Substrate-specific binding and conformational changes involving Ser313 and transmembrane domain 8 of the human reduced folate carrier, as determined by site-directed mutagenesis and protein cross-linking

RFC (reduced folate carrier) is the major transporter for reduced folates and antifolates [e.g. MTX (methotrexate)]. RFC is characterized by two halves, each with six TMD (transmembrane domain) α helices connected by a hydrophilic loop, and cytoplasmic N- and C-termini. We previously identified TMDs 4, 5, 7, 8, 10 and 11 as forming the hydrophilic cavity for translocation of (anti)folates. The proximal end of TMD8 (positions 311–314) was implicated in substrate binding from scanning-cysteine accessibility methods; cysteine replacement of Ser313 resulted in loss of transport. In the present study, Ser313 was mutated to alanine, cysteine, phenylalanine and threonine. Mutant RFCs were expressed in RFC-null R5 HeLa cells. Replacement of Ser313 with cysteine or phenylalanine abolished MTX transport, whereas residual activity was preserved for the alanine and threonine mutants. In stable K562 transfectants, S313A and S313T RFCs showed substantially decreased Vmax values without changes in Kt values for MTX compared with wild-type RFC. S313A and S313T RFCs differentially impacted binding of ten diverse (anti)folate substrates. Cross-linking between TMD8 and TMD5 was studied by expressing cysteine-less TMD1–6 (N6) and TMD7–12 (C6) half-molecules with cysteine insertions spanning these helices in R5 cells, followed by treatment with thiol-reactive homobifunctional cross-linkers. C6–C6 and N6–N6 cross-links were seen for all cysteine pairs. From the N6 and C6 cysteine pairs, Cys175/Cys311 was cross-linked; cross-linking increased in the presence of transport substrates. The results of the present study indicate that the proximal end of TMD8 is juxtaposed to TMD5 and is conformationally active in the presence of transport substrates, and TMD8, including Ser313, probably contributes to the RFC substrate-binding domain.

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Mapping the Contact Sites of the Escherichia coli Division-Initiating Proteins FtsZ and ZapA by BAMG Cross-Linking and Site-Directed Mutagenesis

International Journal of Molecular Sciences ◽

10.3390/ijms19102928 ◽

2018 ◽

Vol 19 (10) ◽

pp. 2928 ◽

Cited By ~ 7

Author(s):

Winfried Roseboom ◽

Madhvi Nazir ◽

Nils Meiresonne ◽

Tamimount Mohammadi ◽

Jolanda Verheul ◽

...

Keyword(s):

Site Directed Mutagenesis ◽

Cross Linking ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Binding Interface ◽

Tetrameric Structure ◽

Z Ring ◽

Cross Links ◽

Chemical Cross Linking

Cell division in bacteria is initiated by the polymerization of FtsZ at midcell in a ring-like structure called the Z-ring. ZapA and other proteins assist Z-ring formation and ZapA binds ZapB, which senses the presence of the nucleoids. The FtsZ–ZapA binding interface was analyzed by chemical cross-linking mass spectrometry (CXMS) under in vitro FtsZ-polymerizing conditions in the presence of GTP. Amino acids residue K42 from ZapA was cross-linked to amino acid residues K51 and K66 from FtsZ, close to the interphase between FtsZ molecules in protofilaments. Five different cross-links confirmed the tetrameric structure of ZapA. A number of FtsZ cross-links suggests that its C-terminal domain of 55 residues, thought to be largely disordered, has a limited freedom to move in space. Site-directed mutagenesis of ZapA reveals an interaction site in the globular head of the protein close to K42. Using the information on the cross-links and the mutants that lost the ability to interact with FtsZ, a model of the FtsZ protofilament–ZapA tetramer complex was obtained by information-driven docking with the HADDOCK2.2 webserver.

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Site-Directed Disulfide Cross-Linking Shows that Cleft Flexibility in the Periplasmic Domain Is Needed for the Multidrug Efflux Pump AcrB of Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.01127-07 ◽

2007 ◽

Vol 189 (23) ◽

pp. 8677-8684 ◽

Cited By ~ 47

Author(s):

Yumiko Takatsuka ◽

Hiroshi Nikaido

Keyword(s):

Escherichia Coli ◽

Conformational Changes ◽

Disulfide Bonds ◽

Drug Transport ◽

Site Directed Mutagenesis ◽

Cross Linking ◽

Cysteine Residues ◽

Multidrug Efflux ◽

Specific Chemical ◽

Periplasmic Domain

ABSTRACT Escherichia coli AcrB is a multidrug efflux transporter that recognizes multiple toxic chemicals having diverse structures. Recent crystallographic studies of the asymmetric trimer of AcrB suggest that each protomer in the trimeric assembly goes through a cycle of conformational changes during drug export. However, biochemical evidence for these conformational changes has not been provided previously. In this study, we took advantage of the observation that the external large cleft in the periplasmic domain of AcrB appears to become closed in the crystal structure of one of the three protomers, and we carried out in vivo cross-linking between cysteine residues introduced by site-directed mutagenesis on both sides of the cleft, as well as at the interface between the periplasmic domains of the AcrB trimer. Double-cysteine mutants with mutations in the cleft or the interface were inactive. The possibility that this was due to the formation of disulfide bonds was suggested by the restoration of transport activity of the cleft mutants in a dsbA strain, which had diminished activity to form disulfide bonds in the periplasm. Furthermore, rapidly reacting, sulfhydryl-specific chemical cross-linkers, methanethiosulfonates, inactivated the AcrB transporter with double-cysteine residues in the cleft expressed in dsbA cells, and this inactivation could be observed within a few seconds after the addition of a cross-linker in real time by increased ethidium influx into the cells. These observations indicate that conformational changes, including the closure of the external cleft in the periplasmic domain, are required for drug transport by AcrB.

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Investigation of the quaternary structure of Neurospora pyruvate kinase by cross-linking with bifunctional reagents: the effect of substrates and allosteric ligands

Canadian Journal of Biochemistry ◽

10.1139/o77-008 ◽

1977 ◽

Vol 55 (1) ◽

pp. 43-49 ◽

Cited By ~ 17

Author(s):

M. Kapoor ◽

M. D. O'Brien

Keyword(s):

Pyruvate Kinase ◽

Functional Groups ◽

Conformational Changes ◽

Quaternary Structure ◽

Polyacrylamide Gel ◽

Cross Linking ◽

Complex Cross ◽

Dimethyl Suberimidate ◽

Cross Links

Pyruvate kinase (EC 2.7.1.40) of Neurospora, a tetramer composed of apparently identical subunits, has been shown to be a dimer of dimers by interprotomeric cross-linking experiments in which bifunctional reagents were used. An analysis of the polyacrylamide gel profiles of the enzyme after cross-linking with glutaraldehyde, dimethyl suberimidate, and dimethyl adipimidate shows that the extent of intersubunit cross-linking is influenced markedly by the ligand bound to the enzyme. Bifunctional cross-linking reagents with a shorter distance between the two functional groups form cross-links effectively in the unliganded enzyme. In the FDP – pyruvate kinase complex, cross-linking was observed over longer distances compared with the unliganded enzyme. It is demonstrated that covalent cross-linkers can be used as sensitive indicators of conformational changes induced in pyruvate kinase by substrates and allosteric ligands.

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Molecular insights into phosphorylation-induced allosteric conformational changes in β2-adrenergic receptor

10.1101/2021.10.01.462841 ◽

2021 ◽

Author(s):

Midhun K Madhu ◽

Annesha Debroy ◽

Rajesh K. Murarka

Keyword(s):

G Protein ◽

Conformational Changes ◽

Adrenergic Receptor ◽

Transmembrane Domain ◽

Conformational Transition ◽

Specific Binding ◽

Experimental Studies ◽

Residue Interaction ◽

Β2 Adrenergic Receptor ◽

Experimental Findings

The large conformational flexibility of G protein-coupled receptors (GPCRs) has been a puzzle in structural and pharmacological studies for the past few decades. Apart from structural rearrangements induced by ligands, enzymatic phosphorylations by GPCR kinases (GRKs) at the carboxy-terminal tail (C-tail) of a GPCR also makes conformational alterations to the transmembrane helices and facilitates the binding of one of its transducer proteins named β-arrestin. Phosphorylation-induced conformational transition of the receptor that causes specific binding to β-arrestin but prevents the association of other transducers such as G proteins lacks atomistic understanding and is elusive to experimental studies. Using microseconds of all-atom conventional and Gaussian accelerated molecular dynamics (GaMD) simulations, we investigate the allosteric mechanism of phosphorylation induced-conformational changes in β2-adrenergic receptor, a well-characterized GPCR model system. Free energy profiles reveal that the phosphorylated receptor samples a new conformational state in addition to the canonical active state corroborating with recent nuclear magnetic resonance experimental findings. The new state has a smaller intracellular cavity that is likely to accommodate β-arrestin better than G protein. Using contact map and inter-residue interaction energy calculations, we found the phosphorylated C-tail adheres to the cytosolic surface of the transmembrane domain of the receptor. Transfer entropy calculations show that the C-tail residues drive the correlated motions of TM residues, and the allosteric signal is relayed via several residues at the cytosolic surface. Our results also illustrate how the redistribution of inter-residue nonbonding interaction couples with the allosteric communication from the phosphorylated C-tail to the transmembrane. Atomistic insight into phosphorylation-induced β-arrestin specific conformation is therapeutically important to design drugs with higher efficacy and fewer side effects. Our results therefore open novel opportunities to fine-tune β-arrestin bias in GPCR signaling.

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Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ

Open Biology ◽

10.1098/rsob.200406 ◽

2021 ◽

Vol 11 (4) ◽

Author(s):

Evelyn Ploetz ◽

Gea K. Schuurman-Wolters ◽

Niels Zijlstra ◽

Amarins W. Jager ◽

Douglas A. Griffith ◽

...

Keyword(s):

Ligand Binding ◽

Single Molecule ◽

Conformational Changes ◽

Protein Interactions ◽

Transmembrane Domain ◽

Substrate Binding ◽

Titration Calorimetry ◽

Uptake System ◽

Conformational States ◽

Binding Domains

The ATP-binding cassette transporter GlnPQ is an essential uptake system that transports glutamine, glutamic acid and asparagine in Gram-positive bacteria. It features two extra-cytoplasmic substrate-binding domains (SBDs) that are linked in tandem to the transmembrane domain of the transporter. The two SBDs differ in their ligand specificities, binding affinities and their distance to the transmembrane domain. Here, we elucidate the effects of the tandem arrangement of the domains on the biochemical, biophysical and structural properties of the protein. For this, we determined the crystal structure of the ligand-free tandem SBD1-2 protein from Lactococcus lactis in the absence of the transporter and compared the tandem to the isolated SBDs. We also used isothermal titration calorimetry to determine the ligand-binding affinity of the SBDs and single-molecule Förster resonance energy transfer (smFRET) to relate ligand binding to conformational changes in each of the domains of the tandem. We show that substrate binding and conformational changes are not notably affected by the presence of the adjoining domain in the wild-type protein, and changes only occur when the linker between the domains is shortened. In a proof-of-concept experiment, we combine smFRET with protein-induced fluorescence enhancement (PIFE–FRET) and show that a decrease in SBD linker length is observed as a linear increase in donor-brightness for SBD2 while we can still monitor the conformational states (open/closed) of SBD1. These results demonstrate the feasibility of PIFE–FRET to monitor protein–protein interactions and conformational states simultaneously.

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Localization of a Substrate Binding Domain of the Human Reduced Folate Carrier to Transmembrane Domain 11 by Radioaffinity Labeling and Cysteine-substituted Accessibility Methods

Journal of Biological Chemistry ◽

10.1074/jbc.m507295200 ◽

2005 ◽

Vol 280 (43) ◽

pp. 36206-36213 ◽

Cited By ~ 26

Author(s):

Zhanjun Hou ◽

Sarah E. Stapels ◽

Christina L. Haska ◽

Larry H. Matherly

Keyword(s):

Hela Cells ◽

Mammalian Cells ◽

Transmembrane Domain ◽

Substrate Binding ◽

Binding Domain ◽

Reduced Folate Carrier ◽

Western Blots ◽

Transmembrane Channel ◽

Functional Components ◽

Substrate Binding Domain

The human reduced folate carrier (hRFC) mediates the membrane transport of reduced folates and classical anti-folates into mammalian cells. RFC is characterized by 12 transmembrane domains (TMDs), internally oriented N and C termini, and a large central linker connecting TMDs 1–6 and 7–12. By co-expression and N-hydroxysuccinimide methotrexate (Mtx) radioaffinity labeling of hRFC TMD 1–6 and TMD 7–12 half-molecules, combined with endoproteinase GluC digestion, a substrate binding domain was previously localized to within TMDs 8–12 (Witt, T. L., Stapels, S. E., and Matherly, L. H. (2004) J. Biol. Chem. 279, 46755–46763). In this report, this region was further refined to TMDs 11–12 by digestion with 2-nitro-5-thiocyanatobenzoic acid. A transportcompetent cysteine-less hRFC was used as a template to prepare single cysteine-replacement mutant constructs in which each residue from Glu-394 to Asp-420 of TMD 11 and Tyr-435 to His-457 of TMD 12 was replaced individually by a cysteine. The mutant constructs were transfected into hRFC-null HeLa cells. Most of the 50 single cysteine-substituted constructs were expressed at high levels on Western blots. With the exception of G401C hRFC, all mutants were active for Mtx transport. Treatment with sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) had no effect on hRFC activity for all of the cysteine mutants within TMD 12 and for the majority of the cysteine mutants within TMD 11. However, MTSES inhibited Mtx uptake by the T404C, A407C, T408C, T412C, F416C, I417C, V418C, and S419C mutants by 25–65%. Losses of activity by MTSES treatment for T404C, A407C, T412C, and I417C hRFCs were appreciably reversed in the presence of excess leucovorin, a hRFC substrate. Our results strongly suggest that residues within TMD 11 are likely critical structural and/or functional components of the putative hRFC transmembrane channel for anionic folate and anti-folate substrates.

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Covalently Linked Trimer of the AcrB Multidrug Efflux Pump Provides Support for the Functional Rotating Mechanism

Journal of Bacteriology ◽

10.1128/jb.01441-08 ◽

2008 ◽

Vol 191 (6) ◽

pp. 1729-1737 ◽

Cited By ~ 89

Author(s):

Yumiko Takatsuka ◽

Hiroshi Nikaido

Keyword(s):

Conformational Changes ◽

Transmembrane Domain ◽

Efflux Pump ◽

Polyacrylamide Gel Electrophoresis ◽

Residual Activity ◽

Salt Bridge ◽

Relay Network ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Intact Cells

ABSTRACT Escherichia coli AcrB is a proton motive force-dependent multidrug efflux transporter that recognizes multiple toxic chemicals having diverse structures. Recent crystallographic studies of the asymmetric trimer of AcrB suggest that each protomer in the trimeric assembly goes through a cycle of conformational changes during drug export (functional rotation hypothesis). In this study, we devised a way to test this hypothesis by creating a giant gene in which three acrB sequences were connected together through short linker sequences. The “linked-trimer” AcrB was expressed well in the inner membrane fraction of ΔacrB ΔrecA strains, as a large protein of ∼300 kDa which migrated at the same rate as the wild-type AcrB trimer in native polyacrylamide gel electrophoresis. The strain expressing the linked-trimer AcrB showed resistance to some toxic compounds that was sometimes even higher than that of the cells expressing the monomeric AcrB, indicating that the linked trimer functions well in intact cells. When we inactivated only one of the three protomeric units in the linked trimer, either with mutations in the salt bridge/H-bonding network (proton relay network) in the transmembrane domain or by disulfide cross-linking of the external cleft in the periplasmic domain, the entire trimeric complex was inactivated. However, some residual activity was seen, presumably as a result of random recombination of monomeric fragments (produced by protease cleavage or by transcriptional/translational truncation). These observations provide strong biochemical evidence for the functionally rotating mechanism of AcrB pump action. The linked trimer will be useful for further biochemical studies of mechanisms of transport in the future.

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UV photoaffinity labeling of Tn3 transposase–DNA complexes: identification of DNA binding domains

Canadian Journal of Microbiology ◽

10.1139/m96-007 ◽

1996 ◽

Vol 42 (1) ◽

pp. 46-59

Author(s):

Geoffrey S. Gottlieb ◽

Michael A. Fennewald

Keyword(s):

Amino Acids ◽

Dna Binding ◽

Specific Binding ◽

Binding Domain ◽

Target Site ◽

Site Directed Mutagenesis ◽

Cross Linking ◽

Dna Binding Domain ◽

Transposition Frequency ◽

Terminal Domain

The prokaryotic transposon Tn3 requires the transposase protein, as well as the cis-acting terminal inverted repeats (IRs), for transposition. The first step in the transposition process requires transposase binding to the IRs, as well as target site selection for element insertion. The primary aim of this study is to define the relationship between the structure of Tru3 transposase and its DNA binding functions. We have defined, by UV cross-linking, two broad regions of transposase that interact with DNA: a 70-kDa N-terminal domain and a 30-kDa C-terminal domain. The 70-kDa N-terminal domain encompasses the IR sequence-specific binding domain, as well as a nonspecific DNA binding domain that has been previously described. We have also defined, by UV cross-linking, a region in the nonspecific DNA binding domain centered at amino acids 376 and 381 that is in contact with DNA. We have used site-directed mutagenesis of amino acids 376 and 381 to help delineate the function of this region of the transposase protein. Mutations in this region reduce transposition frequency to 30–40% of the wild type. These mutations reduce nonspecific DNA binding three- to four-fold but do not appear to affect specific binding to the IR. Transposition immunity is unaffected by mutations in the nonspecific DNA binding domain. This suggests that this region may be involved in target site selection.Key words: transposon, Tn3, DNA–protein cross-linking, UV cross-linking, transposase.

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Identification of Interactions in the E1E2 Heterodimer of Hepatitis C Virus Important for Cell Entry

Journal of Biological Chemistry ◽

10.1074/jbc.m110.213942 ◽

2011 ◽

Vol 286 (27) ◽

pp. 23865-23876 ◽

Cited By ~ 25

Author(s):

Guillemette Maurin ◽

Judith Fresquet ◽

Ophélia Granio ◽

Czeslaw Wychowski ◽

François-Loïc Cosset ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Conformational Changes ◽

Transmembrane Domain ◽

Cell Entry ◽

Site Directed Mutagenesis ◽

Terminal Part ◽

Conserved Domains ◽

Biochemical Analyses

Several conserved domains critical for E1E2 assembly and hepatitis C virus entry have been identified in E1 and E2 envelope glycoproteins. However, the role of less conserved domains involved in cross-talk between either glycoprotein must be defined to fully understand how E1E2 undergoes conformational changes during cell entry. To characterize such domains and to identify their functional partners, we analyzed a set of intergenotypic E1E2 heterodimers derived from E1 and E2 of different genotypes. The infectivity of virions indicated that Con1 E1 did not form functional heterodimers when associated with E2 from H77. Biochemical analyses demonstrated that the reduced infectivity was not related to alteration of conformation and incorporation of Con1 E1/H77 E2 heterodimers but rather to cell entry defects. Thus, we generated chimeric E1E2 glycoproteins by exchanging different domains of each protein in order to restore functional heterodimers. We found that both the ectodomain and transmembrane domain of E1 influenced infectivity. Site-directed mutagenesis highlighted the role of amino acids 359, 373, and 375 in transmembrane domain in entry. In addition, we identified one domain involved in entry within the N-terminal part of E1, and we isolated a motif at position 219 that is critical for H77 function. Interestingly, using additional chimeric E1E2 complexes harboring substitutions in this motif, we found that the transmembrane domain of E1 acts as a partner of this motif. Therefore, we characterized domains of E1 and E2 that have co-evolved inside a given genotype to optimize their interactions and allow efficient entry.

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Cross-linking of bovine rhodopsin with sulfosuccinimidyl 4-(N maleimidomethyl)cyclohexane-1-carboxylate affects its functionality

Biochemical Journal ◽

10.1042/bcj20200376 ◽

2020 ◽

Vol 477 (12) ◽

pp. 2295-2312

Author(s):

Rafael Medina ◽

Deisy Perdomo ◽

Carolina Möller ◽

José Bubis

Keyword(s):

Conformational Changes ◽

Polyacrylamide Gel Electrophoresis ◽

Three Dimensional ◽

Binding Activity ◽

Dimensional Structure ◽

Cross Linking ◽

Bovine Rhodopsin ◽

Rhodopsin Kinase ◽

Cross Links ◽

Guanine Nucleotide Binding

Rhodopsin is the photoreceptor protein involved in visual excitation in retinal rods. The functionality of bovine rhodopsin was determined following treatment with sulfosuccinimidyl 4-(N maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), a bifunctional reagent capable of forming covalent cross-links between suitable placed lysines and cysteines. Denaturing polyacrylamide gel electrophoresis showed that rhodopsin incubated with sulfo-SMCC generated intermolecular dimers, trimers, and higher oligomers, although most of the sulfo-SMCC-treated protein remained as a monomer. Minor alterations on the absorption spectrum of light-activated sulfo-SMCC-treated rhodopsin were observed. However, only ∼2% stimulation of the guanine nucleotide binding activity of transducin was measured in the presence of sulfo-SMCC-cross-linked photolyzed rhodopsin. Moreover, rhodopsin kinase was not able of phosphorylating sulfo-SMCC-cross-linked rhodopsin after illumination. Rhodopsin was purified in the presence of either 0.1% or 1% n-dodecyl β-d-maltoside, to obtain dimeric and monomeric forms of the protein, respectively. Interestingly, no generation of the regular F1 and F2 thermolytic fragments was perceived with sulfo-SMCC-cross-linked rhodopsin either in the dimeric or monomeric state, implying the formation of intramolecular connections in the protein that might thwart the light-induced conformational changes required for interaction with transducin and rhodopsin kinase. Structural analysis of the rhodopsin three-dimensional structure suggested that the following lysine and cysteine pairs: Lys66/Lys67 and Cys316, Cys140 and Lys141, Cys140 and Lys248, Lys311 and Cys316, and/or Cys316 and Lys325 are potential candidates to generate intramolecular cross-links in the protein. Yet, the lack of fragmentation of sulfo-SMCC-treated Rho with thermolysin is consistent with the formation of cross-linking bridges between Lys66/Lys67 and Cys316, and/or Cys140 and Lys248.

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