Structure of human thioredoxin exhibits a large conformational change

Thioredoxin is an oxidoreductase, which is ubiquitously present across phyla from humans to plants and bacteria. Thioredoxin reduces a variety of substrates through active site Cys 32, which is subsequently oxidized to form the intramolecular disulphide with Cys 35. The thioredoxin fold is known to be highly stable and conformational changes in the active site loops and residues Cys 32, Cys 35 have been characterized between ligand bound and free structures. We have determined a novel 2.0 Å resolution crystal structure for a human thioredoxin, which reveals a much larger conformational change than previously characterized. The principal change involves unraveling of a helix to form an extended loop that is linked to secondary changes in further loop regions and the wider area of the active site Cys 32. This gives rise to a more open conformation and an elongated hydrophobic pocket results in place of the helix. Buried residue Cys 62 from this helix becomes exposed in the open conformation. This provides a structural basis for observations that the Cys 62 sidechain can form mixed disulphides and be modified by thiol reactive small molecules.     

A major conformational change in p97 AAA ATPase upon ATP binding

AAA ATPases play central roles in cellular activities. The ATPase p97, a prototype of this superfamily, participates in organelle membrane fusion. Cryoelectron microscopy and single-particle analysis revealed that a major conformational change of p97 during the ATPase cycle occurred upon nucleotide binding and not during hydrolysis as previously hypothesized. Furthermore, our study indicates that six p47 adaptor molecules bind to the periphery of the ring-shaped p97 hexamer. Taken together, these results provide a revised model of how this and possibly other AAA ATPases can translate nucleotide binding into conformational changes of associated binding partners.     

Cytochrome c oxidase exhibits a rapid conformational change upon reduction of CuA: a tryptophan fluorescence study

When cytochrome c oxidase is reduced, it undergoes a conformational change that shifts its tryptophan fluorescence maximum from 329 to 345 nm. Studies of ligand-bound, mixed-valence forms of the enzyme show that this conformational change is dependent on the redox state of the low-potential metal centers, cytochrome a and CuA. The intrinsic fluorescence of oxidized cytochrome c oxidase is not effectively quenched by Cs+; however, marked quenching is observed for the reduced enzyme with a Stern-Volmer constant of 0.69. These observations, together with the significant red shift of the emission maximum, suggest that the emitting tryptophan residues are becoming more solvent accessible in the reduced enzyme. Stopped-flow spectra show that this conformational transition occurs rapidly upon reduction of the low-potential sites with a pseudo-first-order rate constant of 4.07 +/- 0.40 s-1. The conformational change monitored by tryptophan fluorescence is suggested to be related to the previously proposed "open-closed" transition of cytochrome c oxidase. Reductive titration of the cyanide-inhibited enzyme with ferrocytochrome c shows a nonlinear response of the fluorescence shift to added electron equivalents. A theoretical treatment of the reduction of the two interacting sites of the cyanide-inhibited enzyme has been developed that gives the population of each redox state as a function of the total number of electrons accepted by the enzyme. This treatment depends on two parameters: the difference in redox potential between the two metals and the redox interaction between the redox centers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Yeast hexokinase in solution exhibits a large conformational change upon binding glucose or glucose 6-phosphate.

Using small-angle X-ray scattering from solutions of yeast hexokinase, we have measured the radii of gyration of the monomeric B isozyme and its complexes with sugar substrates. We find that the radius of gyration decreases by 0.95 +/- 0.24 A upon binding glucose and 1.25 +/- 0.28 A upon binding glucose 6-phosphate. This observed reduction in radius of gyration in the presence of glucose is the same as that calculated from the coordinates of the high-resolution crystal structures of native hexokinase B and a glucose complex with hexokinase A. Thus, these measurements suggest that the dramatic closing of the slit between the two lobes of hexokinase observed in the crystal structures (Bennett, W.S., & Steitz, T.A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4848--4852) occurs in solution when either glucose or glucose 6-phosphate is bound.     

Fluorescence-monitored conformational change on the 3'-end of tRNA upon aminoacylation.

Fluorescent tRNAs species with formycine in the 3'-terminal position (tRNA-CCF) were derived from Escherichia coli tRNA(Val). Thermus thermophilus tRNA(Aap) and Thermus thermophilus tRNA(Phe). The fluorescence of formycine was used to monitor the conformational changes at the 3'-terminus of tRNA caused by aminoacylation and hydrolysis of aminoacyl residue from aminoacyl-tRNAs. An increase of about 15% in the fluorescence intensity was observed after aminoacylation of the three tRNA-CCF. This change in fluorescence amplitude that is reversed by hydrolysis of the aminoacyl residue, does not depend on the structure of the amino acid or tRNA sequence. A local conformational change at the 3'-terminal formycine probably involving a partial destacking of the base moiety in the ACCF end takes place as a consequence of aminoacylation. A structural change at the 3'-terminus of tRNA induced by attachment and detachment of the acyl residue may be important in controlling the substrate/product relationship in reactions in which tRNA participates during protein biosynthesis.     

Grb2 adaptor undergoes conformational change upon dimerization

Grb2 is an adaptor protein that couples activated receptor tyrosine kinases to downstream effector molecules such as Ras and Akt. Despite being a central player in mitogenic signaling and a target for therapeutic intervention, the role of Grb2 oligomerization in cellular signaling is not well understood. Here, using the techniques of size-exclusion chromatography, mass spectrometry, analytical ultra-centrifugation and isothermal titration calorimetry, we demonstrate that Grb2 exists in monomer-dimer equilibrium in solution and that the dissociation of dimer into monomers is entropically-driven without an unfavorable enthalpic change at physiological temperatures. Our data indicate that enthalpy and entropy of dimer dissociation are highly temperature-dependent and largely compensate each other resulting in negligible effect of temperature on the overall free energy. From the plot of enthalpy change versus temperature, the magnitude of heat capacity change derived is much smaller than that expected from the rather large molecular surfaces becoming solvent-occluded upon Grb2 dimerization, implying that Grb2 monomers undergo conformational rearrangement upon dimerization. 3D structural models of Grb2 dimer and monomers suggest strongly that such conformational rearrangement upon dimerization may arise from domain swapping. Taken together, our study provides novel insights into the role of Grb2 as an adaptor in cellular signaling circuitry and how Grb2 dimerization may impart high fidelity in signal transduction as well as lead to rapid signal amplification upon receptor stimulation.     

Transglutaminase 2 undergoes a large conformational change upon activation

Human transglutaminase 2 (TG2), a member of a large family of enzymes that catalyze protein crosslinking, plays an important role in the extracellular matrix biology of many tissues and is implicated in the gluten-induced pathogenesis of celiac sprue. Although vertebrate transglutaminases have been studied extensively, thus far all structurally characterized members of this family have been crystallized in conformations with inaccessible active sites. We have trapped human TG2 in complex with an inhibitor that mimics inflammatory gluten peptide substrates and have solved, at 2-Å resolution, its x-ray crystal structure. The inhibitor stabilizes TG2 in an extended conformation that is dramatically different from earlier transglutaminase structures. The active site is exposed, revealing that catalysis takes place in a tunnel, bridged by two tryptophan residues that separate acyl-donor from acyl-acceptor and stabilize the tetrahedral reaction intermediates. Site-directed mutagenesis was used to investigate the acyl-acceptor side of the tunnel, yielding mutants with a marked increase in preference for hydrolysis over transamidation. By providing the ability to visualize this activated conformer, our results create a foundation for understanding the catalytic as well as the non-catalytic roles of TG2 in biology, and for dissecting the process by which the autoantibody response to TG2 is induced in celiac sprue patients.     

Human plasminogen catalytic domain undergoes an unusual conformational change upon activation

Activation of the serine protease plasmin from its zymogen, plasminogen, is the key step in fibrinolysis leading to blood clot dissolution. It also plays critical roles in cell migration, such as in tumor metastasis. Here, we report the crystal structure of an inactive S741A mutant of human plasminogen catalytic domain at 2.0 A resolution. This structure permits a direct comparison with that of the plasmin catalytic unit. Unique conformational differences are present between these two structures that are not seen in other zymogen-enzyme pairs of the trypsin family. The functional significance of these differences and the structural basis of plasminogen activation is discussed in the light of this new structure.     

Deoxymyoglobin studied by the conformational normal mode analysis. II. The conformational change upon oxygenation

The conformational change taking place in myoglobin concomitantly with the observed geometrical change at the heme-His(F8) linkage upon oxygenation is studied by normal mode analysis, which is based on the quadratic approximation of the conformational energy function. The heme-globin interaction energy increases for this change by 8.114 kcal/mol when both the heme group and the globin molecule are held rigid. When they are permitted flexibility, the interaction energy relaxes by 7.038 kcal/mol, and the difference (1.076 kcal/mol) is distributed as strain energy within the molecule. This increase is the work necessary for the heme group to move against the force exerted by the globin. If the force is assumed to be invariable during this move, the work is small, 0.276 kcal/mol, meaning that the force is strongly variable. Furthermore, this means that the heme group is located near the equilibrium point of the potential energy of the heme-globin interaction. The change in the localized strain energy stored in the force field at the linkage between the heme and the imidazole of HisF8 is estimated to be of the same order of magnitude as the distributed energy. The largest atomic displacements are observed at the region from the F helix to the beginning of the G helix, and secondary large displacements occur at several regions, i.e, the A helix, from the C helix to the CD corner, the E helix, and the C-terminal side of the H helix. All of these regions have strong dynamic interactions with the heme group, either directly or indirectly. Their secondary structures show complex deformations. In other parts, relatively rigid segments undergo rotational and/or bending changes in a way consistent with the large changes described above and close atomic packing within the molecule. The calculated conformational change is decomposed to vibrational normal modes of deoxymyoglobin. The magnitude of the conformational change, measured by the mass-weighted mean-square atomic displacement, is accounted for up to 92.0% by the 151 normal modes with frequencies lower than 40 cm-1. In descending order of contribution, the first six modes, each of which has a frequency lower than 12 cm-1, account for up to 57.4%. This means that the functionally important conformational change can well be expressed in terms of a relatively small number of collective low frequency normal modes.     

Induced conformational change in human IL‐4 upon binding of a signal‐neutralizing DARPin

The crystal structure of DARPin 44C12V5 that neutralizes IL‐4 signaling has been determined alone and bound to human IL‐4. A significant conformational change occurs in the IL‐4 upon DARPin binding. The DARPin binds to the face of IL‐4 formed by the A and C α‐helices. The structure of the DARPin remains virtually unchanged. The conformational changes in IL‐4 include a reorientation of the C‐helix Trp91 side chain and repositioning of CD‐loop residue Leu96. Both side chains move by >9 Å, becoming buried in the central hydrophobic region of the IL‐4:DARPin interface. This hydrophobic region is surrounded by a ring of hydrophilic interactions comprised of hydrogen bonds and salt bridges and represents a classical “hotspot.” The structures also reveal how the DARPin neutralizes IL‐4 signaling. Comparing the IL‐4:DARPin complex structure with the structures of IL‐4 bound to its receptors (Hage et al., Cell 1999; 97 , 271‐281; La Porte et al., Cell 2008, 132, 259‐272), it is found that the DARPin binds to the same IL‐4 face that interacts with the junction of the D1 and D2 domains of the IL‐4Rα receptors. Signaling is blocked since IL‐4 cannot bind to this receptor, which it must do first before initiating a productive receptor complex with either the IL‐13α1 or the γ_c receptor. Proteins 2015; 83:1191–1197. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Localisation of the human hSuv3p helicase in the mitochondrial matrix and its preferential unwinding of dsDNA

We characterised the human hSuv3p protein belonging to the family of NTPases/helicases. In yeast mitochondria the hSUV3 orthologue is a component of the degradosome complex and participates in mtRNA turnover and processing, while in Caenorhabditis elegans the hSUV3 orthologue is necessary for viability of early embryos. Using immunofluorescence analysis, an in vitro mitochondrial uptake assay and sub-fractionation of human mitochondria we show hSuv3p to be a soluble protein localised in the mitochondrial matrix. We expressed and purified recombinant hSuv3p protein from a bacterial expression system. The purified enzyme was capable of hydrolysing ATP with a K_m of 41.9 µM and the activity was only modestly stimulated by polynucleotides. hSuv3p unwound partly hybridised dsRNA and dsDNA structures with a very strong preference for the latter. The presented analysis of the hSuv3p NTPase/helicase suggests that new functions of the protein have been acquired in the course of evolution.     

Stu2p binds tubulin and undergoes an open-to-closed conformational change

Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat-containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs.     

The first extracellular loop of the Saccharomyces cerevisiae G protein-coupled receptor Ste2p undergoes a conformational change upon ligand binding

In this study of the Saccharomyces cerevisiae G protein-coupled receptor Ste2p, we present data indicating that the first extracellular loop (EL1) of the alpha-factor receptor has tertiary structure that limits solvent accessibility and that its conformation changes in a ligand-dependent manner. The substituted cysteine accessibility method was used to probe the solvent exposure of single cysteine residues engineered to replace residues Tyr(101) through Gln(135) of EL1 in the presence and absence of the tridecapeptide alpha-factor and a receptor antagonist. Surprisingly, many residues, especially those at the N-terminal region, were not solvent-accessible, including residues of the binding-competent yet signal transduction-deficient mutants L102C, N105C, S108C, Y111C, and T114C. In striking contrast, two N-terminal residues, Y101C and Y106C, were readily solvent-accessible, but upon incubation with alpha-factor labeling was reduced, suggesting a pheromone-dependent conformational change limiting solvent accessibility had occurred. Labeling in the presence of the antagonist, which binds Ste2p but does not initiate signal transduction, did not significantly alter reactivity with the Y101C and Y106C receptors, suggesting that the alpha-factor-dependent decrease in solvent accessibility was not because of steric hindrance that prevented the labeling reagent access to these residues. Based on these and previous observations, we propose a model in which the N terminus of EL1 is structured such that parts of the loop are buried in a solvent-inaccessible environment interacting with the extracellular part of the transmembrane domain bundle. This study highlights the essential role of an extracellular loop in activation of a G protein-coupled receptor upon ligand binding.     

Purified Bacillus anthracis lethal toxin complex formed in vitro and during infection exhibits functional and biological activity

Anthrax protective antigen (PA, 83 kDa), a pore-forming protein, upon protease activation to 63 kDa (PA(63)), translocates lethal factor (LF) and edema factor (EF) from endosomes into the cytosol of the cell. The relatively small size of the heptameric PA(63) pore (approximately 12 angstroms) raises questions as to how large molecules such as LF and EF can move through the pore. In addition, the reported high binding affinity between PA and EF/LF suggests that EF/LF may not dissociate but remain complexed with activated PA(63). In this study, we found that purified (PA(63))(7)-LF complex exhibited biological and functional activities similar to the free LF. Purified LF complexed with PA(63) heptamer was able to cleave both a synthetic peptide substrate and endogenous mitogen-activated protein kinase kinase substrates and kill susceptible macrophage cells. Electrophysiological studies of the complex showed strong rectification of the ionic current at positive voltages, an effect similar to that observed if LF is added to the channels formed by heptameric PA(63) pore. Complexes of (PA(63))(7)-LF found in the plasma of infected animals showed functional activity. Identifying active complex in the blood of infected animals has important implications for therapeutic design, especially those directed against PA and LF. Our studies suggest that the individual toxin components and the complex must be considered as critical targets for anthrax therapeutics.     

Gamma-secretase activity is associated with a conformational change of nicastrin

Gamma-secretase is a high molecular weight multicomponent protein complex with an unusual intramembrane-cleaving aspartyl protease activity. Gamma-secretase is intimately associated with Alzheimer disease because it catalyzes the proteolytic cleavage, which leads to the liberation of amyloid beta-peptide. At least presenilin (PS), Nicastrin (Nct), APH-1, and PEN-2 are constituents of the gamma-secretase complex, with PS apparently providing the active site of gamma-secretase. Expression of gamma-secretase complex components is tightly regulated, however little is known about the assembly of the complex. Here we demonstrate that Nct undergoes a major conformational change during the assembly of the gamma-secretase complex. The conformational change is directly associated with gamma-secretase function and involves the entire Nct ectodomain. Loss of function mutations generated by deletions failed to undergo the conformational change. Furthermore, the conformational alteration did not occur in the absence of PS. Our data thus suggest that gamma-secretase function critically depends on the structural "activation" of Nct.