Showing papers on "Conformational change published in 1997"
TL;DR: A clear definition of the binding site will provide a structural basis for developing heparin analogues that are more specific toward their intended target antithrombin and therefore less likely to exhibit side effects.
Abstract: Antithrombin, a plasma serpin, is relatively inactive as an inhibitor of the coagulation proteases until it binds to the heparan side chains that line the microvasculature. The binding specifically occurs to a core pentasaccharide present both in the heparans and in their therapeutic derivative heparin. The accompanying conformational change of antithrombin is revealed in a 2.9-A structure of a dimer of latent and active antithrombins, each in complex with the high-affinity pentasaccharide. Inhibitory activation results from a shift in the main sheet of the molecule from a partially six-stranded to a five-stranded form, with extrusion of the reactive center loop to give a more exposed orientation. There is a tilting and elongation of helix D with the formation of a 2-turn helix P between the C and D helices. Concomitant conformational changes at the heparin binding site explain both the initial tight binding of antithrombin to the heparans and the subsequent release of the antithrombin–protease complex into the circulation. The pentasaccharide binds by hydrogen bonding of its sulfates and carboxylates to Arg-129 and Lys-125 in the D-helix, to Arg-46 and Arg-47 in the A-helix, to Lys-114 and Glu-113 in the P-helix, and to Lys-11 and Arg-13 in a cleft formed by the amino terminus. This clear definition of the binding site will provide a structural basis for developing heparin analogues that are more specific toward their intended target antithrombin and therefore less likely to exhibit side effects.
643 citations
TL;DR: In this paper, the authors demonstrate that the fusogenic structure is formed by the destabilization of hemagglutinin (HA) at neutral pH, with either heat or denaturant urea, triggering a conformational change that is biochemically indistinguishable from the change triggered by low pH.
Abstract: Enveloped viruses enter cells by protein-mediated membrane fusion For influenza virus, membrane fusion is regulated by the conformational state of the hemagglutinin (HA) protein, which switches from a native (nonfusogenic) structure to a fusion-active (fusogenic) conformation when exposed to the acidic environment of the cellular endosome Here we demonstrate that destabilization of HA at neutral pH, with either heat or the denaturant urea, triggers a conformational change that is biochemically indistinguishable from the change triggered by low pH In each case, the conformational change is coincident with induction of membrane-fusion activity, providing strong evidence that the fusogenic structure is formed These results indicate that the native structure of HA is trapped in a metastable state and that the fusogenic conformation is released by destabilization of native structure This strategy may be shared by other enveloped viruses, including those that enter the cell at neutral pH, and could have implications for understanding the membrane-fusion step of HIV infection
398 citations
TL;DR: Findings suggest that IANBD bound to 125Cys and 285Cys are exposed to a more polar environment upon agonist binding, and indicate that movements of transmembrane segments III and VI are involved in activation of G protein‐coupled receptors.
Abstract: Agonist binding to G protein-coupled receptors is believed to promote a conformational change that leads to the formation of the active receptor state. However, the character of this conformational change which provides the important link between agonist binding and G protein coupling is not known. Here we report evidence that agonist binding to the beta2 adrenoceptor induces a conformational change around 125Cys in transmembrane domain (TM) III and around 285Cys in TM VI. A series of mutant beta2 adrenoceptors with a limited number of cysteines available for chemical derivatization were purified, site-selectively labeled with the conformationally sensitive, cysteine-reactive fluorophore IANBD and analyzed by fluorescence spectroscopy. Like the wild-type receptor, mutant receptors containing 125Cys and/or 285Cys showed an agonist-induced decrease in fluorescence, while no agonist-induced response was observed in a receptor where these two cysteines were mutated. These data suggest that IANBD bound to 125Cys and 285Cys are exposed to a more polar environment upon agonist binding, and indicate that movements of transmembrane segments III and VI are involved in activation of G protein-coupled receptors.
387 citations
TL;DR: The crystal structure of an mRNA capping enzyme is solved at 2.5 A resolution and provides direct evidence for a mechanism that involves a significant conformational change in the enzyme during catalysis.
242 citations
TL;DR: The ability of the AAPbeta peptides to disrupt and/or aggregate phospholipid vesicles were found to be mediated predominantly through electrostatic interactions with the phospholIPid headgroup, which has implications for AAPbeta toxicity and senile-plaque formation.
Abstract: Increasing evidence suggests that Alzheimer beta-amyloid peptides (AAPbeta) may be toxic agents in Alzheimer disease. We investigated the possibility that the toxicity may be the result of peptide-lipid interactions, involving either the cell membrane or the intracellular vesicular system. The interaction of the AAPbeta-(1-40), AAPbeta-(1-42), AAPbeta-(9-25) and AAPbeta-(25-35)-peptides with acidic and zwitterionic phospholipids was investigated by means of circular dichroism, vesicle disruption and lipid-aggregation assays. These studies were undertaken at peptide concentrations approaching in vivo levels and at physiological salt concentrations. Circular-dichroism studies demonstrate that acidic phospholipids induce a conformational change from random coil to beta structure in AAPbeta-(1-40)-peptide and AAPbeta-(1-42)-peptide at pH 6.0. In contrast, at pH 7.0, only AAPbeta-(1-42)-peptide was induced to adopt beta structure. Phosphatidylinositol was the most efficient inducer of beta structure in AAPbeta-(1-42)-peptide. To further investigate the peptide-lipid interactions, we examined the ability of the AAPbeta peptides to disrupt and/or aggregate phospholipid vesicles. These properties were found to be mediated predominantly through electrostatic interactions with the phospholipid headgroup. The data presented in this paper have implications for AAPbeta toxicity and senile-plaque formation.
220 citations
TL;DR: The observed hydrogen bonding of these residues to the body of the molecule in the latent form explains the mechanism for the release of newly formed antithrombin-protease complexes into the circulation for catabolic removal.
187 citations
TL;DR: Hydrogen bond exchange between the protein and solvent molecules is found to be important in the transition of conformational changes in ras p21, and a transient hydrogen bonding complex between Arg-68 and Tyr-71 in the switch II region and Glu-37 in switch I region stabilizes the intermediate conformation of alpha2.
Abstract: Conformational changes in ras p21 triggered by the hydrolysis of GTP play an essential role in the signal transduction pathway. The path for the conformational change is determined by molecular dynamics simulation with a holonomic constraint directing the system from the known GTP-bound structure (with the γ-phosphate removed) to the GDP-bound structure. The simulation is done with a shell of water molecules surrounding the protein. In the switch I region, the side chain of Tyr-32, which undergoes a large displacement, moves through the space between loop 2 and the rest of the protein, rather than on the outside of the protein. As a result, the charged residues Glu-31 and Asp-33, which interact with Raf in the homologous RafRBD–Raps complex, remain exposed during the transition. In the switch II region, the conformational changes of α2 and loop 4 are strongly coupled. A transient hydrogen bonding complex between Arg-68 and Tyr-71 in the switch II region and Glu-37 in switch I region stabilizes the intermediate conformation of α2 and facilitates the unwinding of a helical turn of α2 (residues 66–69), which in turn permits the larger scale motion of loop 4. Hydrogen bond exchange between the protein and solvent molecules is found to be important in the transition. Possible functional implications of the results are discussed.
159 citations
01 Feb 1997
TL;DR: Analysis of trans-activation by members of the steroid and thyroid hormone receptor superfamily identifies a unique form of allosteric control, the phantom ligand effect, which mimics exactly the effects observed when hormone is bound to RAR.
Abstract: Regulation of gene expression via allosteric control of transcription is one of the fundamental concepts of molecular biology. Studies in prokaryotes have illustrated that binding of small molecules or ligands to sequence-specific transcription factors can produce conformational changes at a distance from the binding site. These ligand-induced changes can dramatically alter the DNA binding and/or trans-activation abilities of the target transcription factors. In this work, analysis of trans-activation by members of the steroid and thyroid hormone receptor superfamily identifies a unique form of allosteric control, the phantom ligand effect. Binding of a novel ligand (LG100754) to one subunit (RXR) of a heterodimeric transcription factor results in a linked conformational change in the second noncovalently bound subunit of the heterodimer (RAR). This conformational change results in both the dissociation of corepressors and association of coactivators in a fashion mediated by the activation function of the non-liganded subunit. Without occupying the RAR hormone binding pocket, binding of LG100754 to RXR mimics exactly the effects observed when hormone is bound to RAR. Thus, LG100754 behaves as a phantom ligand.
134 citations
TL;DR: The studies suggest a molecular mechanism for cell binding to collagen fibers based on a conformational transition in collagen molecules on the fiber surface because of their location in an anisotropic environment.
Abstract: In its physiological solid state, type I collagen serves as a host for many types of cells. Only the molecules on fiber surface are available for interaction. In this interfacial environment, the conformation of a cell binding domain can be expected to fluctuate between the collagen fold and a distinctive non-collagen molecular marker for recognition and allosteric binding. If the cell binding domain can be localized in contiguous residues within the exposed half of a turn of the triple helix (approximately 15 residues), the need for extensive structural modification and unraveling of the triple helix is avoided. We examined the conformational preferences and biological activity of a synthetic 15-residue peptide (P-15), analogous to the sequence 766GTPGPQGIAGQRGVV780 in the alpha 1 (I) chain. Theoretical studies showed a high potential for a stable beta-bend for the central GIAG sequence. The flanking sequences showed facile transition to extended conformations. Circular dichroism of the synthetic peptide in anisotropic solvents confirmed the presence of beta-strand and beta-bend structures. P-15 inhibited fibroblast binding to collagen in a concentration dependent manner, with near maximal inhibition occurring at a concentration of 7.2 x 10(-6) M. The temporal pattern of cell attachment was altered markedly in the presence of P-15. No inhibition was seen with a peptide P-15(AI), an analogue of P-15 with the central IA residues reversed to AI or with collagen-related peptides (Pro-Pro-Gly)10, (Pro-Hyp-Gly)10, and polyproline, and with several unrelated peptides. Our studies suggest a molecular mechanism for cell binding to collagen fibers based on a conformational transition in collagen molecules on the fiber surface. Since the energy barrier between the collagen fold and beta-strand conformation is low, a local conformational change may be possible in molecules on the fiber surface because of their location in an anisotropic environment. Our observations also suggest that the sequence incorporated in P-15 may be a specific ligand for cells. Unlike other reported cell binding peptides, the residues involved in this interaction are non-polar.
126 citations
TL;DR: Differences in the low-frequency modes of the GTP and GDP-bound forms appear to play a role in ligand binding and the oncogenic mutation G12D leads to a global increase in the rigidity of the protein.
123 citations
TL;DR: The C2 domain is a Ca2+-dependent, membrane-targeting motif originally discovered in protein kinase C and recently identified in numerous eukaryotic signal-transducing proteins, including cytosolic phospholipase A2 (cPLA2) of the vertebrate inflammation pathway.
Abstract: The C2 domain is a Ca2+-dependent, membrane-targeting motif originally discovered in protein kinase C and recently identified in numerous eukaryotic signal-transducing proteins, including cytosolic phospholipase A2 (cPLA2) of the vertebrate inflammation pathway. Intracellular Ca2+ signals recruit the C2 domain of cPLA2 to cellular membranes where the enzymatic domain hydrolyzes specific lipids to release arachidonic acid, thereby initiating the inflammatory response. Equilibrium binding and stopped-flow kinetic experiments reveal that the C2 domain of human cPLA2 binds two Ca2+ ions with positive cooperativity, yielding a conformational change and membrane docking. When Ca2+ is removed, the two Ca2+ ions dissociate rapidly and virtually simultaneously from the isolated domain in solution. In contrast, the Ca2+-binding sites become occluded in the membrane-bound complex such that Ca2+ binding and dissociation are slowed. Dissociation of the two Ca2+ ions from the membrane-bound domain is an ordered sequential process, and release of the domain from the membrane is simultaneous with dissociation of the second ion. Thus, the Ca2+-signaling cycle of the C2 domain passes through an active, membrane-bound state possessing two occluded Ca2+ ions, one of which is essential for maintenance of the protein-membrane complex.
TL;DR: The polarographically measured activity of cytochrome c oxidase is lower after preincubation with high ATP/ADP-ratios than with low, in the presence of Tween 20, and this effect of nucleotides is due to interaction with subunit IV.
Abstract: By equilibrium dialysis of cytochrome c oxidase from bovine heart with [35S]ATPalphaS and [35S]ADPalphaS, seven binding sites for ATP and ten for ADP were determined per monomer of the isolated enzyme. The binding of ATP occurs in a time-dependent manner, as shown by a filtration method, which is apparently due to slow exchange of bound cholate. In the crystallized enzyme 10 mol of cholate were determined and partly identified in the high resolution crystal structure. Binding of ADP leads to conformational changes of the Tween 20-solubilized enzyme, as shown by a 12% decrease of the gamma-band. The conformational change is specific for ADP, since CDP, GDP and UDP showed no effects. The spectral changes are not obtained with the dodecylmaltoside solubilized enzyme. The polarographically measured activity of cytochrome c oxidase is lower after preincubation with high ATP/ADP-ratios than with low, in the presence of Tween 20. This effect of nucleotides is due to interaction with subunit IV, because preincubation of the enzyme with a monoclonal antibody to subunit IV released the inhibition by ATP. In the presence of dodecylmaltoside the enzyme had a 2 to 3-fold higher total activity, but this activity was not influenced by preincubation with ATP or ADP.
TL;DR: The solution structure of a mutant (E41A) of the regulatory domain of skeletal troponin C wherein one bidentate ligand to the calcium in site I is missing is solved, indicating that the linkage between calcium binding and the induced conformational change has been broken.
Abstract: The structural transition in troponin C induced by the binding of two calcium ions involves an “opening” of the structure, an event that triggers skeletal muscle contraction We have solved the solution structure of a mutant (E41A) of the regulatory domain of skeletal troponin C wherein one bidentate ligand to the calcium in site I is missing This structure remains “closed” upon calcium binding, indicating that the linkage between calcium binding and the induced conformational change has been broken This provides a snapshot of skeletal troponin C between the off and on state and thereby valuable insight into the mechanism of regulation within skeletal TnC Although several factors contribute to the triggering mechanism, the opening of the troponin C structure is ultimately dependent on one amino acid, Glu41 Insights into the structure of cardiac troponin C can also be derived from this skeletal mutant
TL;DR: A new structure-based inhibitor design search was begun which involved the recently refined crystal structure of the HA ectodomain, new insights into the conformational change, and improvements in the molecular docking program, DOCK, and as a result, new inhibitors of HA-mediated membrane fusion were identified.
Abstract: Past efforts to employ a structure-based approach to design an inhibitor of the fusion-inducing conformational change in the influenza virus hemagglutinin (HA) yielded a family of small benzoquinones and hydroquinones. The most potent of these, tert-butyl hydroquinone (TBHQ), inhibits both the conformational change in HA from strain X:31 influenza virus and viral infectivity in tissue culture cells with 50% inhibitory concentrations in the micromolar range (D. L. Bodian, R. B. Yamasaki, R. L. Buswell, J. F. Stearns, J. M. White, and I. D. Kuntz, Biochemistry 32:2967-2978, 1993). A new structure-based inhibitor design search was begun which involved (i) the recently refined crystal structure (2.1-A resolution) of the HA ectodomain, (ii) new insights into the conformational change, and (iii) improvements in the molecular docking program, DOCK. As a result, we identified new inhibitors of HA-mediated membrane fusion. Like TBHQ, most of these molecules inhibit the conformational change. One of the new compounds, however, facilitates rather than inhibits the HA conformational change. Nonetheless, the facilitator, diiodofluorescein, inhibits HA-mediated membrane fusion and, irreversibly, infectivity. We further characterized the effects of inhibitors from both searches on the conformational change and membrane fusion activity of HA as well as on viral infectivity. We also isolated and characterized several mutants resistant to each class of inhibitor. The implications of our results for HA-mediated membrane fusion, anti-influenza virus therapy, and structure-based inhibitor design are discussed.
TL;DR: It is suggested that the process of complex formation with alpha1-proteinase inhibitor converts trypsin D189S into an inactive, loose structure, which serves as a "conformational trap" of the enzyme that prevents catalytic deacylation.
Abstract: The binding of human R1-proteinase inhibitor to rat trypsin was shown by NMR spectroscopy to raise the pKaof His 57 in the active site but not to disrupt the hydrogen bond between His 57 and Asp 102 . Similar NMR results were observed for the Asp 189 to serine mutant of rat trypsin, which is much more stable than wild-type trypsin against autoproteolysis as the result of mutation of the residue at the base of the specificity pocket. This mutant was used in further studies aimed at determining the extent of the conformational transition in trypsin that accompanies serpin binding and leads to disruption of the catalytic activity of the proteinase such that the inhibitor complex is trapped at the acyl enzyme intermediate stage. The stability of rat trypsin toward thermal denaturation was found to be lower in the free enzyme than in the complex with R1-proteinase inhibitor. This suggests that the complex contains extensive protein - protein interactions that stabilize overall folding. On the other hand, previous investigations have shown that the proteinase in serpin-proteinase complexes becomes more susceptible to limited proteolysis, suggesting that the conformational change that accompanies binding leads to the exposure of susceptible loops in the enzyme. The existence of this type of conformational change upon complex formation has been confirmed here by investigation of the rate of cleavage of disulfide linkages by added dithiothreitol. This study revealed that, despite the increased stability of trypsin in the complex, one or more of its disulfide bridges becomes much more easily reduced. We suggest that the process of complex formation with R1-proteinase inhibitor converts trypsin D189S into an inactive, loose structure, which serves as a "conformational trap" of the enzyme that prevents catalytic deacylation. It is also proposed that plastic region(s) of the activation domain of trypsin may play a crucial role in this inhibitor-induced structural rearrangement.
TL;DR: Direct interaction of peroxisome proliferators and related compounds with, and the resulting conformational alteration(s) in, mPPARα may facilitate interaction of the receptor with transcriptional intermediary factors and/or the general transcription machinery and, thus, may underlie the molecular basis of ligand-dependent transcriptional activation mediated by m PPARα.
TL;DR: These and previous results suggest a model in which the Asn-135 oligosaccharide of alpha-antithrombin is oriented away from the heparin binding site and does not interfere with the first step of heParin binding.
Abstract: The beta-form of antithrombin, lacking a carbohydrate side chain on Asn-135, is known to bind heparin more tightly than the fully glycosylated alpha-form. The molecular basis for this difference in affinity was elucidated by rapid-kinetic studies of the binding of heparin and the antithrombin-binding heparin pentasaccharide to plasma and recombinant forms of alpha- and beta-antithrombin. The dissociation equilibrium constant for the first step of the two-step mechanism of binding of both heparin and pentasaccharide to alpha-antithrombin was only slightly higher than that for the binding to the beta-form. The oligosaccharide at Asn-135 thus at most moderately interferes with the initial, weak binding of heparin to alpha-antithrombin. In contrast, the rate constant for the conformational change induced by heparin and pentasaccharide in the second binding step was substantially lower for alpha-antithrombin than for beta-antithrombin. Moreover, the rate constant for the reversal of this conformational change was appreciably higher for the alpha-form than for the beta-form. The carbohydrate side chain at Asn-135 thus reduces the heparin affinity of alpha-antithrombin primarily by interfering with the heparin-induced conformational change. These and previous results suggest a model in which the Asn-135 oligosaccharide of alpha-antithrombin is oriented away from the heparin binding site and does not interfere with the first step of heparin binding. This initial binding induces conformational changes involving extension of helix D into the adjacent region containing Asn-135, which are transmitted to the reactive-bond loop. The resulting decreased conformational flexibility of the Asn-135 oligosaccharide and its close vicinity to the heparin binding site destabilize the activated relative to the native conformation. This effect results in a higher energy for inducing the activated conformation in alpha-antithrombin, leading to a decrease in heparin binding affinity.
TL;DR: A novel mechanism to explain the functional interaction of FGF-2 with heparin and its transmembrane receptor is proposed.
Abstract: Interaction of basic fibroblast growth factor (FGF-2) with heparin or heparan sulfate proteoglycans (HSPGs) is required for receptor activation and initiation of biological responses. To gain insight into the mechanism of activation of the FGF receptor by FGF-2 and heparin, we have used NMR, dynamic light scattering, and HSPG-deficient cells and cell-free systems. The first 28 N-terminal residues in FGF-2 were found to be highly mobile and flexible, consistent with the disorder found in both the NMR and X-ray structures. The structure of an FGF-2-heparin-decasaccharide complex that binds to and activates the FGF receptor was compared to a heparin-tetrasaccharide-induced complex that does not promote an interaction with the receptor. The major change observed upon the addition of the tetrasaccharide to FGF-2 was an increase in the correlation time consistent with the formation of an FGF-2 dimer. The NMR line widths of FGF-2 in the presence of the decasaccharide are severely broadened relative to the tetrasaccharide, consistent with dynamic light scattering results which indicate FGF-2 is a tetramer. The interaction of these heparin species with FGF-2 does not induce a significant conformational change in the overall structure of FGF-2, but small chemical shift changes are observed in both heparin and receptor binding sites. A trans-oriented symmetric dimer of FGF-2 is formed in the presence of the tetrasaccharide whereas two cis-oriented dimers in a symmetric tetramer are formed in the presence of the decasaccharide. This suggests that the cis-oriented FGF-2 dimer is the minimal biologically active structural unit of FGF-2. These data allow us to propose a novel mechanism to explain the functional interaction of FGF-2 with heparin and its transmembrane receptor.
TL;DR: The results could help to explain how a polymerase controls and achieves its fidelity with a multiple conformational change mechanism.
Abstract: Stopped-flow fluorescence assay was applied to identify conformational changes in the catalytic cycle of DNA polymerase β (Pol β), using a synthetic DNA primer/template containing 2-aminopurine (2-AP) at the template position opposite the incoming dNTP. Two phases of fluorescence change were observed in the stopped-flow fluorescence assay of the incorporation of the correct nucleotide dTTP. The rate of the slow phase corresponds to that of product formation. This slow phase was identified as the result of a rate-limiting conformational change step before chemistry because this slow phase was also observed with a dideoxynucleotide at the 3‘ end of the primer which prevents chemical bond formation. The fast phase was also attributed to a conformational change step since its dependence on [dTTP] is hyperbolic. The rates of the two phases and their dependence on [dTTP] and [Mg2+] suggest that the fast conformational change is induced by the binding of MgdNTP and the slow conformational change is induced by th...
TL;DR: Observations strongly suggest that this newly defined polyamine-binding domain is involved in the intrasteric regulation of CK2 activity.
TL;DR: A stereochemical mechanism for the open-to-closed transition that involves the electrostatic neutralization of two active site arginine residues by the negative charges of the incoming substrate, a large change in the backbone (φ,ψ) conformational angles of two key glycine residues, and the entropy-driven burial of a stretch of hydrophobic residues on the N-terminal helix is proposed.
TL;DR: The data indicate that the α subunit conformational change involving the cystine loop is key to a series of folding events that allow the addition of unassembled subunits.
TL;DR: A number of indirect observations are summarized that suggest the rate acceleration for the CC-1065 and duocarmycin is driven by a binding-induced twist in the linking N2 amide and requires a rigid extended N2Amide substituent, disrupts the vinylogous amide stabilization and activates the agents for DNA alkylation.
TL;DR: The interaction of heparin with the Ca2+- and phospholipid-binding protein annexin II tetramer (AIIt) was characterized and a region of the p36 subunit of AIIt was found to contain a Cardin-Weintraub consensus sequence for glycosaminoglycan recognition.
TL;DR: It appears that apo-HIV-1 IN exists predominantly in an inactive conformation that is converted into a catalytically competent form upon the addition of metal ions.
TL;DR: It appears that indirect perturbation of the Glu45 conformation via an altered quaternary structure may be a contributing factor to the decreased catalytic efficiency of T93A, and this mechanism may also explain the diminished activities of other active site variants of EcoRV.
TL;DR: Two distinct mechanisms by which the inhibitory activity of PAI-1 can be neutralized are demonstrated, which may have implications for the design of therapeutic or preventive strategies to interfere with PAi-1 activity.
TL;DR: It is suggested that L-antithrombin formation is a two-step process: an initial rapid conformational change, probably including partial incorporation of the reactive loop into the A-sheet (as in the active molecule in the crystal structure) and displacement of s1C from the C-beta-sheet which supports polymer formation, and a much slower transition to complete loop insertion within the A, beta-sheet.
Abstract: The inhibitory mechanism of the serpin family of serine protease inhibitors is characterized by a remarkable degree of conformational flexibility. Various conformational states have been elucidated by X-ray crystallography and indicate that the inhibitory loop, the central A-beta-sheet, and the outside edge of the C-beta-sheet are particularly mobile. However, no crystal structure of a serpin-enzyme complex is yet available, and the likely nature of the protease-complexed serpin remains for biochemical and biophysical researchers to examine. Here, we show that the biochemical induction of the latent state of antithrombin is slow relative to polymer formation, and infer that this may reflect structural features that are important for the regulation of the initial docking and subsequent locking of serpins with cognate proteases. L-Antithrombin was induced by incubation of native antithrombin at 60 degrees C for 10 h in the presence of citrate to prevent polymerization. L-Antithrombin was more stable to denaturation by both heat and urea than native antithrombin. Whereas native antithrombin formed binary complexes with synthetic peptide homologues of the inhibitory loop, biochemically induced L-antithrombin did not, indicating that the inhibitory loop of L-antithrombin is probably fully inserted into the A-beta-sheet as in the crystal structure. This was confirmed by limited proteolysis studies which demonstrated that the inhibitory loop of L-antithrombin could not be cleaved by five proteases which do cleave the loop of native antithrombin. The limited proteolysis studies also indicated that the "gate" region (residues 236-248) of the biochemically induced L-antithrombin was in a conformation substantially different from that of the native antithrombin. This again is similar to L-antithrombin in the crystal structure in which the gate has "opened" away from the body of the molecule by a rotation of 24 degrees to facilitate the relocation of strand 1C from its ordered position in the C-beta-sheet to a disordered surface loop. At 60 degrees C in the absence of citrate, antithrombin (and other serpins) rapidly polymerizes. In the presence of citrate, the formation of L-antithrombin is slow and increases with time, indicating that the inhibition of polymer formation by citrate allows the time necessary for the much slower formation of the L form. We therefore suggest that L-antithrombin formation is a two-step process: an initial rapid conformational change, probably including partial incorporation of the reactive loop into the A-sheet (as in the active molecule in the crystal structure) and displacement of s1C from the C-beta-sheet which supports polymer formation, and a much slower transition to complete loop insertion within the A-beta-sheet. It is likely that both the first rapid transitional step and the structural features that impose resistance to the second more extensive conformational change reflect the optimization of the unique inhibitory function in the serpins.
TL;DR: It is concluded that the conserved arginine and glutamate residues at the cytoplasmic surface of the glucose transporter GLUT4 are essential for its appropriate conformation, and that it is the interaction of charged residues which mediates the oscillation between outward and inward facing states.
Abstract: The role of conserved arginine and glutamic acid residues at the cytoplasmic surface of the GLUT4 for transporter function was investigated by site-directed mutagenesis and expression of the constructs in COS-7 cells. Reconstituted glucose transport activity, cytochalasin B binding, and photolabeling with the exofacial label 2-N4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(d-mannosyloxy)-2-propylamine (ATB-BMPA) was assayed in membranes from transfected cells and corrected for immunoreactivity of expressed transporters. Exchange of Arg 92 (R92L amino acid residues are numbered according to the corresponding residues in the GLUT1) or Arg 333/334 (RR333/4LA) reduced or suppressed transport activity with no or very little effect on photolabeling with ATB-BMPA and cytochalasin B binding. It is suggested that the lack of these residues selectively disturbes the substrate-induced conformational change of the carrier during transport. Exchange of Glu 146 (E146D) or Arg 153 (R153L) markedly reduced transport acti...