Unliganded GroEL at 2.8 Å: structure and functional implications

1995 ◽  
Vol 348 (1323) ◽  
pp. 113-119 ◽  
Keyword(s):  
Three Dimensional ◽  
Binding Pocket ◽  
Dimensional Structure ◽  
Outer Diameter ◽  
Central Channel ◽  
Crystal Forms ◽  
Monoclinic Form ◽  
C Terminus ◽  
Ligand Free ◽  
Dyad Symmetry

The three-dimensional structure of the E. coli chaperonin, GroEL, has been determined crystallo-graphically and refined to 2.7 Å in two crystal forms: an orthorhombic form from high salt and a monoclinic form from polyethylene glycol. The former is ligand free, the latter is both liganded with ATP analogues and ligand free. These structures provide a structural scaffold upon which to interpret extensive mutagenesis and biochemical studies. GroEL contains two sevenfold rotationally symmetric rings of identical 547-amino acid subunits. The rings are arranged ‘back-to-back’ with exact dyad symmetry to form a stubby cylinder that is 146 Å high with an outer diameter of about 143 Å. The cylinder has a substantial central channel that is unobstructed for the entire length of the cylinder and has a diameter of about 45 Å except for large bulges that lead into a sevenfold symmetric array of elliptical side windows in each ring. Each subunit is composed of three distinct domains: (i) an ‘equatorial’ domain that contains the N- and C-terminus and the ATP-binding pocket, .(ii) an ‘apical domain’ that forms the opening of the central channel and contains poorly ordered segments that mutational studies implicate in binding unfolded polypeptides and GroES, and (iii) an intermediate domain tht connects the other two domains and may serve to transmit allosteric adjustments.

scholarly journals Crystallization of FcpA fromLeptospira, a novel flagellar protein that is essential for pathogenesis

2017 ◽  
Vol 73 (3) ◽  
pp. 123-129 ◽  
Author(s):  
Fabiana San Martin ◽  
Ariel E. Mechaly ◽  
Nicole Larrieux ◽  
Elsio A. Wunder ◽  
Albert I. Ko ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
X Rays ◽  
The Novel ◽  
X Ray Diffraction ◽  
Crystal Forms ◽  
X Ray ◽  
Monoclinic Form ◽  
Flagellar Filament ◽  
Flagellar Protein

The protein FcpA is a unique component of the flagellar filament of spirochete bacteria belonging to the genusLeptospira. Although it plays an essential role in translational motility and pathogenicity, no structures of FcpA homologues are currently available in the PDB. Its three-dimensional structure will unveil the novel motility mechanisms that render pathogenicLeptospiraparticularly efficient at invading and disseminating within their hosts, causing leptospirosis in humans and animals. FcpA fromL. interroganswas purified and crystallized, but despite laborious attempts no useful X ray diffraction data could be obtained. This challenge was solved by expressing a close orthologue from the related saprophytic speciesL. biflexa. Three different crystal forms were obtained: a primitive and a centred monoclinic form, as well as a hexagonal variant. All forms diffracted X-rays to suitable resolutions for crystallographic analyses, with the hexagonal type typically reaching the highest limits of 2.0 Å and better. A variation of the quick-soaking procedure resulted in an iodide derivative that was instrumental for single-wavelength anomalous diffraction methods.

scholarly journals Structure of the Lectin Mannose 6-Phosphate Receptor Homology (MRH) Domain of Glucosidase II, an Enzyme That Regulates Glycoprotein Folding Quality Control in the Endoplasmic Reticulum

2013 ◽  
Vol 288 (23) ◽  
pp. 16460-16475 ◽  
Author(s):  
Linda J. Olson ◽  
Ramiro Orsi ◽  
Solana G. Alculumbre ◽  
Francis C. Peterson ◽  
Ivan D. Stigliano ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Dimensional Structure ◽  
Mannose Residue ◽  
Glucosidase Ii ◽  
Mannose 6 Phosphate ◽  
First Time ◽  
Conserved Residue

Here we report for the first time the three-dimensional structure of a mannose 6-phosphate receptor homology (MRH) domain present in a protein with enzymatic activity, glucosidase II (GII). GII is involved in glycoprotein folding in the endoplasmic reticulum. GII removes the two innermost glucose residues from the Glc3Man9GlcNAc2 transferred to nascent proteins and the glucose added by UDP-Glc:glycoprotein glucosyltransferase. GII is composed of a catalytic GIIα subunit and a regulatory GIIβ subunit. GIIβ participates in the endoplasmic reticulum localization of GIIα and mediates in vivo enhancement of N-glycan trimming by GII through its C-terminal MRH domain. We determined the structure of a functional GIIβ MRH domain by NMR spectroscopy. It adopts a β-barrel fold similar to that of other MRH domains, but its binding pocket is the most shallow known to date as it accommodates a single mannose residue. In addition, we identified a conserved residue outside the binding pocket (Trp-409) present in GIIβ but not in other MRHs that influences GII glucose trimming activity.

Three-Dimensional Structure of Ribosome-Sec61p Translocation Complexes From Mammals

2000 ◽  
Vol 6 (S2) ◽  
pp. 264-265
Author(s):  
J-F. Ménétret ◽  
D. G. Morgan ◽  
M. Radermacher ◽  
A. Neuhof ◽  
T. A. Rapoport ◽  
...  
Keyword(s):  
Critical Role ◽  
Three Dimensional ◽  
Channel Morphology ◽  
Dimensional Structure ◽  
Central Channel ◽  
Mass Analysis ◽  
Biologically Relevant ◽  
Ribosome Binding ◽  
Physical Mechanisms ◽  
3D Architecture

Co-translational translocation at the endoplasmic reticulum (ER) plays a critical role in the targeting of both soluble and membrane proteins to their correct intra- and intercellular compartments. We are studying the 3D architecture of the ribosome-Sec61p complex (translocon), with the aim of understanding the physical mechanisms of gating and transport. To this end, we are using single particle electron cryo-microscopy and 3D reconstruction of frozen hydrated channel complexes, to obtain interpretable and biologically relevant maps.Previously, we have shown that both co- and post-translational translocation utilize a common central channel comprised of a ring-like Sec61p oligomer. Moreover, this channel morphology is shared with the related Sec YE complex from B. subtilus. Mass analysis, volume calculations and ribosome binding experiments suggest a stoichiometry of 3-4 Sec61p heterotrimers per ring. We currently favor 4 copies of the Sec61p complex per channel, as projection maps demonstrate 4 nearly equi-spaced peaks around the central pore.

Hemoglobin Biosynthesis in Vitreoscilla stercoraria DW: Cloning, Expression, and Characterization of a New Homolog of a Bacterial Globin Gene

1998 ◽  
Vol 64 (6) ◽  
pp. 2220-2228 ◽  
Author(s):  
Meenal Joshi ◽  
Shekhar Mande ◽  
Kanak L. Dikshit
Keyword(s):  
Globin Gene ◽  
Three Dimensional ◽  
Fold Increase ◽  
Binding Pocket ◽  
Growth Conditions ◽  
Dimensional Structure ◽  
Strictly Aerobic ◽  
Coding Region ◽  
Oxygen Control ◽  
Constitutive Production

ABSTRACT In the strictly aerobic, gram-negative bacteriumVitreoscilla strain C1, oxygen-limited growth conditions create a more than 50-fold increase in the expression of a homodimeric heme protein which was recognized as the first bacterial hemoglobin (Hb). The recently determined crystal structure ofVitreoscilla Hb has indicated that the heme pocket of microbial globins differs from that of eukaryotic Hbs. In an attempt to understand the diverse functions of Hb-like proteins in prokaryotes, we have cloned and characterized the gene (vgb) encoding an Hb-like protein from another strain of Vitreoscilla,V. stercoraria DW. Several silent changes were observed within the coding region of the V. stercoraria vgb gene. Apart from that, V. stercoraria Hb exhibited interesting differences between the A and E helices. Compared to its Hb counterpart from Vitreoscilla strain C1, the purified preparation ofV. stercoraria Hb displays a slower autooxidation rate. The differences between Vitreoscilla Hb and V. stercoraria Hb were mapped onto the three-dimensional structure of Vitreoscilla Hb, which indicated that the four changes, namely, Ile7Val, Ile9Thr, Ile10Ser, and Leu62Val, present within theV. stercoraria Hb fall in the region where the A and E helices contact each other. Therefore, alteration in the relative orientation of the A and E helices and the corresponding conformational change in the heme binding pocket of V. stercoraria Hb can be correlated to its slower autooxidation rate. In sharp contrast to the oxygen-regulated biosynthesis of Hb in Vitreoscillastrain C1, production of Hb in V. stercoraria has been found to be low and independent of oxygen control, which is supported by the absence of a fumarate and nitrate reductase regulator box within the V. stercoraria vgb promoter region. Thus, the regulation mechanisms of the Hb-encoding gene appear to be quite different in the two closely related species ofVitreoscilla. The relatively slower autooxidation rate ofV. stercoraria Hb, lack of oxygen sensitivity, and constitutive production of Hb suggest that it may have some other function(s) in the cellular physiology of V. stercorariaDW, together with facilitated oxygen transport, predicted for earlier reported Vitreoscilla Hb.

scholarly journals Three-dimensional EM structure of the ectodomain of integrin αVβ3 in a complex with fibronectin

2005 ◽  
Vol 168 (7) ◽  
pp. 1109-1118 ◽  
Author(s):  
Brian D. Adair ◽  
Jian-Ping Xiong ◽  
Catherine Maddock ◽  
Simon L. Goodman ◽  
M. Amin Arnaout ◽  
...  
Keyword(s):  
Particle Image ◽  
Three Dimensional ◽  
Integrin Αvβ3 ◽  
Dimensional Structure ◽  
Soluble Complex ◽  
Surface Receptors ◽  
Difference Map ◽  
Transmission Electron ◽  
Ligand Free ◽  
Site Binding

Integrins are αβ heterodimeric cell surface receptors that mediate transmembrane signaling by binding extracellular and cytoplasmic ligands. The ectodomain of integrin αVβ3 crystallizes in a bent, genuflexed conformation considered to be inactive (unable to bind physiological ligands in solution) unless it is fully extended by activating stimuli. We generated a stable, soluble complex of the Mn2+-bound αVβ3 ectodomain with a fragment of fibronectin (FN) containing type III domains 7 to 10 and the EDB domain (FN7-EDB-10). Transmission electron microscopy and single particle image analysis were used to determine the three-dimensional structure of this complex. Most αVβ3 particles, whether unliganded or FN-bound, displayed compact, triangular shapes. A difference map comparing ligand-free and FN-bound αVβ3 revealed density that could accommodate the RGD-containing FN10 in proximity to the ligand-binding site of β3, with FN9 just adjacent to the synergy site binding region of αV. We conclude that the ectodomain of αVβ3 manifests a bent conformation that is capable of stably binding a physiological ligand in solution.

Molecular and cellular biology of thyrotropin-releasing hormone receptors

1996 ◽  
Vol 76 (1) ◽  
pp. 175-191 ◽  
Author(s):  
M. C. Gershengorn ◽  
R. Osman
Keyword(s):  
Hormone Receptors ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Amino Acid Sequences ◽  
Thyrotropin Releasing Hormone ◽  
Cellular Biology ◽  
Dimensional Structure ◽  
Releasing Hormone ◽  
G Protein Coupled ◽  
Trh Receptor

Thyrotropin-releasing hormone (TRH) receptor (TRH-R) complementary DNAs have been cloned from several species. The deduced amino acid sequences are compatible with TRH-R being a seven-transmembrane-spanning G protein-coupled receptor. These complementary DNAs and reagents derived from them have permitted detailed study of TRH-R biology at the molecular and cellular levels. Studies that have been performed since 1990 are reviewed in this article under the following headings: TRH-R gene, tissue distribution of TRH-R, primary structure of TRH-Rs, three-dimensional structure of the TRH-R binding pocket, TRH-R and G proteins, TRH-R activation, TRH desensitization, TRH-R endocytosis, and regulation of TRH-R number. It is evident that many new insights into the structure, function, and regulation of TRH-Rs have been gained in the last several years but that our understanding of these processes is incomplete. We look forward to even greater progress in the future.

scholarly journals FpvA-Mediated Ferric Pyoverdine Uptake in Pseudomonas aeruginosa: Identification of Aromatic Residues in FpvA Implicated in Ferric Pyoverdine Binding and Transport

2005 ◽  
Vol 187 (24) ◽  
pp. 8511-8515 ◽  
Author(s):  
Jiang-Sheng Shen ◽  
Valérie Geoffroy ◽  
Shadi Neshat ◽  
Zongchao Jia ◽  
Allison Meldrum ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Dimensional Structure ◽  
Aromatic Residues ◽  
Three Dimensional Structure

ABSTRACT A number of aromatic residues were seen to cluster in the upper portion of the three-dimensional structure of the FpvA ferric pyoverdine receptor of Pseudomonas aeruginosa, reminiscent of the aromatic binding pocket for ferrichrome in the FhuA receptor of Escherichia coli. Alanine substitutions in three of these, W362, W391, and F795, markedly compromised ferric pyoverdine binding and transport, consistent with a role of FpvA in ferric pyoverdine recognition.

scholarly journals Cloning, Baeyer-Villiger Biooxidations, and Structures of the Camphor Pathway 2-Oxo-Δ3-4,5,5-Trimethylcyclopentenylacetyl-Coenzyme A Monooxygenase of Pseudomonas putida ATCC 17453

2012 ◽  
Vol 78 (7) ◽  
pp. 2200-2212 ◽  
Author(s):  
Hannes Leisch ◽  
Rong Shi ◽  
Stephan Grosse ◽  
Krista Morley ◽  
Hélène Bergeron ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Coenzyme A ◽  
Free Acid ◽  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Dimensional Structure ◽  
Content Type ◽  
Crystal Forms

ABSTRACTA dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor byPseudomonas putidaATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140–152, 1983). Here we cloned and overexpressed the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) inEscherichia coliand determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP+at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP+. A comparison of several crystal forms of OTEMO bound to FAD and NADP+revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (kcat/Km) favors 2-n-hexyl cyclopentanone (4.3 × 105M−1s−1) as a substrate, although its affinity (Km= 32 μM) was lower than that of the CoA-activated substrate (Km= 18 μM). In whole-cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO) and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members.

Molecular cloning, sequence characteristics, and tissue expression analysis of glucagon receptor gene in Bama minipig

2020 ◽  
Vol 100 (3) ◽  
pp. 536-546
Author(s):  
Cuiping An ◽  
Kaiyi Zhang ◽  
Wenjuan Zhu ◽  
Yanzhen Bi ◽  
Tianwen Wu ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Receptor Gene ◽  
Three Dimensional ◽  
Salt Bridge ◽  
Binding Pocket ◽  
Tissue Expression ◽  
Dimensional Structure ◽  
Glucagon Receptor ◽  
Tissue Expression Analysis ◽  
Glucose And Lipid Metabolism

Recent studies have shown that the glucagon receptor (GCGR) plays an important role in the development of type 2 diabetes mellitus. Both pigs and humans exhibit significantly similar behaviors in their glucose and lipid metabolism. In this study, the obtained Bama minipig GCGR coding sequence was 1437 bp encoding 479 amino acids (AA), which demonstrated higher sequence homology with humans than other species. It showed the highest expression profile in the liver, followed by the lung and kidney. In addition, the three-dimensional structure analysis showed that the porcine GCGR protein also had a classic sevenfold transmembrane region and a stalk region at the N-terminus for ligand binding. The stalk region of GCGR possessed five AA variations. The ligand binding pocket of GCGR has one AA variation in the key region, none of which affected the glucagon binding verified by the crystal structure mutagenesis in humans. There was no variation found in the region of membrane anchoring, hydrophobic bond, salt bridge, and hydrogen bond. However, the Gly40Ser mutation in mice resulted in major diseases, meaning that pigs are more suitable for the evaluation of GCGR-related drugs than mice.

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