scholarly journals The first putative transmembrane helix of the 16 kDa proteolipid lines a pore in the Vo sector of the vacuolar H+-ATPase

1995 ◽  
Vol 312 (3) ◽  
pp. 739-747 ◽  
Author(s):  
P C Jones ◽  
M A Harrison ◽  
Y I Kim ◽  
M E Finbow ◽  
J B C Findlay
Keyword(s):  
Structural Model ◽  
Proton Translocation ◽  
Transmembrane Helix ◽  
Structural Features ◽  
Lipid Phase ◽  
Protein Chemistry ◽  
Alpha Helices ◽  
Multimeric Complex ◽  
Disulphide Bond Formation ◽  
Single Face

The 16 kDa proteolipid is the major component of the vacuolar H(+)-ATPase membrane sector, responsible for proton translocation. Expression of a related proteolipid from the arythropod Nephrops norvegicus in a Saccharomyces strain in which the VMA3 gene for the endogenous proteolipid has been disrupted results in restored vacuolar H(+)-ATPase function. We have used this complementation system, coupled to cysteine substitution mutagenesis and protein chemistry, to investigate structural features of the proteolipid. Consecutive cysteines were introduced individually into putative transmembrane segment 1 of the proteolipid, and at selected sites in extramembranous regions and in segment 3 and 4. Analysis of restored vacuolar H(+)-ATPase function showed that segment 1 residues sensitive to mutation to cysteine were clustered on a single face, but only if the segment was helical. Only residues insensitive to mutation could be covalently modified by the cysteine-specific reagent fluorescein 5-maleimide. A cysteine introduced into segment 3 was the only residue accessible to a relatively hydrophobic reagent, suggesting accessibility to the lipid phase. Analysis of disulphide bond formation between introduced cysteines indicates that the first transmembrane alpha-helices of each monomer are adjacent to each other at the centre of the proteolipid multimeric complex. The data are consistent with a model in which the fluorescein maleimide-accessible face of helix I lines a pore at the centre of a hexameric complex formed by the proteolipid, with the mutationally sensitive face oriented into the protein core. The implications for ion-transport function in this family of proteins are discussed in the context of this structural model.

scholarly journals Construction and validation of a homology model of the human voltage-gated proton channel hHV1

2013 ◽  
Vol 141 (4) ◽  
pp. 445-465 ◽  
Author(s):  
Kethika Kulleperuma ◽  
Susan M.E. Smith ◽  
Deri Morgan ◽  
Boris Musset ◽  
John Holyoake ◽  
...  
Keyword(s):  
Structural Model ◽  
Proton Translocation ◽  
External Solution ◽  
Homology Model ◽  
Structural Features ◽  
Proton Channel ◽  
Dimensional Structure ◽  
Internal Solution ◽  
Voltage Gated ◽  
Homology Models

The topological similarity of voltage-gated proton channels (HV1s) to the voltage-sensing domain (VSD) of other voltage-gated ion channels raises the central question of whether HV1s have a similar structure. We present the construction and validation of a homology model of the human HV1 (hHV1). Multiple structural alignment was used to construct structural models of the open (proton-conducting) state of hHV1 by exploiting the homology of hHV1 with VSDs of K+ and Na+ channels of known three-dimensional structure. The comparative assessment of structural stability of the homology models and their VSD templates was performed using massively repeated molecular dynamics simulations in which the proteins were allowed to relax from their initial conformation in an explicit membrane mimetic. The analysis of structural deviations from the initial conformation based on up to 125 repeats of 100-ns simulations for each system reveals structural features consistently retained in the homology models and leads to a consensus structural model for hHV1 in which well-defined external and internal salt-bridge networks stabilize the open state. The structural and electrostatic properties of this open-state model are compatible with proton translocation and offer an explanation for the reversal of charge selectivity in neutral mutants of Asp112. Furthermore, these structural properties are consistent with experimental accessibility data, providing a valuable basis for further structural and functional studies of hHV1. Each Arg residue in the S4 helix of hHV1 was replaced by His to test accessibility using Zn2+ as a probe. The two outermost Arg residues in S4 were accessible to external solution, whereas the innermost one was accessible only to the internal solution. Both modeling and experimental data indicate that in the open state, Arg211, the third Arg residue in the S4 helix in hHV1, remains accessible to the internal solution and is located near the charge transfer center, Phe150.

scholarly journals Residues within the Transmembrane Domain of the Glucagon-Like Peptide-1 Receptor Involved in Ligand Binding and Receptor Activation: Modelling the Ligand-Bound Receptor

2011 ◽  
Vol 25 (10) ◽  
pp. 1804-1818 ◽  
Author(s):  
K. Coopman ◽  
R. Wallis ◽  
G. Robb ◽  
A. J. H. Brown ◽  
G. F. Wilkinson ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Structural Model ◽  
Transmembrane Domain ◽  
Transmembrane Helix ◽  
Receptor Activation ◽  
Agonist Affinity ◽  
Camp Generation ◽  
N Terminus ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1

The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9–39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9–39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues.

scholarly journals Structural characterization of the N-terminal part of the MERS-CoV nucleocapsid by X-ray diffraction and small-angle X-ray scattering

2016 ◽  
Vol 72 (2) ◽  
pp. 192-202 ◽  
Author(s):  
Nicolas Papageorgiou ◽  
Julie Lichière ◽  
Amal Baklouti ◽  
François Ferron ◽  
Marion Sévajol ◽  
...  
Keyword(s):  
Structural Model ◽  
Structural Features ◽  
Terminal Region ◽  
X Ray Diffraction ◽  
Terminal Part ◽  
Multifunctional Protein ◽  
X Ray ◽  
N Protein ◽  
Intrinsically Disordered ◽  
X Ray Scattering

The N protein of coronaviruses is a multifunctional protein that is organized into several domains. The N-terminal part is composed of an intrinsically disordered region (IDR) followed by a structured domain called the N-terminal domain (NTD). In this study, the structure determination of the N-terminal region of the MERS-CoV N proteinviaX-ray diffraction measurements is reported at a resolution of 2.4 Å. Since the first 30 amino acids were not resolved by X-ray diffraction, the structural study was completed by a SAXS experiment to propose a structural model including the IDR. This model presents the N-terminal region of the MERS-CoV as a monomer that displays structural features in common with other coronavirus NTDs.

Wave propagation simulation on a 3D model of the Ruhr district for locations of seismic monitoring.

2021 ◽  
Author(s):  
Gabriela de los Angeles Gonzalez de Lucio ◽  
Claudia Finger ◽  
Erik Saenger
Keyword(s):  
Wave Propagation ◽  
Seismic Risk ◽  
Structural Model ◽  
Three Dimensional ◽  
Seismic Source ◽  
Industrial Area ◽  
Structural Features ◽  
Maximum Energy ◽  
Seismic Events ◽  
Ruhr District

<p>The Ruhr district meets the necessary elements to carry out geothermal projects due to its geothermal potential and demand, as it is a densely populated industrial area. Currently, there are projects for direct use, whereas projects for electricity generation are planned. The latter, due to greater depths, reservoir enhancement techniques are required in some cases. This may increase the associated seismic risk which should be elaborated in detail.</p><p>With available data, a three-dimensional geological and structural model was created. The shallower parts have been widely studied and documented by mining activity in the Ruhr region during the last century.  Below a depth of 1 km, data are scarce, and uncertainties increase. The full elastic wavefield emitted by a realistic seismic source has been simulated using a finite differences scheme and the derived geological model. The elastic properties were estimated with well data. The source has common characteristics of real seismic events in the area.</p><p>The wave propagation simulations let us analyze the seismic response with different sources and velocities models. Three cases are considered, two seismic events with distinct depths based on real events. The third case is based on the proposed location of a deep geothermal project.</p><p>Especially for the case with the deeper source, the areas with relatively high amplitudes of displacement correlated with structural features of the model. Applying the imaging condition of maximum energy density allows us to define zones with a potentially increased seismic risk that should be monitored more closely.</p>

scholarly journals Membrane Protein Complex ExbB4-ExbD1-TonB1from Escherichia coli Demonstrates Conformational Plasticity

2015 ◽  
Vol 197 (11) ◽  
pp. 1873-1885 ◽  
Author(s):  
Aleksandr Sverzhinsky ◽  
Jacqueline W. Chung ◽  
Justin C. Deme ◽  
Lucien Fabre ◽  
Kristian T. Levey ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Iron Acquisition ◽  
Proton Translocation ◽  
Three Dimensional ◽  
Structural Data ◽  
Structural Features ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type

ABSTRACTIron acquisition at the outer membrane (OM) of Gram-negative bacteria is powered by the proton motive force (PMF) of the cytoplasmic membrane (CM), harnessed by the CM-embedded complex of ExbB, ExbD, and TonB. Its stoichiometry, ensemble structural features, and mechanism of action are unknown. By panning combinatorial phage libraries, periplasmic regions of dimerization between ExbD and TonB were predicted. Using overexpression of full-length His6-taggedexbB-exbDand S-taggedtonB, we purified detergent-solubilized complexes of ExbB-ExbD-TonB fromEscherichia coli. Protein-detergent complexes of ∼230 kDa with a hydrodynamic radius of ∼6.0 nm were similar to previously purified ExbB4-ExbD2complexes. Significantly, they differed in electronegativity by native agarose gel electrophoresis. The stoichiometry was determined to be ExbB4-ExbD1-TonB1. Single-particle electron microscopy agrees with this stoichiometry. Two-dimensional averaging supported the phage display predictions, showing two forms of ExbD-TonB periplasmic heterodimerization: extensive and distal. Three-dimensional (3D) particle classification showed three representative conformations of ExbB4-ExbD1-TonB1. Based on our structural data, we propose a model in which ExbD shuttles a proton across the CM via an ExbB interprotein rearrangement. Proton translocation would be coupled to ExbD-mediated collapse of extended TonB in complex with ligand-loaded receptors in the OM, followed by repositioning of TonB through extensive dimerization with ExbD. Here we present the first report for purification of the ExbB-ExbD-TonB complex, molar ratios within the complex (4:1:1), and structural biology that provides insights into 3D organization.IMPORTANCEReceptors in the OM of Gram-negative bacteria allow entry of iron-bound siderophores that are necessary for pathogenicity. Numerous iron-acquisition strategies rely upon a ubiquitous and unique protein for energization: TonB. Complexed with ExbB and ExbD, the Ton system links the PMF to OM transport. Blocking iron uptake by targeting a vital nanomachine holds promise in therapeutics. Despite much research, the stoichiometry, structural arrangement, and molecular mechanism of the CM-embedded ExbB-ExbD-TonB complex remain unreported. Here we demonstratein vitroevidence of ExbB4-ExbD1-TonB1complexes. Using 3D EM, we reconstructed the complex in three conformational states that show variable ExbD-TonB heterodimerization. Our structural observations form the basis of a model for TonB-mediated iron acquisition.

scholarly journals The Lactococcin G Immunity Protein Recognizes Specific Regions in Both Peptides Constituting the Two-Peptide Bacteriocin Lactococcin G

2009 ◽  
Vol 76 (4) ◽  
pp. 1267-1273 ◽  
Author(s):  
Camilla Oppegård ◽  
Linda Emanuelsen ◽  
Lisbeth Thorbek ◽  
Gunnar Fimland ◽  
Jon Nissen-Meyer
Keyword(s):  
Structural Model ◽  
Transmembrane Helix ◽  
Cellular Component ◽  
Expression Vectors ◽  
Wild Type ◽  
Terminal Part ◽  
Gene Encoding ◽  
Immunity Protein ◽  
Hybrid Peptides ◽  
Helix Structure

ABSTRACT Lactococcin G and enterocin 1071 are two homologous two-peptide bacteriocins. Expression vectors containing the gene encoding the putative lactococcin G immunity protein (lagC) or the gene encoding the enterocin 1071 immunity protein (entI) were constructed and introduced into strains sensitive to one or both of the bacteriocins. Strains that were sensitive to lactococcin G became immune to lactococcin G when expressing the putative lactococcin G immunity protein, indicating that the lagC gene in fact encodes a protein involved in lactococcin G immunity. To determine which peptide or parts of the peptide(s) of each bacteriocin that are recognized by the cognate immunity protein, combinations of wild-type peptides and hybrid peptides from the two bacteriocins were assayed against strains expressing either of the two immunity proteins. The lactococcin G immunity protein rendered the enterococcus strain but not the lactococcus strains resistant to enterocin 1071, indicating that the functionality of the immunity protein depends on a cellular component. Moreover, regions important for recognition by the immunity protein were identified in both peptides (Lcn-α and Lcn-β) constituting lactococcin G. These regions include the N-terminal end of Lcn-α (residues 1 to 13) and the C-terminal part of Lcn-β (residues 14 to 24). According to a previously proposed structural model of lactococcin G, these regions will be positioned adjacent to each other in the transmembrane helix-helix structure, and the model thus accommodates the present results.

scholarly journals Insights into substrate-mediated assembly of the chloroplast TAT receptor complex

2020 ◽  
Author(s):  
Qianqian Ma ◽  
Christopher Paul New ◽  
Carole Dabney-Smith
Keyword(s):  
Structural Model ◽  
Substrate Recognition ◽  
Transmembrane Helix ◽  
Complex Function ◽  
Transmembrane Helices ◽  
Translocation Event ◽  
Active Substrate ◽  
Tat System ◽  
Folded Proteins ◽  
Transport Complex

AbstractThe Twin Arginine Transport (TAT) system translocates fully folded proteins across the thylakoid membrane in the chloroplast (cp) and the cytoplasmic membrane of bacteria. In chloroplasts, cpTAT transport is achieved by three components: Tha4, Hcf106, and cpTatC. Hcf106 and cpTatC function as the substrate recognition/binding complex while Tha4 is thought to play a significant role in forming the translocation pore. Recent studies challenged this idea by suggesting that cpTatC-Hcf106-Tha4 function together in the active translocase. Here, we have mapped the inter-subunit contacts of cpTatC-Hcf106 during the resting state and built a cpTatC-Hcf106 structural model based on our crosslinking data. In addition, we have identified a substrate-mediated reorganization of cpTatC-Hcf106 contact sites during active substrate translocation. The proximity of Tha4 to the cpTatC-Hcf106 complex was also identified. Our data suggest a model for cpTAT function in which the transmembrane helices of Hcf106 and Tha4 may each contact the fifth transmembrane helix of cpTatC while the insertion of the substrate signal peptide may rearrange the cpTatC-Hcf106-Tha4 complex and initiate the translocation event.One sentence summaryProtein subunits of the thylakoidal twin arginine transport complex function together during substrate recognition and translocase assembly.

scholarly journals Conformational aspects of N-glycosylation of proteins. Studies with linear and cyclic peptides as probes

1982 ◽  
Vol 203 (3) ◽  
pp. 761-768 ◽  
Author(s):  
Ernst Bause ◽  
Harald Hettkamp ◽  
Günter Legler
Keyword(s):  
Hydrogen Bond ◽  
Cyclic Peptides ◽  
Degrees Of Freedom ◽  
Attachment Site ◽  
Structural Features ◽  
Peptide Chain ◽  
Hydrogen Bond Interaction ◽  
Hydrogen Bond Acceptor ◽  
Disulphide Bond Formation ◽  
Partial Fixation

Conformational aspects of N-glycosylation of glycoproteins have been studied by using a series of peptides which contained, in addition to the ‘marker sequence’ Asn-Gly-Thr, two cysteine residues in various positions of the peptide chain. The presence of two cysteines permitted a partial fixation of the above triplet sequence in cyclic structures of various size by intramolecular disulphide bond formation. Comparison of the glycosyl acceptor properties of the linear peptides and their corresponding cyclic analogues allows the following statements. The considerably lower acceptor capabilities of the cyclic derivatives indicate that the restriction of rotational degrees of freedom imposed by disulphide bonding results in a conformation which hinders a favourable interaction of the peptide substrate with the N-glycosyltransferase. On the other hand, the glycosylation rate of linear peptides increases with increasing chain length, suggesting that the amino acids on both the N- and C-terminal side of the ‘marker sequence’ may contribute to a considerable extent to the induction of an ‘active’ conformation. Realization of a potential sugar attachment site requires a hydrogen bond interaction within the ‘marker sequence’ between the oxygen of threonine (serine) as the hydrogen bond acceptor and the β-amide of asparagine as the donor [Bause & Legler (1981) Biochem. J.195, 639–644]. This interaction is obviously facilitated when the peptide chain can adopt a conformation which resembles a β-turn or other loop structure. The available experimental and statistical data are discussed in terms of possible structural features for N-glycosylation, with the aid of space-filling models.

Infrared Crystallography: Structural Refinement through Spectroscopy

1997 ◽  
Vol 51 (4) ◽  
pp. 568-573 ◽  
Author(s):  
J. Timothy Sage
Keyword(s):  
Single Crystals ◽  
Protein Function ◽  
Structural Changes ◽  
Structural Model ◽  
Structural Features ◽  
Heme Iron ◽  
Structural Refinement ◽  
Crystal Forms ◽  
X Ray ◽  
Quantitative Understanding

A method for determining the orientation of individual bonds within complex macromolecules from polarized IR measurements on oriented single crystals is described. At present, X-ray diffraction is the principal technique used to define the global structure and orientation of macromolecules in the crystalline state. However, resolution limitations and conformational disorder limit the accuracy of the resulting structural model. A quantitative understanding of protein function often requires a more precise description of structural features at a localized active site. Polarized IR measurements of internal stretching bands of N−3, CN−, OCN−, and SCN− bound at the ferric heme iron of single crystals of myoglobin are presented. A synthesis of such measurements on different crystal forms leads to an N−3 orientation consistent with IR measurements on both P21 and P212121 crystals, but significantly different from the orientation reported in X-ray crystal structures. Subtle structural changes between these two crystal forms of the CN− complex may account for small differences in the stretching frequency and orientation of the C–N bond.

scholarly journals Investigation of the catalytic triad of arylamine N-acetyltransferases: essential residues required for acetyl transfer to arylamines

2005 ◽  
Vol 390 (1) ◽  
pp. 115-123 ◽  
Author(s):  
James Sandy ◽  
Adeel Mushtaq ◽  
Simon J. Holton ◽  
Pamela Schartau ◽  
Martin E. M. Noble ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Structural Model ◽  
Sequence Data ◽  
Structural Features ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
X Ray ◽  
X Ray Crystallography ◽  
Acetyl Transfer ◽  
Insight Into

The NATs (arylamine N-acetyltransferases) are a well documented family of enzymes found in both prokaryotes and eukaryotes. NATs are responsible for the acetylation of a range of arylamine, arylhydrazine and hydrazine compounds. We present here an investigation into the catalytic triad of residues (Cys-His-Asp) and other structural features of NATs using a variety of methods, including site-directed mutagenesis, X-ray crystallography and bioinformatics analysis, in order to investigate whether each of the residues of the catalytic triad is essential for catalytic activity. The catalytic triad of residues, Cys-His-Asp, is a well defined motif present in several families of enzymes. We mutated each of the catalytic residues in turn to investigate the role they play in catalysis. We also mutated a key residue, Gly126, implicated in acetyl-CoA binding, to examine the effects on acetylation activity. In addition, we have solved the structure of a C70Q mutant of Mycobacterium smegmatis NAT to a resolution of 1.45 Å (where 1 Å=0.1 nm). This structure confirms that the mutated protein is correctly folded, and provides a structural model for an acetylated NAT intermediate. Our bioinformatics investigation analysed the extent of sequence conservation between all eukaryotic and prokaryotic NAT enzymes for which sequence data are available. This revealed several new sequences, not yet reported, of NAT paralogues. Together, these studies have provided insight into the fundamental core of NAT enzymes, and the regions where sequence differences account for the functional diversity of this family. We have confirmed that each of the three residues of the triad is essential for acetylation activity.

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