scholarly journals The Second Extracellular Loop of Pore-Forming Subunits of ATP-Binding Cassette Transporters for Basic Amino Acids Plays a Crucial Role in Interaction with the Cognate Solute Binding Protein(s)

2010 ◽  
Vol 192 (8) ◽  
pp. 2150-2159 ◽  
Author(s):  
Viola Eckey ◽  
Daniela Weidlich ◽  
Heidi Landmesser ◽  
Ulf Bergmann ◽  
Erwin Schneider
Keyword(s):  
Amino Acids ◽  
Atpase Activity ◽  
Crucial Role ◽  
Mutational Analysis ◽  
Binding Protein ◽  
Substrate Binding ◽  
Extracellular Loop ◽  
Basic Amino Acids ◽  
Charged Residues ◽  
Positively Charged

ABSTRACT In the thermophile Geobacillus stearothermophilus, the uptake of basic amino acids is mediated by an ABC transporter composed of the substrate binding protein (receptor) ArtJ and a homodimer each of the pore-forming subunit, ArtM, and the nucleotide-binding subunit, ArtP. We recently identified two putative binding sites in ArtJ that might interact with the Art(MP)2 complex, thereby initiating the transport cycle (A. Vahedi-Faridi et al., J. Mol. Biol. 375:448-459, 2008). Here we investigated the contribution of charged amino acid residues in the second extracellular loop of ArtM to contact with ArtJ. Our results demonstrate a crucial role for residues K177, R185, and E188, since mutations to oppositely charged amino acids or glutamine led to a complete loss of ArtJ-stimulated ATPase activity of the complex variants in proteoliposomes. The defects could not be suppressed by ArtJ variants carrying mutations in site I (K39E and K152E) or II (E163K and D170K), suggesting a more complex interplay than that by a single salt bridge. These findings were supported by cross-linking assays demonstrating physical proximity between ArtJ(N166C) and ArtM(E182C). The importance of positively charged residues for receptor-transporter interaction was underscored by mutational analysis of the closely related transporter HisJ/LAO-HisQMP2 of Salmonella enterica serovar Typhimurium. While transporter variants with mutated positively charged residues in HisQ displayed residual ATPase activities, corresponding mutants of HisM could no longer be stimulated by HisJ/LAO. Interestingly, the ATPase activity of the HisQM(K187E)P2 variant was inhibited by l- and d-histidine in detergent, suggesting a role of the residue in preventing free histidine from gaining access to the substrate binding site within HisQM.

scholarly journals The Vaccine Candidate Substrate Binding Protein SBP2 Plays a Key Role in Arginine Uptake, Which Is Required for Growth of Moraxella catarrhalis

2015 ◽  
Vol 84 (2) ◽  
pp. 432-438 ◽  
Author(s):  
Taketo Otsuka ◽  
Charmaine Kirkham ◽  
Aimee Brauer ◽  
Mary Koszelak-Rosenblum ◽  
Michael G. Malkowski ◽  
...  
Keyword(s):  
Amino Acids ◽  
Respiratory Tract ◽  
Abc Transporter ◽  
Moraxella Catarrhalis ◽  
Vaccine Candidate ◽  
Binding Protein ◽  
Substrate Binding ◽  
Content Type ◽  
Basic Amino Acids ◽  
Substrate Binding Protein

Moraxella catarrhalisis an exclusively human pathogen that is an important cause of otitis media in children and lower respiratory tract infections in adults with chronic obstructive pulmonary disease. A vaccine to preventM. catarrhalisinfections would have an enormous global impact in reducing morbidity resulting from these infections. Substrate binding protein 2 (SBP2) of an ABC transporter system has recently been identified as a promising vaccine candidate antigen on the bacterial surface ofM. catarrhalis. In this study, we showed that SBP1, -2, and -3 individually bind different basic amino acids with exquisite specificity. We engineered mutants that each expressed a single SBP from this gene cluster and showed in growth experiments that SBP1, -2, and -3 serve a nutritional function through acquisition of amino acids for the bacterium. SBP2 mediates uptake of arginine, a strict growth requirement ofM. catarrhalis. Adherence and invasion assays demonstrated that SBP1 and SBP3 play a role in invasion of human respiratory epithelial cells, consistent with a nutritional role in intracellular survival in the human respiratory tract. This work demonstrates that the SBPs of an ABC transporter system function in the uptake of basic amino acids to support growth ofM. catarrhalis. The critical role of SBP2 in arginine uptake may contribute to its potential as a vaccine antigen.

scholarly journals Substrate recognition and ATPase activity of the E. coli cysteine/cystine ABC transporter YecSC-FliY

2020 ◽  
Vol 295 (16) ◽  
pp. 5245-5256 ◽  
Author(s):  
Siwar Sabrialabed ◽  
Janet G. Yang ◽  
Elon Yariv ◽  
Nir Ben-Tal ◽  
Oded Lewinson
Keyword(s):  
Atpase Activity ◽  
Abc Transporter ◽  
Conformational Changes ◽  
Binding Protein ◽  
Substrate Binding ◽  
Transporter Family ◽  
Wide Range ◽  
Substrate Binding Protein ◽  
Sulfur Containing ◽  
Sulfur Containing Compounds

Sulfur is essential for biological processes such as amino acid biogenesis, iron–sulfur cluster formation, and redox homeostasis. To acquire sulfur-containing compounds from the environment, bacteria have evolved high-affinity uptake systems, predominant among which is the ABC transporter family. Theses membrane-embedded enzymes use the energy of ATP hydrolysis for transmembrane transport of a wide range of biomolecules against concentration gradients. Three distinct bacterial ABC import systems of sulfur-containing compounds have been identified, but the molecular details of their transport mechanism remain poorly characterized. Here we provide results from a biochemical analysis of the purified Escherichia coli YecSC-FliY cysteine/cystine import system. We found that the substrate-binding protein FliY binds l-cystine, l-cysteine, and d-cysteine with micromolar affinities. However, binding of the l- and d-enantiomers induced different conformational changes of FliY, where the l- enantiomer–substrate-binding protein complex interacted more efficiently with the YecSC transporter. YecSC had low basal ATPase activity that was moderately stimulated by apo FliY, more strongly by d-cysteine–bound FliY, and maximally by l-cysteine– or l-cystine–bound FliY. However, at high FliY concentrations, YecSC reached maximal ATPase rates independent of the presence or nature of the substrate. These results suggest that FliY exists in a conformational equilibrium between an open, unliganded form that does not bind to the YecSC transporter and closed, unliganded and closed, liganded forms that bind this transporter with variable affinities but equally stimulate its ATPase activity. These findings differ from previous observations for similar ABC transporters, highlighting the extent of mechanistic diversity in this large protein family.

scholarly journals Mutational Analysis of Plant Cap-Binding Protein eIF4E Reveals Key Amino Acids Involved in Biochemical Functions and Potyvirus Infection

2008 ◽  
Vol 82 (15) ◽  
pp. 7601-7612 ◽  
Author(s):  
Sylvie German-Retana ◽  
Jocelyne Walter ◽  
Bénédicte Doublet ◽  
Geneviève Roudet-Tavert ◽  
Valérie Nicaise ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mutational Analysis ◽  
Binding Protein ◽  
Point Mutations ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Natural Resistance ◽  
Initiation Factor ◽  
Amino Acid Residues ◽  
Eukaryotic Translation

ABSTRACT The eukaryotic translation initiation factor 4E (eIF4E) (the cap-binding protein) is involved in natural resistance against several potyviruses in plants. In lettuce, the recessive resistance genes mo1 1 and mo1 2 against Lettuce mosaic virus (LMV) are alleles coding for forms of eIF4E unable, or less effective, to support virus accumulation. A recombinant LMV expressing the eIF4E of a susceptible lettuce variety from its genome was able to produce symptoms in mo1 1 or mo1 2 varieties. In order to identify the eIF4E amino acid residues necessary for viral infection, we constructed recombinant LMV expressing eIF4E with point mutations affecting various amino acids and compared the abilities of these eIF4E mutants to complement LMV infection in resistant plants. Three types of mutations were produced in order to affect different biochemical functions of eIF4E: cap binding, eIF4G binding, and putative interaction with other virus or host proteins. Several mutations severely reduced the ability of eIF4E to complement LMV accumulation in a resistant host and impeded essential eIF4E functions in yeast. However, the ability of eIF4E to bind a cap analogue or to fully interact with eIF4G appeared unlinked to LMV infection. In addition to providing a functional mutational map of a plant eIF4E, this suggests that the role of eIF4E in the LMV cycle might be distinct from its physiological function in cellular mRNA translation.

scholarly journals Structural Determinants for Heparin Binding in Human Coagulation Factor XI.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2705-2705
Author(s):  
Sergei Shikov ◽  
Wenman Wu ◽  
Peter N. Walsh
Keyword(s):  
Catalytic Domain ◽  
Mutational Analysis ◽  
Solid Phase ◽  
Coagulation Factor ◽  
Hek293 Cells ◽  
Heparin Binding ◽  
Factor Xi ◽  
Dimeric Structure ◽  
Charged Residues ◽  
Positively Charged

Abstract Previous studies from our laboratory and others have demonstrated that zymogen factor XI (FXI) binds to heparin with moderate (KD ∼110 nM) affinity via residues (K252, K253 and K255) located in the Apple 3 (A3) domain. In contrast, the enzyme, FXIa, was shown to bind to heparin (Biochemistry40: 7569–7580, 2001) with significantly higher affinity (KD ∼9 nM by SPR and ∼1.5 nM by ELISA) via residues (K529, R530 and R532) within the catalytic domain (CD). This interaction potentiates by ∼10-fold the inhibition of FXIa by protease nexin-2. Also, polyanions heparin and dextran sulfate inhibit the catalytic activity of the enzyme factor XIa. The present study was designed to determine the relative contributions of positively charged residues as well as the dimeric structure of FXI to heparin binding. Mutational analysis of full-length FXI expressed in HEK293 cells was based on the following criteria: Conservation of the positively charged residues in FXI among various species; Surface exposure of the residues based on the X-ray crystal structure of FXI (Papagrigoriou E, McEwan P, Walsh PN, Emsley J,Nat. Struct. & Mol. Biol. 2006; 13:557–558); and comparison with human plasma prekallikrein (PK), which does not bind heparin. Two positively charged residues Arg507 (147, chymotrypsin numbering) and Arg532 (173) are conserved in FXI genes of all species for which sequences are available. In human PK, Arg507 is replaced by lysine, while Arg532 is replaced by a neutral glutamine. We have expressed and purified wtFXI, R507A, R532A as well as monomeric C321S/K331A and C321A/I290A. While wtFXI, R507A and R532A demonstrated normal activity in APTT assays; monomeric FXI mutants retained 60-70% activity. The R532A and R507A mutants demonstrated ∼75% decrease in total number of heparin binding sites based on the solid phase ELISA assay using 5F7 monoclonal antibody. Also, the apparent dissociation constants for R507 (11 nM) and R532A (22 nM) were 7 and 11-fold increased respectively compared with 1.6 nM for the wtFXI. We also characterized monomeric FXI C321S/K331A and C321A/I290A proteins for their ability to bind to heparin compared with wtFXI using surface plasmon resonance (SPR). Surprisingly, the monomeric FXI mutants, C321S/K331A and C321A/I290A, which had no mutations in any heparin-binding regions, displayed major defects in binding to heparin by SPR. Although kinetic analysis is challenging due to complex binding kinetics, while Rmax is about 10-fold lower, the off-rate for the binding of the monomeric FXI mutants is drastically increased when compared to that of wtFXI. These results suggest the possibility that the unique dimeric structure of FXI is required for cooperative binding to heparin. Thus, the dimeric structure of FXI and basic residues R507 and R532 in the catalytic domain of factor XI are both necessary for high-affinity heparin binding.

scholarly journals Arginine mutations within a transmembrane domain of Tar, an Escherichia coli aspartate receptor, can drive homodimer dissociation and heterodimer association in vivo

2004 ◽  
Vol 385 (1) ◽  
pp. 29-36 ◽  
Author(s):  
Neta SAL-MAN ◽  
Yechiel SHAI
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Genetic Diseases ◽  
Ion Pairs ◽  
Aspartate Receptor ◽  
Charged Residues ◽  
Arginine Residues ◽  
Tm Domain ◽  
Positively Charged

The interactions between the TM (transmembrane) domains of many membrane proteins are important for their proper functioning. Mutations of residues into positively charged ones within TM domains were reported to be involved in many genetic diseases, possibly because these mutations affect the self- and/or hetero-assembly of the corresponding proteins. To our knowledge, despite significant progress in understanding the role of various amino acids in TM–TM interactions in vivo, the direct effect of positively charged residues on these interactions has not been studied. To address this issue, we employed the N-terminal TM domain of the aspartate receptor (Tar-1) as a dimerization model system. We expressed within the ToxR TM assembly system several Tar-1 constructs that dimerize via polar- or non-polar amino acid motifs, and mutated these by replacement with a single arginine residue. Our results have revealed that a mutation in each of the motifs significantly reduced the ability of the TMs to dimerize. Furthermore, a Tar-1 construct that contained two arginine residues was unable to correctly integrate itself into the membrane. Nevertheless, an exogenous synthetic Tar-1 peptide containing these two arginine residues was able to inhibit in vivo the marked dimerization of a mutant Tar-1 construct that contained two glutamate residues at similar positions. This indicates that hetero-assembly of TM domains can be mediated by the interaction of two oppositely charged residues, probably by formation of ion pairs. This study broadens our knowledge regarding the effect of positively charged residues on TM–TM interactions in vivo, and provides a potential therapeutic approach to inhibit uncontrolled dimerization of TM domains caused by mutations of polar amino acids.

scholarly journals OppA, the Substrate-Binding Subunit of the Oligopeptide Permease, Is the Major Ecto-ATPase of Mycoplasma hominis

2004 ◽  
Vol 186 (4) ◽  
pp. 1021-1028 ◽  
Author(s):  
Miriam Hopfe ◽  
Birgit Henrich
Keyword(s):  
Atpase Activity ◽  
Binding Protein ◽  
Substrate Binding ◽  
Mycoplasma Hominis ◽  
Disulfonic Acid ◽  
Atp Binding ◽  
Atpase Inhibitor ◽  
Atp Binding Site ◽  
Oligopeptide Permease ◽  
Substrate Binding Protein

ABSTRACT Most ATPases, involved in energy-driven processes, act in the cytoplasm. However, external membrane-bound ATPases have also been described in parasites and eukaryotic cells. In Mycoplasma hominis, a bacterium lacking a cell wall, the surface-exposed substrate-binding protein OppA of an oligopeptide permease (Opp) contains an ATP binding P-loop structure in the C-terminal region. With ATP affinity chromatography and tryptic digestion in the presence or absence of ATP, the functionality of the Mg2+-dependent ATP binding site is demonstrated. In addition to ATP, ADP also could bind to OppA. The presence of an ATPase activity on the surface of M. hominis is indicated by the inactivation of ATP hydrolyzing activity of intact mycoplasma cells by the impermeable ATPase inhibitor 4′,4′-diisothiocyanostilbene-2′,2′-disulfonic acid and influenced by the ATP analog 5′-fluorosulfonyl-benzoyladenosine. Comparing equimolar amounts of OppA in intact mycoplasma cells and in the purified form indicated that more than 80% of the surface-localized ATPase activity is derived from OppA, implying that OppA is the main ATPase on the surface of mycoplasma cells. Together, these data present the first evidence that the cytoadhesive substrate binding protein OppA of the oligopeptide permease also functions as an ecto-ATPase in Mycoplasma hominis.

scholarly journals The carboxy-terminal domain of IGF-binding protein-5 inhibits heparin binding to a site in the central domain

2001 ◽  
Vol 26 (3) ◽  
pp. 229-239 ◽  
Author(s):  
H Song ◽  
JH Shand ◽  
J Beattie ◽  
DJ Flint ◽  
GJ Allan
Keyword(s):  
Amino Acids ◽  
Binding Protein ◽  
Binding Activity ◽  
Heparin Binding ◽  
Central Domain ◽  
Basic Amino Acids ◽  
Terminal Domain ◽  
Igf Binding ◽  
Igf Binding Protein

The IGF-binding protein (IGFBP)-5 protein contains consensus heparin binding motifs in both its carboxy (C)-terminal and central domains, although only the C-terminal site has previously been shown to be functional. We have made two chimeric IGFBP proteins by switching domains between rat IGFBP-5 and -2, named BP552 and BP522 to reflect the domains present, and a truncated rat IGFBP-5 mutant (1-168), named BP550. The ability of these proteins and wild-type (wt) IGFBPs-5 and -2 to bind to either IGFs or heparin was determined using biosensor real-time analysis and heparin ligand blotting respectively. We report that the chimeric molecules have IGF binding affinities comparable to those of the native IGFBPs from which they were derived and, as expected, the binding of BP550 to IGFs was greatly compromised. More surprising was the finding that the ability of BP552 and BP550 to bind to heparin was equivalent to that of wtIGFBP-5, whereas wtIGFBP-2 and BP522 failed to bind. These results demonstrate that the active heparin binding site in BP552 and BP550 is contained within the central domain of IGFBP-5, and that this site is active only in the absence of the C-terminal domain. We subsequently mutated two basic amino acids (R136A:R137A) in the central consensus binding sites between residues 132-140. This resulted in the loss of heparin binding for BP550, confirming the importance of these two basic amino acids in the central domain heparin binding activity. In light of these findings, we suggest that C-terminally truncated fragments of IGFBP-5 generated in vivo by proteolysis could retain heparin/extracellular matrix binding properties.

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