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1.
Starting with nine plaques of influenza A/Kamata/14/91(H3N2) virus, we selected mutants in the presence of monoclonal antibody 203 (mAb203). In total, amino acid substitutions were found at nine positions (77, 80, 131, 135, 141, 142, 143, 144 and 146), which localized in the antigenic site A of the hemagglutinin (HA). The escape mutants differed in the extent to which they had lost binding to mAb203. HA protein with substitutions of some amino acid residues created by site-directed mutagenesis in the escape mutants retained the ability to bind to mAb203. Changes in the amino acid character affecting charge or hydrophobicity accounted for the binding capacity to the antibody of the HA with most of the substitutions in the escape mutants and binding-positive mutants. However, the effect of some amino acid substitutions remained unexplained. A three-dimensional model of the 1991 HA was constructed and used to analyze substituted amino acids in these mutants for the accessible surface hydrophobic and hydrophilic characters. One amino acid substitution in an escape mutant and another amino acid substitution in a binding-positive mutant seemed to be explained by the changes noted on this model.  相似文献   

2.
MhsT of Bacillus halodurans is a transporter of hydrophobic amino acids and a homologue of the eukaryotic SLC6 family of Na+‐dependent symporters for amino acids, neurotransmitters, osmolytes, or creatine. The broad range of transported amino acids by MhsT prompted the investigation of the substrate recognition mechanism. Here, we report six new substrate‐bound structures of MhsT, which, in conjunction with functional studies, reveal how the flexibility of a Gly‐Met‐Gly (GMG) motif in the unwound region of transmembrane segment 6 (TM6) is central for the recognition of substrates of different size by tailoring the binding site shape and volume. MhsT mutants, harboring substitutions within the unwound GMG loop and substrate binding pocket that mimick the binding sites of eukaryotic SLC6A18/B0AT3 and SLC6A19/B0AT1 transporters of neutral amino acids, exhibited impaired transport of aromatic amino acids that require a large binding site volume. Conservation of a general (G/A/C)ΦG motif among eukaryotic members of SLC6 family suggests a role for this loop in a common mechanism for substrate recognition and translocation by SLC6 transporters of broad substrate specificity.  相似文献   

3.
The global spread of highly pathogenic avian influenza A H5N1 viruses raises concerns about more widespread infection in the human population. Pre-pandemic vaccine for H5N1 clade 1 influenza viruses has been produced from the A/Viet Nam/1194/2004 strain (VN1194), but recent prevalent avian H5N1 viruses have been categorized into the clade 2 strains, which are antigenically distinct from the pre-pandemic vaccine. To understand the antigenicity of H5N1 hemagglutinin (HA), we produced a neutralizing monoclonal antibody (mAb12-1G6) using the pre-pandemic vaccine. Analysis with chimeric and point mutant HAs revealed that mAb12-1G6 bound to the loop (amino acid positions 140-145) corresponding to an antigenic site A in the H3 HA. mAb12-1G6 failed to bind to the mutant VN1194 HA when only 3 residues were substituted with the corresponding residues of the clade 2.1.3.2 A/Indonesia/5/05 strain (amino acid substitutions at positions Q142L, K144S, and S145P), suggesting that these amino acids are critical for binding of mAb12-1G6. Escape mutants of VN1194 selected with mAb12-1G6 carried a S145P mutation. Interestingly, mAb12-1G6 cross-neutralized clade 1 and clade 2.2.1 but not clade 2.1.3.2 or clade 2.3.4 of the H5N1 virus. We discuss the cross-reactivity, based on the amino acid sequence of the epitope.  相似文献   

4.
Earlier studies of a group of monoclonal antibody-resistant (mar) mutants of herpes simplex virus type 1 glycoprotein C (gC) operationally defined two distinct antigenic sites on this molecule, each consisting of numerous overlapping epitopes. In this report, we further define epitopes of gC by sequence analysis of the mar mutant gC genes. In 18 mar mutants studied, the mar phenotype was associated with a single nucleotide substitution and a single predicted amino acid change. The mutations were localized to two regions within the coding sequence of the external domain of gC and correlated with the two previously defined antigenic sites. The predicted amino acid substitutions of site I mutants resided between residues Gln-307 and Pro-373, whereas those of site II mutants occurred between amino acids Arg-129 and Glu-247. Of the 12 site II mutations, 9 induced amino acid substitutions within an arginine-rich segment of 8 amino acids extending from residues 143 to 151. The clustering of the majority of substituted residues suggests that they contribute to the structure of the affected sites. Moreover, the patterns of substitutions which affected recognition by antibodies with similar epitope specificities provided evidence that epitope structures are physically linked and overlap within antigenic sites. Of the nine epitopes defined on the basis of mutations, three were located within site I and six were located within site II. Substituted residues affecting the site I epitopes did not overlap substituted residues of site II, supporting our earlier conclusion that sites I and II reside in spatially distinct antigenic domains. A computer analysis of the distribution of charged residues and the predicted secondary structural features of wild-type gC revealed that the two antigenic sites reside within the most hydrophilic regions of the molecule and that the antigenic residues are likely to be organized as beta sheets which loop out from the surface of the molecule. Together, these data and our previous studies support the conclusion that the mar mutations identified by sequence analysis very likely occur within or near the epitope structures themselves. Thus, two highly antigenic regions of gC have now been physically and genetically mapped to well-defined domains of the protein molecule.  相似文献   

5.
6.
Abstract: MAP 1B is a microtubule-associated phosphoprotein that is expressed early in neurons and plays a role in axon growth. MAP 1B has two types of phosphoisoforms, one of which is developmentally down-regulated after neuronal maturation and one of which persists into adulthood. Because phosphorylation regulates MAP 1B binding activity, characterisation of the phosphorylation sites and identification of the corresponding kinases/phosphatases are important goals. We have characterised the developmentally down-regulated phosphorylation sites recognised by monoclonal antibody (mAb) SMI-31. We purified MAP 1B from neonatal rat brain and mapped the mAb SMI-31 sites to specific MAP 1B fragments after chemical cleavage. We then developed an in vitro kinase assay by using a high-speed spin supernatant from neonatal rat brain in the presence of ATP and recombinant proteins encoding selective regions of the MAP 1B molecule. Phosphorylation of the recombinant protein was detected on western blots using mAb SMI-31. This analysis showed that mAb SMI-31 recognises two recombinant proteins corresponding to residues 1,109–1,360 and 1,836–2,076 of rat MAP 1B after in vitro phosphorylation. The former phosphorylation site was further defined in the in vitro kinase assay by inhibition with peptides and antibodies from candidate regions of the MAP 1B sequence. This approach identified a region of 20 amino acids, from 1,244 to 1,264, characterised by a high concentration of serines immediately upstream of prolines, indicating that the kinase responsible is a proline-directed serine kinase.  相似文献   

7.
The transport function of the rat type IIa Na(+)/P(i) cotransporter is inhibited after binding the cysteine modifying reagent 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) to a cysteine residue substituted for a serine at position 460 (S460C) in the predicted third extracellular loop. This suggests that Ser-460 lies in a functionally important region of the protein. To establish a "structure-function" profile for the regions that flank Ser-460, the substituted cysteine accessibility method was employed. 18 mutants were constructed in which selected amino acids from Arg-437 through Leu-465 were substituted one by one for a cysteine. Mutants were expressed in Xenopus oocytes and transport function (cotransport and slippage) and kinetics were assayed by electrophysiology with or without prior treatment with cysteine modifying (methanethiosulfonate, MTS) reagents. Except for mutant I447C, mutants with cysteines at sites from Arg-437 through Thr-449, as well as Pro-461, were inactive. Cotransport function of mutants with Cys substitutions at sites Arg-462 through Leu-465 showed low sensitivity to MTS reagents. The preceding mutants (Cys substitution at Thr-451 to Ser-460) showed a periodic accessibility pattern that would be expected for an alpha-helix motif. Apart from loss of transport function, exposure of mutants A453C and A455C to MTSEA or 2-(triethylammonium)ethyl MTS bromide (MTSET) increased the uncoupled slippage current, which implicated the mutated sites in the leak pathway. Mutants from Ala-453 through Ala-459 showed less pH dependency, but generally stronger voltage dependency compared with the wild type, whereas those flanking this group were more sensitive to pH and showed weaker voltage dependence of cotransport mode kinetics. Our data indicate that parts of the third extracellular loop are involved in the translocation of the fully loaded carrier and show a membrane-associated alpha-helical structure.  相似文献   

8.
The relationships between the bacteriophage lambda binding site, the starch binding site and the pore formed by maltoporin (LamB protein, lambda receptor protein) were investigated. Bacteria with single amino acid substitutions in the maltoporin sequence, which were previously shown to be strongly reduced in phage lambda sensitivity, were assayed for maltose- (and maltodextrin) selective pore functions. Maltose transport assays was performed at low substrate concentrations, under conditions where LamB is limiting for transport. It revealed three classes of mutants. Class A is composed of mutants with no effect on transport (substitutions at amino acid residues 154, 155, 259, 382 and 401); class B corresponds to mutants with a significant but variable reduction in transport (sites 148, 151, 152, 163, 164, 245, 247 and 250); class C is represented by a single mutant for which transport is almost completely abolished (site 18). Starch binding was assayed by two different methods that gave compatible results. In class A mutants, binding was normal, while no binding was observed in the class C mutant. Binding was impaired to various extents in category B mutants. There was a correlation between the level of impairment of starch binding and impairment of maltose transport, consistent with the notion that the residues influencing starch binding are inside, or in close proximity to, the pore. These results, together with previous data on starch-binding mutants that were not affected in phage binding (substitutions at residues 8, 74, 82, 118 and 121), suggest that the binding sites for starch and phage lambda overlap but are distinct. Mutations affecting transport and starch binding are located in the first third of the protein and in the region of residues 245 to 250. Mutations affecting phage adsorption are located mainly in the last two-thirds of the protein. The topological constraints suggested by the results with the available mutants altered in the lamB gene were used to propose a revised model of maltoporin folding across the outer membrane as well as to define the outlines of footprints of macromolecular binding sites (phage, starch and monoclonal antibodies) on the surface of the protein.  相似文献   

9.
This investigation describes the design, synthesis and evaluation of chimeric peptides related to the bovine thyrotropin beta-subunit, bTSHbeta. The structures of these chimeric peptides were derived from investigations with linear peptides and sequence alignment studies, in association with a homology model of TSHbeta developed from the hCG X-ray crystallographic structure. The structures of these chimeric peptides comprised beta-turn regions of loop L1 [bTSHbeta(14-20)] and loop L3 [bTSHbeta(65-72)] held in close proximity by a bis-beta-alanine linker and the disulfide bond bTSHbeta[Cys16-Cys67]. Linear and cyclic chimeric peptides were evaluated in immunochemical assays for their ability to inhibit the binding of radio-iodinated bTSHbeta [125I-bTSHbeta] to the monoclonal antibodies, mAb279 and mAb299. Previously, mAb279 and mAb299 have been shown to recognize epitopes accessible on the surface of TSHbeta that lie in close proximity to the TSH receptor-binding site. The results indicate that these chimeric peptides can specifically inhibit in a dose-dependent manner the binding of 125I-bTSHbeta to mAb299, while having a lesser effect on the binding with mAb279. Based on these results, it can be concluded that the bTSHbeta-epitope recognized by mAb299 involves contributions from amino residues from the beta-turn regions of the L1 and L3 loops of TSHbeta, and that these loop regions flank part of the receptor binding site of the bTSH beta-subunit.  相似文献   

10.
The membrane topology of the human reduced folate carrier protein (591 amino acids) was assessed by single insertions of the hemagglutinin epitope into nine sites of the protein. Reduced folate carrier-deficient Chinese hamster ovary cells expressing each of these constructs were probed with anti-hemagglutinin epitope monoclonal antibodies to assess whether the insertion was exposed to the external environment or to the cytoplasm. The results are consistent with the 12-transmembrane topology predicted for this protein. The hemagglutinin epitope insertion mutants were also tested for their effects on the function of the reduced folate carrier. For these studies, each of the constructs had a carboxyl-terminal fusion of the enhanced green fluorescent protein to monitor and quantitate expression. Insertions into the external loop between transmembrane regions 7 and 8 (Pro-297), the cytoplasmic loop between transmembrane regions 6 and 7 (Ser-225), and near the cytoplasmic amino and carboxyl termini (Pro-20 and Gly-492, respectively) had minor effects on methotrexate binding and uptake. The insertion into the cytoplasmic loop between transmembrane regions 10 and 11 (Gln-385) greatly reduced both binding and uptake of methotrexate, whereas the insertion into the external loop between transmembrane regions 11 and 12 (Pro-427) selectively interfered with uptake but not binding.  相似文献   

11.
Aims:  To establish the role of maltoporin (LamB) in adherence of enteropathogenic Escherichia coli (EPEC) to epithelial cells in vitro.
Methods and Results:  Three strains, wild type (WT) EPEC, a maltoporin (LamB) mutant ΔlamB , and DH5α were used to study adherence to cultured HEp-2 cells. Mutant ΔlamB was found to be deficient in adherence compared to WT EPEC. Adherence of ΔlamB was restored to wild type levels when complemented with the cloned lamB gene. The non–adherent strain DH5α also adhered to HEp-2 cells when it harboured the cloned lamB gene. The LamB protein was isolated from WT EPEC by electroelution and antibodies were raised in rabbits. The specificity of the antibodies was analysed by Western blotting. Anti-LamB antiserum reduced adherence of WT EPEC to HEp-2 cells. The LamB protein was coated on latex beads and the beads adhered to HEp-2 cells. Anti-LamB antiserum prevented bead adherence to HEp-2 cells. Multiple sequence alignment showed that the L9 loop of EPEC LamB had four amino acids different from the L9 loop of LamB from several other related pathogens.
Conclusions:  LamB serves as an alternative or additional adherence factor for EPEC.
Significance and Impact of the Study:  Adherence is an important component of the pathogenesis of noninvasive pathogens like EPEC. A putative adhesin such as LamB, which has already been found to be co-expressed with virulence factor EspB may be a potential vaccine candidate for control of EPEC and related pathogens.  相似文献   

12.
13.
Recent crystallographic studies reveal loops in human AP endonuclease 1 (APE1) that interact with the major and minor grooves of DNA containing apurinic/apyrimidinic (AP) sites. These loops are postulated to stabilize the DNA helix and the flipped out AP residue. The loop alpha8 interacts with the major groove on the 3' side of the AP site. To determine the essentiality of the amino acids that constitute the alpha8 loop, we created a mutant library containing random nucleotides at codons 222-229 that, in wild-type APE1, specify the sequence NPKGNKKN. Upon expression of the library (2 x 10(6) different clones) in Escherichia coli and multiple rounds of selection with the alkylating agent methyl-methane sulfonate (MMS), we obtained approximately 2 x 10(5) active mutants that complemented the MMS sensitivity of AP endonuclease-deficient E. coli. DNA sequencing showed that active mutants tolerated amino acid substitutions at all eight randomized positions. Basic and uncharged polar amino acids together comprised the majority of substitutions, reflecting the positively charged, polar character of the wild-type loop. Asn-222, Asn-226, and Asn-229 exhibited the least mutability, consistent with x-ray data showing that each asparagine contacts a DNA phosphate. Substitutions at residues 226-229, located nearer to the AP site, that reduced basicity or hydrogen bonding potential, increased Km 2- to 6-fold and decreased AP site binding; substitutions at residues 222-225 exhibited lesser effects. This initial mutational analysis of the alpha8 loop supports and extends the conclusion of crystallographic studies that the loop is important for binding of AP.DNA and AP site incision.  相似文献   

14.
The consequences of alphaB-crystallin phosphorylation on its chaperone activity were investigated using a detailed analysis of the recognition and binding of destabilized T4 lysozyme (T4L) mutants by alphaB-crystallin phosphorylation mimics containing combinations of serine to aspartate substitutions. The T4L site-directed mutants were selected to constitute an energetic ladder of progressively destabilized proteins having similar structures in the folded state. alphaB-crystallin and its variants differentially recognize the T4L mutants, binding the more destabilized ones to a larger extent. Furthermore, the aspartate substitutions result in an increase in the extent of binding to the same T4L mutant and in the appearance of biphasic binding isotherms. The latter indicates the presence of two modes of binding characterized by different affinities and different numbers of binding sites. The transition to two-mode binding can also be induced by temperature or pH activation of the second mode. The similarity between the phosphorylation, pH, and temperature effects suggests a common structural origin. The location of the phosphorylation sites in the N-terminal domain and the hypothesized burial of this domain in the core of the oligomeric structure are consistent with a critical role for the destabilization of the quaternary structure in the process of recognition and binding by small heat-shock proteins.  相似文献   

15.
Pseudomonas syringae pathovars expressing avrPto are avirulent on plants expressing the resistance gene Pto. Over 85 mutants of avrPto were generated with multiple strategies, and several assays were used to characterize AvrPto function. Only a core of 95 amino acids of the 164 residues was sufficient for binding Pto in the yeast two-hybrid system. Only nine of 65 mutant proteins of AvrPto with amino acid substitutions, created in planta and in vitro, did not interact with Pto in the Gal4 yeast two-hybrid system, suggesting that AvrPto can tolerate many nonconservative substitutions and still interact with Pto. These nine and 12 additional substitution mutants of AvrPto were characterized further. The ability to elicit a hypersensitive response and the effect on pathogenesis in planta for these 21 mutants of AvrPto were strongly correlated with recognition by Pto in the yeast two-hybrid system. Analyses of two proteins with substitutions H54P or D52G/L65P indicated that these residues may be required for delivery into the host cell and protein stability in the bacterial cytoplasm, respectively. The mutants that no longer interacted with Pto and had modified activities in planta were predicted to have changes in their secondary structure.  相似文献   

16.
Neutralizing antibodies that recognize the human immunodeficiency virus gp120 exterior envelope glycoprotein and are directed against either the third variable (V3) loop or conserved, discontinuous epitopes overlapping the CD4 binding region have been described. Here we report several observations that suggest a structural relationship between the V3 loop and amino acids in the fourth conserved (C4) gp120 region that constitute part of the CD4 binding site and the conserved neutralization epitopes. Treatment of the gp120 glycoprotein with ionic detergents resulted in a V3 loop-dependent masking of both linear C4 epitopes and discontinuous neutralization epitopes overlapping the CD4 binding site. Increased recognition of the native gp120 glycoprotein by an anti-V3 loop monoclonal antibody, 9284, resulted from from single amino acid changes either in the base of the V3 loop or in the gp120 C4 region. These amino acid changes also resulted in increased exposure of conserved epitopes overlapping the CD4 binding region. The replication-competent subset of these mutants exhibited increased sensitivity to neutralization by antibody 9284 and anti-CD4 binding site antibodies. The implied relationship of the V3 loop, which mediates post-receptor binding steps in virus entry, and components of the CD4 binding region may be important for the interaction of these functional gp120 domains and for the observed cooperativity of neutralizing antibodies directed against these regions.  相似文献   

17.
To study the active site(s) of IL-6 we combined mutagenesis of IL-6 with epitope mapping of IL-6 specific mAb. In addition to amino-terminal deletion mutants we described previously, carboxyl-terminal deletion mutants were prepared. Functional analysis showed that deletion of only five carboxyl-terminal amino acids already reduced the bioactivity 1000-fold. A panel of mAb to IL-6 was subsequently analyzed by antibody competition experiments and binding to the amino- and carboxyl-terminal deletion mutants. On the basis of the competition experiments the six neutralizing mAb were divided in two groups (I and II). The binding pattern with the deletion mutants suggested that the region recognized by the four mAb in group I is composed of residues of amino- and carboxyl-terminus: binding of two mAb was abolished after deletion of amino acid Ala I-Ile26, of the third mAb after deletion of the four carboxyl-terminal amino acids whereas the fourth mAb did not bind to either mutant. Group II mAb retained binding to these mutants. Taken together these data suggest that in the native IL-6 molecule amino acid residues of amino and carboxyl terminus are in close proximity and that together they constitute an active site. Furthermore our data suggest that the part of the molecule recognized by group II antibodies is a second site involved in biologic activity.  相似文献   

18.
The class I MHC molecule HLA-B27 bears an unpaired Cys residue at position 67, which is predicted to face the Ag binding pocket, based on the x-ray crystallographic model of HLA-A2. To investigate the potential of this residue in the antigenic structure of HLA-B27, a panel of 11 mutant HLA-B27 genes has been created, each bearing a separate amino acid substitution at position 67. The genes were transfected into mouse L cells and the resulting cells analyzed by cytofluorography with a panel of antibodies reactive with the wild-type B27 molecule. Although previous studies had indicated that all mAb that bound the B27 molecule on human lymphocytes bound comparably to L cells transfected with the wild-type B27 gene in the absence of h beta 2-m (human beta 2-microglobulin), the first of the mutant B27 genes was found to express several mAb epitopes in the presence but not in the absence of a h beta 2-m gene. Therefore, subsequent analysis of the B27 mutant panel was conducted in L cells coexpressing the h beta 2-m gene. Under these circumstances, all of the mutants bound the monomorphic anti-class I HLA mAb W6/32 and B.9.12.1, as well as the broadly polymorphic mAb B.1.23.2. Binding to the mutant transfectants of three anti-B27 mAb that cross-react with HLA-B7, ME1, GS145.2, and GSP5.3, was directly proportional to the size of the substituted amino acid side chain. The binding of another anti-B27 mAb, B27M2, that recognizes a B27 determinant that includes the region of amino acids 77-81, was not affected by the Cys67- greater than Tyr67 substitution. Rabbit antibodies to a synthetic peptide composed of B27 amino acids 61-84 bound to both the wild-type B27 and to the Tyr67 mutant. This binding, but not the binding of ME1 or B27M2, was inhibited by the synthetic peptide. These data are interpreted as suggesting that the large amino acid substitutions at position 67 induce a limited conformational change that disrupts the epitopes of the three anti-B27, B7 mAb, that are themselves at least partially conformational. The potential implications of these findings for the role of HLA-B27 in disease pathogenesis are discussed.  相似文献   

19.
The VP7 and VP4 genes of seven antigenic mutants of simian rotavirus SA11 4fM (serotype 3) selected after 39 passages in the presence of SA11 4fM hyperimmune antiserum, were sequenced. Nucleotide sequence analysis indicated the following. (i) Twice as many amino acid substitutions occurred in the VP7 protein than in VP4, which has a molecular weight twice that of VP7. (ii) Most amino acid changes that occurred clustered in six variable regions of VP7 and in two variable regions of VP4; these variable regions may represent immunodominant epitopes. (iii) Most amino acid substitutions that occurred in VP7 and VP4 of these mutants were also observed in antigenic mutants selected with neutralizing monoclonal antibodies (NMAbs); however, some amino acid substitutions occurred that were not selected for NMAbs. (iv) On VP7, some of the neutralization epitopes appeared to be interrelated because amino acid substitution in one site affected binding of specific NMAbs to other sites, while other neutralization epitopes on VP7 appeared to be independent, in that amino acid substitution in one site did not affect the binding of NMAbs to another distant site.  相似文献   

20.
Four monoclonal antibodies (mAb) to non-human primate hemoglobin referred to as Cap-4, Cap-5, Rh-2, and Rh-4, and two mAb to human hemoglobin, referred to as H-1 and H-3 were isolated and were partially characterized. Binding studies with these mAb on a panel of hemoglobins and isolated alpha and beta globin chains revealed a unique reactivity pattern for each mAb. Amino acid sequence analysis of the antigens used to generate the binding data suggests that the specific recognition of certain hemoglobin antigens by each mAb is controlled by the presence of a particular amino acid at a specific position within the epitope. The use of synthetic peptides as antigens confirmed this observation for five of the mAb. No synthetic peptides were tested with the sixth mAb, Rh-2. The amino acids required for binding of mAb Cap-4, Cap-5, Rh-4, and Rh-2 to hemoglobin are alanine at beta 5, threonine at beta 13, glutamine at beta 125, and leucine at alpha 68. The non-human primate hemoglobin antibodies require a specific amino acid that is not present in human hemoglobin. The amino acid required for binding of Cap-4, Cap-5, and Rh-4 could arise by a single base change in the beta globin gene, whereas the amino acid required for Rh-2 binding would only occur if two base changes occurred. Thus these mAb are candidate probes for a somatic cell mutation assay on the basis of the detection of peripheral blood red cells that possess single amino acid substituted hemoglobin as a result of single base substitutions in the globin genes of precursor cells.  相似文献   

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