scholarly journals Intergenic Suppression between the Flagellar MS Ring Protein FliF of Salmonella and FlhA, a Membrane Component of Its Export Apparatus

2001 ◽  
Vol 183 (5) ◽  
pp. 1655-1662 ◽  
Author(s):  
May Kihara ◽  
Tohru Minamino ◽  
Shigeru Yamaguchi ◽  
Robert M. Macnab
Keyword(s):  
Amino Acid ◽  
Amino Acid Level ◽  
Rare Event ◽  
Amino Acid Position ◽  
Partial Recovery ◽  
Wild Type ◽  
Membrane Domain ◽  
Flagellar Assembly ◽  
Negative Dominance ◽  
Region Ii

ABSTRACT The MS ring of the flagellar basal body of Salmonellais an integral membrane structure consisting of about 26 subunits of a 61-kDa protein, FliF. Out of many nonflagellate fliFmutants tested, three gave rise to intergenic suppressors in flagellar region II. The pseudorevertants swarmed, though poorly; this partial recovery of motile function was shown to be due to partial recovery of export function and flagellar assembly. The three parental mutants were all found to carry the same mutation, a six-base deletion corresponding to loss of Ala-174 and Ser-175 in the predicted periplasmic domain of the FliF protein. The 19 intergenic suppressors identified all lay inflhA, and they consisted of 10 independent examples at the nucleotide level or 9 at the amino acid level. Since two of the nine corresponded to different substitutions at the same amino acid position, only eight positions in the FlhA protein have given rise to suppressors. Thus, FliF-FlhA intergenic suppression is a fairly rare event. FlhA is a component of the flagellar protein export apparatus, with an integral membrane domain encompassing the N-terminal half of the sequence and a cytoplasmic C-terminal domain. All of the suppressing mutations lay within the integral membrane domain. These mutations, when placed in a wild-type fliF background, had no mutant phenotype. In the fliF mutant background, mutant FlhA was dominant, yielding a pseudorevertant phenotype. Wild-type FlhA did not exert significant negative dominance in the pseudorevertant background, indicating that it does not compete effectively with mutant FlhA for interaction with mutant FliF. Mutant FliF was partially dominant over wild-type FliF in both the wild-type and second-site FlhA backgrounds. Membrane fractionation experiments indicated that thefliF mutation, though preventing export, was mild enough to permit assembly of the MS ring itself, and also assembly of the cytoplasmic C ring onto the MS ring. The data from this study provide genetic support for a model in which at least the FlhA component of the export apparatus physically interacts with the MS ring within which it is housed.

scholarly journals Fine mapping MHC associations in Graves’ disease and its clinical subtypes in Han Chinese

2018 ◽  
Vol 55 (10) ◽  
pp. 685-692 ◽  
Author(s):  
Xun Chu ◽  
Minjun Yang ◽  
Zhen-Ju Song ◽  
Yan Dong ◽  
Chong Li ◽  
...  
Keyword(s):  
Amino Acid ◽  
Molecular Mechanisms ◽  
Amino Acid Level ◽  
Antithyroid Drug ◽  
Amino Acid Position ◽  
Thyroid Stimulating Hormone ◽  
Han Chinese ◽  
Graves Disease ◽  
Hla Genes ◽  
Antithyroid Drug Therapy

BackgroundThe classical human leucocyte antigen (HLA) genes were the most important genetic determinant for Graves’ disease (GD). The aim of the study was to fine map causal variants of the HLA genes.MethodsWe applied imputation with a Pan-Asian HLA reference panel to thoroughly investigate themajor histocompatibility complex (MHC) associations with GD down to the amino acid level of classical HLA genes in 1468 patients with GD and 1490 controls of Han Chinese.ResultsThe strongest finding across the HLA genes was the association with HLA-DPβ1 position 205 (Pomnibus=2.48×10−33). HLA-DPA1*02:02 was the strongest association among the classical HLA alleles, which was in perfect linkage disequilibrium with HLA-DPα1 residue Met11 (OR=1.90, Pbinary=1.76×10−31). Applying stepwise conditional analysis, we identified amino acid position 205 in HLA-DPβ1, position 66 and 99 in HLA-B and position 28 in HLA-DRβ1 explain majority of the MHC association to GD risk. We further evaluated risk of two clinical subtypes of GD, namely persistent thyroid stimulating hormone receptor antibody -positive (pTRAb+) group and ‘non-persistent TRAb positive’ (pTRAb−) group after antithyroid drug therapy. We found that HLA-B residues Lys66-Arg69-Val76 could drive pTRAb− GD risk alone, while HLA-DPβ1 position 205, HLA-B position 69 and 199 and HLA-DRβ1 position 28 drive pTRAb+ GD risk. The risk heterogeneity between pTRAb+ and pTRAb− GD might be driven by HLA-DPα1 Met11.ConclusionsFour amino acid positions could account for the associations of MHC with GD in Han Chinese. These distinct HLA association patterns indicated the two subtypes have distinct molecular mechanisms of pathogenesis.

Molecular Characterization of Genes Coding for Wild-Type and Sulfonylurea-Resistant Acetolactate Synthase in the Cyanobacterium Synechococcus PC C7942

1990 ◽  
Vol 45 (5) ◽  
pp. 538-543 ◽  
Author(s):  
D. Friedberg ◽  
J. Seijffers
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Characterization ◽  
Amino Acid Level ◽  
Acetolactate Synthase ◽  
Mutant Gene ◽  
Wild Type ◽  
E Coli

We present here the isolation and molecular characterization of acetolactate synthase (ALS) genes from the cyanobacterium Synechococcus PCC7942 which specify a sulfonylurea-sensitive enzyme and from the sulfonylurea-resistant mutant SM3/20, which specify resistance to sulfonylurea herbicides. The ALS gene was cloned and mapped by complementation of an Escherichia coli ilv auxotroph that requires branched-chain amino acids for growth and lacks ALS activity. The cyanobacterial gene is efficiently expressed in this heterologous host. The ALS gene codes for 612 amino acids and shows high sequence homology (46%) at the amino acid level with ALS III of E. coli and with the tobacco ALS. The resistant phenotype is a consequence of proline to serine substitution in residue 115 of the deduced amino acid sequence. Functional expression of the mutant gene in wild-type Synechococcus and in E. coli confirmed that this amino-acid substitution is responsible for the resistance. Yet the deduced amino-acid sequence as compared with othjer ALS proteins supports the notion that the amino-acid context of the substitution is important for the resistance.

Calmodulin-dependent binding to the NHE1 cytosolic tail mediates activation of the Na+/H+ exchanger by Ca2+ and endothelin

2013 ◽  
Vol 305 (11) ◽  
pp. C1161-C1169 ◽  
Author(s):  
Xiuju Li ◽  
Daniel Prins ◽  
Marek Michalak ◽  
Larry Fliegel
Keyword(s):  
Amino Acid ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Dependent ◽  
Wild Type ◽  
Membrane Domain ◽  
Plasma Membrane Protein ◽  
Nhe1 Activity

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a ubiquitous plasma membrane protein that regulates intracellular pH by removing a single proton (H+) in exchange for one extracellular Na+. The human protein contains a ∼500-amino acid membrane domain and a regulatory, ∼315-amino acid cytosolic domain. NHE1 is activated by a number of hormones including endothelin (ET) and by Ca2+. The regulatory tail possesses an inhibitory calmodulin (CaM)-binding domain, and inhibition of NHE1 is relieved by binding of a Ca2+-CaM complex. We examined the dynamics of ET-1 and Ca2+ regulation of binding to NHE1 in vivo. CFP was linked to the NHE1 protein cytoplasmic COOH terminus. This was stably transfected into AP-1 cells that are devoid of their own NHE1 protein. The protein was expressed and targeted properly and retained NHE1 activity comparable to the wild-type protein. We examined the in vivo coupling of NHE1 to CaM by Förster resonance energy transfer using CaM linked to the fluorescent protein Venus. CaM interaction with NHE1 was dynamic. Removal of serum reduced CaM interaction with NHE1. Addition of the Ca2+ ionophore ionomycin increased the interaction between CaM and NHE1. We expressed an ET receptor in AP-1 cells and also found a time-dependent association of NHE1 with CaM in vivo that was dependent on ET treatment. The results are the first demonstration of the in vivo association of NHE1 and CaM through ET-dependent signaling pathways.

scholarly journals Variants of β-Lactamase KPC-2 That Are Resistant to Inhibition by Avibactam

2015 ◽  
Vol 59 (7) ◽  
pp. 3710-3717 ◽  
Author(s):  
Krisztina M. Papp-Wallace ◽  
Marisa L. Winkler ◽  
Magdalena A. Taracila ◽  
Robert A. Bonomo
Keyword(s):  
Amino Acid ◽  
Molecular Modeling ◽  
Clavulanic Acid ◽  
Amino Acid Position ◽  
Proton Donor ◽  
Single Amino Acid ◽  
Wild Type ◽  
Content Type ◽  
Mechanistic Basis ◽  
Dynamics Simulations

ABSTRACTKPC-2 is the most prevalent class A carbapenemase in the world. Previously, KPC-2 was shown to hydrolyze the β-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam. In addition, substitutions at amino acid position R220 in the KPC-2 β-lactamase increased resistance to clavulanic acid. A novel bridged diazabicyclooctane (DBO) non-β-lactam β-lactamase inhibitor, avibactam, was shown to inactivate the KPC-2 β-lactamase. To better understand the mechanistic basis for inhibition of KPC-2 by avibactam, we tested the potency of ampicillin-avibactam and ceftazidime-avibactam against engineered variants of the KPC-2 β-lactamase that possessed single amino acid substitutions at important sites (i.e., Ambler positions 69, 130, 234, 220, and 276) that were previously shown to confer inhibitor resistance in TEM and SHV β-lactamases. To this end, we performed susceptibility testing, biochemical assays, and molecular modeling.Escherichia coliDH10B carrying KPC-2 β-lactamase variants with the substitutions S130G, K234R, and R220M demonstrated elevated MICs for only the ampicillin-avibactam combinations (e.g., 512, 64, and 32 mg/liter, respectively, versus the MICs for wild-type KPC-2 at 2 to 8 mg/liter). Steady-state kinetics revealed that the S130G variant of KPC-2 resisted inactivation by avibactam; thek2/Kratio was significantly lowered 4 logs for the S130G variant from the ratio for the wild-type enzyme (21,580 M−1s−1to 1.2 M−1s−1). Molecular modeling and molecular dynamics simulations suggested that the mobility of K73 and its ability to activate S70 (i.e., function as a general base) may be impaired in the S130G variant of KPC-2, thereby explaining the slowed acylation. Moreover, we also advance the idea that the protonation of the sulfate nitrogen of avibactam may be slowed in the S130G variant, as S130 is the likely proton donor and another residue, possibly K234, must compensate. Our findings show that residues S130 as well as K234 and R220 contribute significantly to the mechanism of avibactam inactivation of KPC-2. Fortunately, the emergence of S130G, K234R, and R220M variants of KPC in the clinic should not result in failure of ceftazidime-avibactam, as the ceftazidime partner is potent againstE. coliDH10B strains possessing all of these variants.

scholarly journals Delta plus variant with neutral mutation is less virulent compared to wild type SARS-CoV2

2021 ◽  
Author(s):  
Sonal Upadhyay ◽  
Anuj Kumar ◽  
Pradeep Kumar ◽  
Pawan K. Dubey ◽  
Anima Tripathi ◽  
...  
Keyword(s):  
Protein Structure ◽  
Amino Acid ◽  
3D Structure ◽  
Amino Acid Position ◽  
Docking Study ◽  
Docking Studies ◽  
Wild Type ◽  
Neutral Mutation ◽  
New Variant ◽  
Antigen Antibody

Abstract BackgroundWith the start of the Coronavirus disease19 (COVID19) pandemic, the Coronavirus has mutated constantly. Recently a new variant called Delta plus has been reported in few countries, including South Africa, Brazil and India. The Delta plus variant contains an additional mutation called K417N on the coronavirus spike. The present study aims to determine the virulence and transmissibility of the Delta plus variant and to check the efficiency of different antibodies on its neutralization.Materials and MethodsDifferent computational tools such as PROVEAN, an online tool, HOPE server, simulation using CABS Flex, Clus pro, an online docking tool, were used to predict the structure and function of Delta plus variant by performing a comparative study with wild type protein. Also, to find an effective antibody against Delta plus variant, antigen-antibody docking studies were conducted through Clus pro server. Furthermore, we performed a 2D interaction diagram analysis to find the amino acid residue's interaction against antibodies.Results PROVEAN and HOPE showed the mutation (K417N) in the S-glycoprotein of Delta plus as NEUTRAL mutation. This mutation causes the loss of cysteine bonds leading to the destabilization of the 3D structure of spike protein. Furthermore, the RMSF plot emphasizing the 17th amino acid position of wild and Delta plus mutant revealed the high fluctuation of mutant protein structure compared to the wild protein structure. Further, a comparative docking study against hACE2 shows higher binding energy of wild-type RBD (-751.7 kcal/mol) than mutant RBD (-750.1 kcal/mol). Moreover, antigen-antibody docking study revealed higher affinity of BD-23 Fab antibodies with greater interaction energy ( -997 kcal/mol) compared to other antibodies and thus may prove to be a promising therapeutic against Delta plus variant.ConclusionDelta plus variant is less stable, has a lower binding affinity to hACE2 and has less virulence than wild type. However, the BD-23 Fab antibody has shown a more significant association for this variant and can be used in its treatment.

scholarly journals The FLA3 KAP Subunit Is Required for Localization of Kinesin-2 to the Site of Flagellar Assembly and Processive Anterograde Intraflagellar Transport

2005 ◽  
Vol 16 (3) ◽  
pp. 1341-1354 ◽  
Author(s):  
Joshua Mueller ◽  
Catherine A. Perrone ◽  
Raqual Bower ◽  
Douglas G. Cole ◽  
Mary E. Porter
Keyword(s):  
Amino Acid ◽  
Basal Body ◽  
Intraflagellar Transport ◽  
Body Region ◽  
Wild Type ◽  
Gene Encoding ◽  
Flagellar Assembly ◽  
Cilia And Flagella ◽  
Preferential Incorporation ◽  
Dic Microscopy

Intraflagellar transport (IFT) is a bidirectional process required for assembly and maintenance of cilia and flagella. Kinesin-2 is the anterograde IFT motor, and Dhc1b/Dhc2 drives retrograde IFT. To understand how either motor interacts with the IFT particle or how their activities might be coordinated, we characterized a ts mutation in the Chlamydomonas gene encoding KAP, the nonmotor subunit of Kinesin-2. The fla3-1 mutation is an amino acid substitution in a conserved C-terminal domain. fla3-1 strains assemble flagella at 21°C, but cannot maintain them at 33°C. Although the Kinesin-2 complex is present at both 21 and 33°C, the fla3-1 Kinesin-2 complex is not efficiently targeted to or retained in the basal body region or flagella. Video-enhanced DIC microscopy of fla3-1 cells shows that the frequency of anterograde IFT particles is significantly reduced. Anterograde particles move at near wild-type velocities, but appear larger and pause more frequently in fla3-1. Transformation with an epitope-tagged KAP gene rescues all of the fla3-1 defects and results in preferential incorporation of tagged KAP complexes into flagella. KAP is therefore required for the localization of Kinesin-2 at the site of flagellar assembly and the efficient transport of anterograde IFT particles within flagella.

scholarly journals An Amino Acid Substitution at Position 740 in σ70 of Ralstonia solanacearum Strain OE1-1 Affects Its In Planta Growth

2008 ◽  
Vol 74 (18) ◽  
pp. 5841-5844 ◽  
Author(s):  
Ayami Kanda ◽  
Kazuhiro Tsuneishi ◽  
Ai Mori ◽  
Kouhei Ohnishi ◽  
Akinori Kiba ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Ralstonia Solanacearum ◽  
Wild Type ◽  
In Planta ◽  
Solanacearum Strain ◽  
Type Protein ◽  
Wild Type Protein ◽  
Negative Dominance

ABSTRACT Growth of Ralstonia solanacearum strain OE1-1 in roots after invasion is required for virulence. An Arg740Cys substitution in σ70 of OE1-1 resulted in loss of in planta growth and virulence. The negative dominance of mutant σ70 over the wild-type protein suggested that the amino acid substitution may affect the in planta growth of OE1-1, leading to a lack of virulence.

scholarly journals Influenza virus site recognized by a murine helper T cell specific for H1 strains. Localization to a nine amino acid sequence in the hemagglutinin molecule.

1983 ◽  
Vol 158 (2) ◽  
pp. 294-302 ◽  
Author(s):  
C J Hackett ◽  
B Dietzschold ◽  
W Gerhard ◽  
B Ghrist ◽  
R Knorr ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
T Cell ◽  
Antigen Presenting Cells ◽  
Amino Acid Position ◽  
H1n1 Influenza ◽  
Wild Type ◽  
Helper T Cell ◽  
Human Influenza Virus ◽  
Antigen Presenting

The functional helper T cell line Vir-2, derived from a PR8 (H1N1) influenza virus-immunized BALB/c mouse, proliferates in response to syngeneic antigen-presenting cells and naturally occurring strains of subtype H1 human influenza virus from 1934-1957 and 1977-1980 isolates. A conserved region of the hemagglutinin molecule around amino acid position 115 in the heavy chain (HA1) was implicated as being important in this recognition by the lack of stimulatory activity associated with a glutamic acid to lysine substitution at position 115 in the laboratory mutant RV6, derived from wild-type PR8. Characterization of the stimulatory determinant on the wild-type hemagglutinin molecule was then undertaken using cleavage products and synthetic peptides. Vir-2 cells recognized the reduced and alkylated purified HA1 of PR8 virus, and this reactivity was retained after cleavage at methionine and tryptophan residues. High-pressure liquid chromatography separation of cleavage fragments indicated that a short sequence of the HA1 containing residue 115 was being recognized. This recognition was localized to a nine amino acid segment (positions 111-119) by assaying stimulation with synthetic peptide homologues of different lengths from that region. As with native hemagglutinin, Vir-2 cells responded to active peptides when presented by H-2d but not H-2k antigen-presenting cells.

Recombinant Variants of Tissue-Type Plasminogen Activator Containing Amino Acid Substitutions in the Finger Domain

1992 ◽  
Vol 68 (06) ◽  
pp. 672-677 ◽  
Author(s):  
Hitoshi Yahara ◽  
Keiji Matsumoto ◽  
Hiroyuki Maruyama ◽  
Tetsuya Nagaoka ◽  
Yasuhiro Ikenaka ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasminogen Activator ◽  
Half Life ◽  
Tissue Type ◽  
Clot Lysis ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Plasma Clot ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

SummaryTissue-type plasminogen activator (t-PA) is a fibrin-specific agent which has been used to treat acute myocardial infarction. In an attempt to clarify the determinants for its rapid clearance in vivo and high affinity for fibrin clots, we produced five variants containing amino acid substitutions in the finger domain, at amino acid residues 7–9, 10–14, 15–19, 28–33, and 37–42. All the variants had a prolonged half-life and a decreased affinity for fibrin of various degrees. The 37–42 variant demonstrated about a 6-fold longer half-life with a lower affinity for fibrin. Human plasma clot lysis assay estimated the fibrinolytic activity of the 37–42 variant to be 1.4-fold less effective than that of the wild-type rt-PA. In a rabbit jugular vein clot lysis model, doses of 1.0 and 0.15 mg/kg were required for about 70% lysis in the wild-type and 37–42 variant, respectively. Fibrinogen was degraded only when the wild-type rt-PA was administered at a dose of 1.0 mg/kg. These findings suggest that the 37–42 variant can be employed at a lower dosage and that it is a more fibrin-specific thrombolytic agent than the wild-type rt-PA.

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