport and utilization showed that the elevated pools of glutamine in the Gln- mutant resulted from a threefold increase in glutamine permease and a fivefold decrease in glutamate synthase. The pleiotropic effect of the gln-22 mutation on the expression of several genes suggests that either the glutamine synthetase protein or its enzymatic product, glutamine, is involved in the regulation of several metabolic pathways in B. subtilis.

Among water channel proteins (aquaporins), aquaporin-collecting duct (AQP-CD) is the vasopressin-regulated water channel. Vasopressin causes cAMP production in the renal collecting duct cells, and this is believed to lead to exocytic insertion of water channel into the apical membrane (shuttle hypothesis). AQP-CD contains a consensus sequence for cAMP-dependent protein kinase, residues at positions 253-256 (Arg-Arg-Gln-Ser). To determine the role of this site, Ser-256 was substituted for Ala, Leu, Thr, Asp, or Glu by site-directed mutagenesis. In Xenopus oocytes injected with wild-type or mutated AQP-CD cRNAs, osmotic water permeability (Pf) was 4.8-7.7 times higher than Pf of water-injected oocytes. Incubation with cAMP plus forskolin or direct cAMP injection into the oocytes increased Pf of wild-type, but not mutated, AQP-CD-expressing oocytes, whereas the amounts of AQP-CD expression were similar in wild and mutated types as identified by Western blot analysis. In vitro phosphorylation studies of AQP-CD proteins expressed in oocyte showed that cAMP-dependent protein kinase phosphorylated wild-type, but not mutated, AQP-CD proteins. Phosphoamino acid analysis revealed that this phosphorylation occurred at the serine residue. Moreover, phosphorylation of AQP-CD protein in intact rat kidney medulla tissues was stimulated by incubation with cAMP. Our data suggest that cAMP stimulates water permeability of AQP-CD by phosphorylation. This process may contribute to the vasopressin-regulated water permeability of collecting duct in addition to the apical insertion of AQP-CD by exocytosis.

In order to understand the functional implication of residues constituting the dNTP-binding pocket of human immunodeficiency virus type 1 reverse transcriptase, we performed site-directed mutagenesis at positions 65, 72, 113, 115, 151, 183, 184, and 219, and the resulting mutant enzymes were examined for their biochemical properties and nucleotide selectivity on RNA and DNA templates. Mutations at positions 65, 115, 183, 184, and 219 had negligible to moderate influence on the polymerase activity, while Ala substitution at positions 72 and 151 as well as substitution with Ala or Glu at position 113 severely impaired the polymerase function of the enzyme. The K219A, Y115F, and Q151M mutants had no influence on the fidelity; Y183A, Y183F, K65A, and Q151N mutants exhibited higher fidelity on both RNA and DNA templates, while Y115A was less error-prone selectively on a DNA template. Analysis of the three-dimensional model of the enzyme-template primer-dNTP ternary complex suggests that residues Tyr-183, Lys-65, and Gln-151 may have impact on the flexibility of the dNTP-binding pocket by virtue of their multiple interactions with the dNTP, template, primer, and other neighboring residues constituting the pocket. Recruitment of the correct versus incorrect nucleotides may be a function of the flexibility of this pocket. A relatively rigid pocket would provide greater stringency, resulting in higher fidelity of DNA synthesis in contrast to a flexible pocket. Substitution of a residue having multiple interactions with a residue having reduced interaction capability will alter the internal geometry of the pocket, thus directly influencing the fidelity.

Polymorphisms in the prion protein (PrP) gene are associated with phenotypic expression differences of transmissible spongiform encephalopathies in animals and humans. In sheep, at least 10 different mutually exclusive polymorphisms are present in PrP. In this study, we determined the efficiency of the in vitro formation of protease-resistant PrP of nine sheep PrP allelic variants in order to gauge the relative susceptibility of sheep for scrapie. No detectable spontaneous protease-resistant PrP formation occurred under the cell-free conditions used. All nine host-encoded cellular PrP (PrP(C)) variants had distinct conversion efficiencies induced by PrP(Sc) isolated from sheep with three different homozygous PrP genotypes. In general, PrP allelic variants with polymorphisms at either codon 136 (Ala to Val) or codon 141 (Leu to Phe) and phylogenetic wild-type sheep PrP(C) converted with highest efficiency to protease-resistant forms, which indicates a linkage with a high susceptibility of sheep for scrapie. PrP(C) variants with polymorphisms at codons 171 (Gln to Arg), 154 (Arg to His), and to a minor extent 112 (Met to Thr) converted with low efficiency to protease-resistant isoforms. This finding indicates a linkage of these alleles with a reduced susceptibility or resistance for scrapie. In addition, PrP(Sc) with the codon 171 (Gln-to-His) polymorphism is the first variant reported to induce higher conversion efficiencies with heterologous rather than homologous PrP variants. The results of this study strengthen our views on polymorphism barriers and have further implications for scrapie control programs by breeding strategies.

Mutations were identified in the catalytic subunit (C) of the cAMP-dependent protein kinase (EC 2.7.1.37) that block inactivation by regulatory subunit (R) without compromising catalytic activity. Randomly mutagenized mouse C expression vectors were screened functionally for clones that stimulated gene induction in the presence of excess R. Point mutations in the C coding sequence were identified that result in a His----Gln substitution at amino acid 87 (His87Gln) and a Trp----Arg change at amino acid 196 (Trp196Arg). In contrast to wild-type C, both mutants retained partial activity in the presence of excess R isoform RI alpha, although only Trp196Arg retained partial activity in the presence of excess R isoform RII alpha. A C expression vector that included both mutations was fully active in promoting gene induction and was virtually unaffected by an 80-fold excess of either RI alpha or RII alpha. These results demonstrate that mutations at His-87 and Trp-196 alter R interactions with C at a site that is not involved in substrate recognition or enzymatic activity. In contrast to these randomly generated mutations, a site-specific alteration of the autophosphorylated Thr-197 to an Ala resulted in an 80% loss of biological activity and partial resistance to R inhibition. The location and proximity of His-87 and Trp-196 in the crystal structure of C suggest a surface domain that may interact with a region of R that is outside of the substrate/pseudosubstrate site.

Tobacco smoking and occupational exposures are the main known risk factors for bladder cancer, causing direct and indirect damage to DNA. Repair of DNA damage is under genetic control, and DNA repair genes may play a key role in maintaining genome integrity and preventing cancer development. Polymorphisms in DNA repair genes resulting in variation of DNA repair efficiency may therefore be associated with bladder cancer risk. A hospital-based case-control study was conducted in Brescia, Italy, to assess the relationship between polymorphisms in DNA repair genes XRCC1 (Arg(399)Gln), XRCC3 (Thr(241)Met), and XPD (Lys(751)Gln) and bladder cancer risk. A total of 201 male incident bladder cancer cases and 214 male controls with urological nonneoplastic diseases were recruited and frequency-matched on age, period, and hospital of recruitment. Detailed information was collected using a semistructured questionnaire on demographic, dietary, environmental, and occupational factors. Genotypes were determined by PCR-RFLP analysis. The XRCC3 codon 241 variant genotype exhibited a protective effect against bladder cancer [odds ratio (OR), 0.63; 95% confidence interval (CI), 0.42-0.93], which was prominent among heavy smokers (OR, 0.49; 95% CI, 0.28-0.88) but not among never and light smokers. No overall impact of the XRCC1 codon 399 polymorphism was found (OR, 0.86; 95% CI, 0.59-1.28), but a protective influence of the homozygous variant was suggested among heavy smokers (OR, 0.38; 95% CI, 0.14-1.02). XPD polymorphisms did not show an association with bladder cancer (OR, 0.92; 95% CI, 0.62-1.37). There was no statistical evidence of an interaction between these three genetic polymorphisms and either tobacco smoking or occupational exposure to polycyclic aromatic hydrocarbons and aromatic amines. The XRCC3 codon 241 polymorphism had an overall protective effect against bladder cancer that was most apparent among heavy smokers. Similarly, the XRCC1 codon 399 polymorphism also had a protective effect on bladder cancer among heavy smokers. The XPD polymorphism was not, however, associated with bladder cancer risk.

The particulate fraction from a lymphoma cell line, LSTRA, was found to contain an apparent high level of tyrosine protein kinase activity. When this fraction was incubated with [gamma-32P]ATP in the presence of 10 mM MnCl2, hydrolyzed, and assayed, 70--80% of the radioactivity recovered in phosphoamino acids was in phosphotyrosine. Gel electrophoresis of the proteins showed that a large portion of the 32P was in a single protein with a molecular weight of approximately 58,000. The phosphorylated residue in this protein was identified as phosphotyrosine. Detergent extracts of the particulate fraction from LSTRA cells contained both the Mr 58,000 protein and the enzyme responsible for its phosphorylation. These extracts were found to catalyze the phosphorylation of the tyrosine residue in the synthetic peptide, Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg-Gln-Gly, corresponding to the sequence around the tyrosine that is phosphorylated in pp60src; the Km for the peptide in this reaction was 5 mM. High-performance liquid chromatography was used to assay for this phosphorylation. A second peptide was synthesized that contained two additional arginine residues whose presence permitted the phosphorylation of the peptide to be measured by a simple assay using phosphocellulose paper. The Km for this peptide was 3--4 mM, indicating that the presence of the additional arginine residues did not alter the apparent affinity of the kinase for the peptide.

Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs both ensure accurate RNA recognition and prevent the binding of noncognate substrates. Here we show for Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) that the accuracy of tRNA recognition also determines the efficiency of cognate amino acid recognition. Steady-state kinetics revealed that interactions between tRNA identity nucleotides and their recognition sites in the enzyme modulate the amino acid affinity of GlnRS. Perturbation of any of the protein-RNA interactions through mutation of either component led to considerable changes in glutamine affinity with the most marked effects seen at the discriminator base, the 10:25 base pair, and the anticodon. Reexamination of the identity set of tRNA(Gln) in the light of these results indicates that its constituents can be differentiated based upon biochemical function and their contribution to the apparent Gibbs' free energy of tRNA binding. Interactions with the acceptor stem act as strong determinants of tRNA specificity, with the discriminator base positioning the 3' end. The 10:25 base pair and U35 are apparently the major binding sites to GlnRS, with G36 contributing both to binding and recognition. Furthermore, we show that E. coli tryptophanyl-tRNA synthetase also displays tRNA-dependent changes in tryptophan affinity when charging a noncognate tRNA. The ability of tRNA to optimize amino acid recognition reveals a novel mechanism for maintaining translational fidelity and also provides a strong basis for the coevolution of tRNAs and their cognate synthetases.

OBJECTIVE

Deamidation is a common posttranslational modification in human lens crystallins and may be a key factor in the age-related denaturation of such lifelong proteins. The aim of this study was to identify the sites of deamidation in older lenses.

METHODS

High-performance liquid chromatography/mass spectrometry of tryptic digests was used to identify sites of deamidation in the major human lens crystallins. Older normal and age-matched cataractous lenses were compared with fetal lenses.

RESULTS

Approximately equal numbers of glutamine and asparagine residues were deamidated in older lenses; however, the extent of deamidation of Asn was three times greater than that of Gln (Asn, 22.6% +/- 3.6%; Gln, 6.6% +/- 1.3%). Individual crystallins differed markedly in their extent of deamidation, and deamidated residues were typically localized within discrete regions of the polypeptides. A large percentage (42%) of the sites of deamidation were characterized by the presence of a basic amino acid one residue removed from the original Gln or Asn. At nine such sites, the extent of Asn deamidation averaged 50% in aged lenses. There were few differences in deamidation between crystallins of aged normal and nuclear cataractous lenses.

CONCLUSIONS

Equal numbers of Asn and Gln residues are deamidated in crystallins from aged normal and cataractous lenses. Deamidation of Asn/Gln in lifelong proteins, such as those in the lens, may be governed to a significant degree by base-catalyzed processes.

The P2X(7) receptor is a ligand-gated channel that is highly expressed on mononuclear cells of the immune system and that mediates ATP-induced apoptosis. Wide variations in the function of the P2X receptor have been observed, explained in part by (7)loss-of-function polymorphisms that change Glu(496) to Ala (E496A) and Ile(568) to Asn (I568N). In this study, a third polymorphism, which substitutes an uncharged glutamine for the highly positively charged Arg(307) (R307Q), has been found in heterozygous dosage in 12 of 420 subjects studied. P2X(7) function was measured by ATP-induced fluxes of Rb(+), Ba(2+), and ethidium(+) into peripheral blood monocytes or various lymphocyte subsets and was either absent or markedly decreased. Transfection experiments showed that P2X(7) carrying the R307Q mutation lacked either channel or pore function despite robust protein synthesis and surface expression of the receptor. The monoclonal antibody (clone L4) that binds to the extracellular domain of wild type P2X(7) and blocks P2X(7) function failed to bind to the R307Q mutant receptor. Differentiation of monocytes to macrophages up-regulated P2X(7) function in cells heterozygous for the R307Q to a value 10-40% of that for wild type macrophages. However, macrophages from a subject who was double heterozygous for R307Q/I568N remained totally non-functional for P2X(7), and lymphocytes from the same subject also lacked ATP-stimulated phospholipase D activity. These data identify a third loss-of-function polymorphism affecting the human P2X(7) receptor, and since the affected Arg(307) is homologous to those amino acids essential for ATP binding to P2X(1) and P2X(2), it is likely that this polymorphism abolishes the binding of ATP to the extracellular domain of P2X(7).

Agonist binding to the inhibitory glycine receptor (GlyR) initiates the opening of a chloride-selective channel that modulates the neuronal membrane potential. Point mutations of the GlyR, substituting Arg-271 with either Leu or Gln, have been shown to underlie the inherited neurological disorder startle disease (hyperekplexia). We show that these substitutions result in the redistribution of GlyR single-channel conductances to lower conductance levels. Additionally, the binding of the glycinergic agonists beta-alanine and taurine to mutated GlyRs does not initiate a chloride current, but instead competitively antagonizes currents activated by glycine. These findings are consistent with mutations of Arg-271 resulting in the uncoupling of the agonist binding process from the channel activation mechanism of the receptor.

The relative affinities of all Escherichia coli amino-acyl-tRNAs for E. coli elongation factor (EF) Tu-GTP have been measured by two independent applications of the competition form of the ribonuclease resistance assay. The set of aminoacyl-tRNAs includes at least one tRNA for each of the 20 amino acids as well as purified isoacceptor tRNA species for arginine, glycine, leucine, lysine, and tyrosine. In the first competition study, [3H]Phe-tRNA was used as the competing aminoacyl-tRNA against [14C]aminoacyl-tRNA in the set of all tRNAs; in the second study, [3H]Leu-tRNALeu4 was used as the competing aminoacyl-tRNA. The relative order of aminoacyl-tRNA affinities for EF-Tu-GTP was the same in each study. The results indicate that the affinity of EF-Tu-GTP at 4 degrees C, pH 7.4, is strongest for Gln-tRNA and weakest for Val-tRNA. Both Gly-tRNA and Pro-tRNA bind very strongly to EF-Tu-GTP relative to other aminoacyl-tRNAs. Various models of ternary complex interactions are discussed in light of the new data. Although the properties of the amino acid substituent are primarily responsible for the differences in relative affinities among the noninitiator aminoacyl-tRNAs, the results for the four isoacceptor species of Leu-tRNALeu indicate that the secondary structural features of the tRNA are also influential.

We report on the identification of mutations associated with streptomycin resistance in Mycobacterium tuberculosis. Two isolates (3656 and 3976) showed a wild-type ribosomal protein, S12, but exhibited a single point mutation at 16S rRNA position 491 (C->>T) or 512 (C->>T), respectively. Sequence analysis of a third isolate (2438) revealed a single base change at 16S rRNA position 904 (A->>G). This position is equivalent to invariant position 913 of the Escherichia coli 16S rRNA gene, an A->>G transition of which has been shown previously to impair streptomycin binding and streptomycin-induced misreading in vivo. Surprisingly, strain 2438 harbors an additional mutation in the ribosomal protein S12 (Lys-88->>Gln).

Polarization of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel to the apical plasma membrane in epithelial cells is critical for vectorial chloride transport. Previously, we reported that the C terminus of CFTR constitutes a PDZ-interacting domain that is required for CFTR polarization to the apical plasma membrane and interaction with the PDZ domain-containing protein EBP50 (NHERF). PDZ-interacting domains are typically composed of the C-terminal three to five amino acids, which in CFTR are QDTRL. Our goal was to identify the key amino acid(s) in the PDZ-interacting domain of CFTR with regard to its apical polarization, interaction with EBP50, and ability to mediate transepithelial chloride secretion. Point substitution of the C-terminal leucine (Leu at position 0) with alanine abrogated apical polarization of CFTR, interaction between CFTR and EBP50, efficient expression of CFTR in the apical membrane, and chloride secretion. Point substitution of the threonine (Thr at position -2) with alanine or valine had no effect on the apical polarization of CFTR, but reduced interaction between CFTR and EBP50, efficient expression of CFTR in the apical membrane as well as chloride secretion. By contrast, individual point substitution of the other C-terminal amino acids (Gln at position -4, Asp at position -3 and Arg at position -1) with alanine had no effect on measured parameters. We conclude that the PDZ-interacting domain, in particular the leucine (position 0) and threonine (position -2) residues, are required for the efficient, polarized expression of CFTR in the apical plasma membrane, interaction of CFTR with EBP50, and for the ability of CFTR to mediate chloride secretion. Mutations that delete the C terminus of CFTR may cause cystic fibrosis because CFTR is not polarized, complexed with EBP50, or efficiently expressed in the apical membrane of epithelial cells.

The role of the second shell in the process of metal binding and selectivity in metalloproteins has been elucidated by combining Protein Data Bank (PDB) surveys of Mg, Mn, Ca, and Zn binding sites with density functional theory/continuum dielectric methods (DFT/CDM). Peptide backbone groups were found to be the most common second-shell ligand in Mg, Mn, Ca, and Zn binding sites, followed (in decreasing order) by Asp/Glu, Lys/Arg, Asn/Gln, and Ser/Thr side chains. Aromatic oxygen- or nitrogen-containing side chains (Tyr, His, and Trp) and sulfur-containing side chains (Cys and Met) are seldom found in the second coordination layer. The backbone and Asn/Gln side chain are ubiquitous in the metal second coordination layer as their carbonyl oxygen and amide hydrogen can act as a hydrogen-bond acceptor and donor, respectively, and can therefore partner practically every first-shell ligand. The second most common outer-shell ligand, Asp/Glu, predominantly hydrogen bonds to a metal-bound water or Zn-bound histidine and polarizes the H-O or H-N bond. In certain cases, a second-shell Asp/Glu could affect the protonation state of the metal ligand. It could also energetically stabilize a positively charged metal complex more than a neutral ligand such as the backbone and Asn/Gln side chain. As for the first shell, the second shell is predicted to contribute to the metal selectivity of the binding site by discriminating between metal cations of different ionic radii and coordination geometries. The first-shell-second-shell interaction energies decay rapidly with increasing solvent exposure of the metal binding site. They are less favorable but are of the same order of magnitude as compared to the respective metal-first-shell interaction energies. Altogether, the results indicate that the structure and properties of the second shell are dictated by those of the first layer. The outer shell is apparently designed to stabilize/protect the inner-shell and complement/enhance its properties.