scholarly journals Roles of Specific Amino Acids in the N Terminus of Pseudomonas aeruginosa Flagellin and of Flagellin Glycosylation in the Innate Immune Response

2005 ◽  
Vol 73 (12) ◽  
pp. 8237-8246 ◽  
Author(s):  
Amrisha Verma ◽  
Shiwani K. Arora ◽  
Sudha K. Kuravi ◽  
Reuben Ramphal
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Binding Site ◽  
A549 Cells ◽  
Single Amino Acid ◽  
Wild Type ◽  
Binding Region ◽  
N Terminus ◽  
Flagellin Glycosylation

ABSTRACT The Toll-like receptor 5 (TLR5) binding site has been predicted to be in the N terminus of the flagellin molecule. In order to better define the interaction between the N-terminal amino acids of Pseudomonas aeruginosa flagellin and TLR5, site-specific mutations were generated between residues 88 and 97 of P. aeruginosa PAK flagellin as well as outside of this region. The mutant flagellins were expressed in Escherichia coli BL21(plysS), purified by affinity chromatography, and passed through a polymyxin B column to remove contaminating lipopolysaccharide (LPS). Their ability to stimulate interleukin-8 (IL-8) release from A549 cells was examined. The cloned mutated genes were used to complement a PAK fliC mutant in order to test for effects on motility and on IL-8 release by purified flagellar preparations. All the mutations, single or double, in the predicted TLR5 binding region reduced IL-8 signaling to less than 95% of the wild-type flagellin levels, but the single mutation outside the binding region had no effect. Changes made at two amino acid sites resulted in loss/reduction of motility; however, changes made at single sites, i.e., Q83A, L88A, R90A, M91A, L94A, and Q97A, had no effect on motility. The mutated genes encoding two of the motile but poorly signaling flagellins had no compensatory mutations to allow motility. Thus, while it is speculated that pathogen-associated molecular patterns (PAMPs) have evolved in locations that are essential to maintain function, it appears that there is tolerance for at least single amino acid changes in the PAMP of P. aeruginosa flagellin. The purpose of flagellin glycosylation in P. aeruginosa is unknown. In order to examine its role, if any, in signaling an inflammatory response, we used whole flagella from the motile chromosomal mutant strains PAKrfbC and PAO1rfbC, which are defective in flagellin glycosylation. IL-8 release from A549 cells stimulated with nonglycosylated flagellar preparations (having less then 1 picogram of LPS/μg) was significantly reduced compared to their respective wild-type flagellar preparations, indicating a role of flagellar glycosylation in the proinflammatory action of Pseudomonas flagellin. The basis of the latter activity is unknown, since the glycosylation sites are found in the D3 domain of flagellins and the TLR5 binding site is located in the D1 domain. Thus, P. aeruginosa flagellin has evolved additional flagellar signaling mechanisms over that described for Salmonella flagellin.

Single amino acid substitution of rat MRP2 results in acquired transport activity for taurocholate

2001 ◽  
Vol 281 (4) ◽  
pp. G1034-G1043 ◽  
Author(s):  
Kousei Ito ◽  
Hiroshi Suzuki ◽  
Yuichi Sugiyama
Keyword(s):  
Amino Acids ◽  
Multidrug Resistance ◽  
Amino Acid ◽  
Membrane Vesicles ◽  
Single Amino Acid ◽  
Sf9 Cells ◽  
Transport Activity ◽  
Wild Type ◽  
Cationic Amino Acids ◽  
The Difference

Multidrug resistance-associated protein 3 (MRP3), unlike other MRPs, transports taurocholate (TC). The difference in TC transport activity between rat MRP2 and MRP3 was studied, focusing on the cationic amino acids in the transmembrane domains. For analysis, transport into membrane vesicles from Sf9 cells expressing wild-type and mutated MRP2 was examined. Substitution of Arg at position 586 with Leu and Ile and substitution of Arg at position 1096 with Lys, Leu, and Met resulted in the acquisition of TC transport activity, while retaining transport activity for glutathione and glucuronide conjugates. Substitution of Leu at position 1084 of rat MRP3 (which corresponds to Arg-1096 in rat MRP2) with Lys, but not with Val or Met, resulted in the loss of transport activity for TC and glucuronide conjugates. These results suggest that the presence of the cationic charge at Arg-586 and Arg-1096 in rat MRP2 prevents the transport of TC, whereas the presence of neutral amino acids at the corresponding position of rat MRP3 is required for the transport of substrates.

scholarly journals Loss of Function in Zeaxanthin Epoxidase of Dunaliella tertiolecta Caused by a Single Amino Acid Mutation within the Substrate-Binding Site

Marine Drugs ◽  
2018 ◽  
Vol 16 (11) ◽  
pp. 418 ◽  
Author(s):  
Minjae Kim ◽  
Jisu Kang ◽  
Yongsoo Kang ◽  
Beom Kang ◽  
EonSeon Jin
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Conformational Change ◽  
Substrate Binding ◽  
Single Amino Acid ◽  
Wild Type ◽  
Amino Acid Mutation ◽  
Zeaxanthin Epoxidase ◽  
Substrate Binding Site ◽  
Single Amino Acid Mutation

The zea1 mutant of marine microalga Dunaliella tertiolecta accumulates zeaxanthin under normal growth conditions, and its phenotype has been speculated to be related to zeaxanthin epoxidase (ZEP). In this study, we isolated the ZEP gene from both wild-type D. tertiolecta and the mutant. We found that the zea1 mutant has a point mutation of the 1337th nucleotide of the ZEP sequence (a change from guanine to adenine), resulting in a change of glycine to aspartate in a highly conserved region in the catalytic domain. Similar expression levels of ZEP mRNA and protein in both wild-type and zea1 were confirmed by using qRT-PCR and western blot analysis, respectively. Additionally, the enzyme activity analysis of ZEPs in the presence of cofactors showed that the inactivation of ZEP in zea1 was not caused by deficiency in the levels of cofactors. From the predicted three-dimensional ZEP structure of zea1, we observed a conformational change on the substrate-binding site in the ZEP. A comparative analysis of the ZEP structures suggested that the conformational change induced by a single amino acid mutation might impact the interaction between the substrate and substrate-binding site, resulting in loss of zeaxanthin epoxidase function.

scholarly journals Structural and functional analyses of Saccharomyces cerevisiae wild-type and mutant RNA1 genes.

1989 ◽  
Vol 9 (7) ◽  
pp. 2989-2999 ◽  
Author(s):  
H M Traglia ◽  
N S Atkinson ◽  
A K Hopper
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Open Reading Frame ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Reading Frame ◽  
Genomic Deletions ◽  
Single Amino Acid Change ◽  
Carboxy Terminal

The yeast gene RNA1 has been defined by the thermosensitive rna1-1 lesion. This lesion interferes with the processing and production of all major classes of RNA. Each class of RNA is affected at a distinct and presumably unrelated step. Furthermore, RNA does not appear to exit the nucleus. To investigate how the RNA1 gene product can pleiotropically affect disparate processes, we undertook a structural analysis of wild-type and mutant RNA1 genes. The wild-type gene was found to contain a 407-amino-acid open reading frame that encodes a hydrophilic protein. No clue regarding the function of the RNA1 protein was obtained by searching banks for similarity to other known gene products. Surprisingly, the rna1-1 lesion was found to code for two amino acid differences from wild type. We found that neither single-amino-acid change alone resulted in temperature sensitivity. The carboxy-terminal region of the RNA1 open reading frame contains a highly acidic domain extending from amino acids 334 to 400. We generated genomic deletions that removed C-terminal regions of this protein. Deletion of amino acids 397 to 407 did not appear to affect cell growth. Removal of amino acids 359 to 397, a region containing 24 acidic residues, caused temperature-sensitive growth. This allele, rna1-delta 359-397, defines a second conditional lesion of the RNA1 locus. We found that strains possessing the rna1-delta 359-397 allele did not show thermosensitive defects in pre-rRNA or pre-tRNA processing. Removal of amino acids 330 to 407 resulted in loss of viability.

scholarly journals Mutational Analysis of Quinolone Resistance Protein QnrB1

2013 ◽  
Vol 57 (11) ◽  
pp. 5733-5736 ◽  
Author(s):  
George A. Jacoby ◽  
Marian A. Corcoran ◽  
Debra M. Mills ◽  
Caitlin M. Griffin ◽  
David C. Hooper
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Mutational Analysis ◽  
Essential Amino Acids ◽  
Quinolone Resistance ◽  
Single Amino Acid ◽  
Protective Activity ◽  
N Terminus ◽  
Resistance Protein

ABSTRACTAlanine substitutions and selected deletions have been used to localize amino acids in QnrB essential for its protective activity. Essential amino acids are found at positions i and i−2in the pentapeptide repeat module and in the larger of two loops, where deletion of only a single amino acid compromises activity. Deletion of 10 amino acids at the N terminus is tolerated, but removal of 3 amino acids in the C-terminal dimerization unit destroys activity.

scholarly journals Structural characterization of human aryl sulphotransferases

1999 ◽  
Vol 337 (2) ◽  
pp. 337-343 ◽  
Author(s):  
Lulu A. BRIX ◽  
Ronald G. DUGGLEBY ◽  
Andrea GAEDIGK ◽  
Michael E. McMANUS
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Change ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Wild Type ◽  
Substrate Specificities ◽  
Loop Region ◽  
Single Amino Acid Change

Human aryl sulphotransferase (HAST) 1, HAST3, HAST4 and HAST4v share greater than 90% sequence identity, but vary markedly in their ability to catalyse the sulphonation of dopamine and p-nitrophenol. In order to investigate the amino acid(s) involved in determining differing substrate specificities of HASTs, a range of chimaeric HAST proteins were constructed. Analysis of chimaeric substrate specificities showed that enzyme affinities are mainly determined within the N-terminal end of each HAST protein, which includes two regions of high sequence divergence, termed Regions A (amino acids 44–107) and B (amino acids 132–164). To investigate the substrate-binding sites of HASTs further, site-directed mutagenesis was performed on HAST1 to change 13 individual residues within these two regions to the HAST3 equivalent. A single amino acid change in HAST1 (A146E) was able to change the specificity for p-nitrophenol to that of HAST3. The substrate specificity of HAST1 towards dopamine could not be converted into that of HAST3 with a single amino acid change. However, compared with wild-type HAST1, a number of the mutations resulted in interference with substrate binding, as shown by elevated Ki values towards the co-substrate 3´-phosphoadenosine 5´-phosphosulphate, and in some cases loss of activity towards dopamine. These findings suggest that a co-ordinated change of multiple amino acids in HAST proteins is needed to alter the substrate specificities of these enzymes towards dopamine, whereas a single amino acid at position 146 determines p-nitrophenol affinity. A HAST1 mutant was constructed to express a protein with four amino acids deleted (P87–P90). These amino acids were hypothesized to correspond to a loop region in close proximity to the substrate-binding pocket. Interestingly, the protein showed substrate specificities more similar to wild-type HAST3 than HAST1 and indicates an important role of these amino acids in substrate binding.

scholarly journals Identification of High-Affinity PB1-Derived Peptides with Enhanced Affinity to the PA Protein of Influenza A Virus Polymerase

2010 ◽  
Vol 55 (2) ◽  
pp. 696-702 ◽  
Author(s):  
Kerstin Wunderlich ◽  
Mindaugas Juozapaitis ◽  
Charlene Ranadheera ◽  
Ulrich Kessler ◽  
Arnold Martin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Binding Affinity ◽  
Influenza A Virus ◽  
Influenza A ◽  
Single Amino Acid ◽  
Binding Region ◽  
Binding Studies ◽  
Relationship Analysis ◽  
The Core

ABSTRACTThe influenza A virus polymerase complex, consisting of the subunits PB1, PB2, and PA, represents a promising target for the development of new antiviral drugs. We have previously demonstrated the feasibility of targeting the protein-protein interaction domain between PA and PB1 using peptides derived from the extreme N terminus of PB1 (amino acids [aa] 1 to 15), comprising the PA-binding domain of PB1. To increase the binding affinity of these peptides, we performed a systematic structure-affinity relationship analysis. Alanine and aspartic acid scans revealed that almost all amino acids in the core binding region (aa 5 to 11) are indispensable for PA binding. Using a library of immobilized peptides representing all possible single amino acid substitutions, we were able to identify amino acid positions outside the core PA-binding region (aa 1, 3, 12, 14, and 15) that are variable and can be replaced by affinity-enhancing residues. Surface plasmon resonance binding studies revealed that combination of several affinity-enhancing mutations led to an additive effect. Thus, the feasibility to enhance the PA-binding affinity presents an intriguing possibility to increase antiviral activity of the PB1-derived peptide and one step forward in the development of an antiviral drug against influenza A viruses.

scholarly journals A single amino acid difference is sufficient to elicit vegetative incompatibility in the fungus Podospora anserina.

Genetics ◽  
1993 ◽  
Vol 135 (1) ◽  
pp. 45-52 ◽  
Author(s):  
C Deleu ◽  
C Clavé ◽  
J Bégueret
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Podospora Anserina ◽  
Single Amino Acid ◽  
Amino Acid Difference ◽  
S Locus ◽  
Wild Type ◽  
Vegetative Incompatibility ◽  
Site Specific ◽  
S Genes

Abstract Vegetative incompatibility is known to limit heterokaryosis in filamentous fungi. It results from genetic differences between incompatible strains at specific loci. The proteins encoded by the two incompatible alleles het-s and het-S of the fungus Podospora anserina differ from each other by 14 amino acids. Two approaches have been used to identify how many and which of these differences are necessary to elicit incompatibility. Twelve alleles of the het-s locus of wild-type isolates of P. anserina and of the related species Podospora comata have been sequenced to determine the extent of the variability of genes controlling s and S specificities. Expression of hybrid het-s/het-S genes and site-specific mutagenesis revealed that the specificities of het-s and het-S are under the control of a limited number of amino acid differences. The results show that vegetative incompatibility between s and S strains can be attributed to a single amino acid difference in the proteins encoded by the het-s locus.

scholarly journals Activating mutations for transformation by p53 produce a gene product that forms an hsc70-p53 complex with an altered half-life.

1988 ◽  
Vol 8 (2) ◽  
pp. 531-539 ◽  
Author(s):  
C A Finlay ◽  
P W Hinds ◽  
T H Tan ◽  
D Eliyahu ◽  
M Oren ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibody ◽  
Amino Acid ◽  
Heat Shock ◽  
Half Life ◽  
P53 Protein ◽  
P53 Gene ◽  
Single Amino Acid ◽  
Ras Oncogene ◽  
Wild Type

The 11-4 p53 cDNA clone failed to transform primary rat fibroblasts when cotransfected with the ras oncogene. Two linker insertion mutations at amino acid 158 or 215 (of 390 amino acids) activated this p53 cDNA for transformation with ras. These mutant cDNAs produced a p53 protein that lacked an epitope, recognized by monoclonal antibody PAb246 (localized at amino acids 88 to 110 in the protein) and preferentially bound to a heat shock protein, hsc70. In rat cells transformed by a genomic p53 clone plus ras, two populations of p53 proteins were detected, PAb246+ and PAb246-, which did or did not bind to this monoclonal antibody, respectively. The PAb246- p53 preferentially associated with hsc70, and this protein had a half-life 4- to 20-fold longer than free p53 (PAb246+). These data suggest a possible functional role for hsc70 in the transformation process. cDNAs for p53 derived from methylcholanthrene-transformed cells transform rat cells in cooperation with the ras oncogene and produce a protein that bound with the heat shock proteins. Recombinant clones produced between a Meth A cDNA and 11-4 were tested for the ability to transform rat cells. A single amino acid substitution at residue 132 was sufficient to activate the 11-4 p53 cDNA for transformation. These studies have identified a region between amino acids 132 and 215 in the p53 protein which, when mutated, can activate the p53 cDNA. These results also call into question what the correct p53 wild-type sequence is and whether a wild-type p53 gene can transform cells in culture.

Activating mutations for transformation by p53 produce a gene product that forms an hsc70-p53 complex with an altered half-life

1988 ◽  
Vol 8 (2) ◽  
pp. 531-539 ◽  
Author(s):  
C A Finlay ◽  
P W Hinds ◽  
T H Tan ◽  
D Eliyahu ◽  
M Oren ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibody ◽  
Amino Acid ◽  
Heat Shock ◽  
Half Life ◽  
P53 Protein ◽  
P53 Gene ◽  
Single Amino Acid ◽  
Ras Oncogene ◽  
Wild Type

The 11-4 p53 cDNA clone failed to transform primary rat fibroblasts when cotransfected with the ras oncogene. Two linker insertion mutations at amino acid 158 or 215 (of 390 amino acids) activated this p53 cDNA for transformation with ras. These mutant cDNAs produced a p53 protein that lacked an epitope, recognized by monoclonal antibody PAb246 (localized at amino acids 88 to 110 in the protein) and preferentially bound to a heat shock protein, hsc70. In rat cells transformed by a genomic p53 clone plus ras, two populations of p53 proteins were detected, PAb246+ and PAb246-, which did or did not bind to this monoclonal antibody, respectively. The PAb246- p53 preferentially associated with hsc70, and this protein had a half-life 4- to 20-fold longer than free p53 (PAb246+). These data suggest a possible functional role for hsc70 in the transformation process. cDNAs for p53 derived from methylcholanthrene-transformed cells transform rat cells in cooperation with the ras oncogene and produce a protein that bound with the heat shock proteins. Recombinant clones produced between a Meth A cDNA and 11-4 were tested for the ability to transform rat cells. A single amino acid substitution at residue 132 was sufficient to activate the 11-4 p53 cDNA for transformation. These studies have identified a region between amino acids 132 and 215 in the p53 protein which, when mutated, can activate the p53 cDNA. These results also call into question what the correct p53 wild-type sequence is and whether a wild-type p53 gene can transform cells in culture.

Structural and functional analyses of Saccharomyces cerevisiae wild-type and mutant RNA1 genes

1989 ◽  
Vol 9 (7) ◽  
pp. 2989-2999
Author(s):  
H M Traglia ◽  
N S Atkinson ◽  
A K Hopper
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Open Reading Frame ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Reading Frame ◽  
Genomic Deletions ◽  
Single Amino Acid Change ◽  
Carboxy Terminal

The yeast gene RNA1 has been defined by the thermosensitive rna1-1 lesion. This lesion interferes with the processing and production of all major classes of RNA. Each class of RNA is affected at a distinct and presumably unrelated step. Furthermore, RNA does not appear to exit the nucleus. To investigate how the RNA1 gene product can pleiotropically affect disparate processes, we undertook a structural analysis of wild-type and mutant RNA1 genes. The wild-type gene was found to contain a 407-amino-acid open reading frame that encodes a hydrophilic protein. No clue regarding the function of the RNA1 protein was obtained by searching banks for similarity to other known gene products. Surprisingly, the rna1-1 lesion was found to code for two amino acid differences from wild type. We found that neither single-amino-acid change alone resulted in temperature sensitivity. The carboxy-terminal region of the RNA1 open reading frame contains a highly acidic domain extending from amino acids 334 to 400. We generated genomic deletions that removed C-terminal regions of this protein. Deletion of amino acids 397 to 407 did not appear to affect cell growth. Removal of amino acids 359 to 397, a region containing 24 acidic residues, caused temperature-sensitive growth. This allele, rna1-delta 359-397, defines a second conditional lesion of the RNA1 locus. We found that strains possessing the rna1-delta 359-397 allele did not show thermosensitive defects in pre-rRNA or pre-tRNA processing. Removal of amino acids 330 to 407 resulted in loss of viability.

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