Structural basis of the transactivation deficiency of the human PPARγ F360L mutant associated with familial partial lipodystrophy

2014 ◽  
Vol 70 (7) ◽  
pp. 1965-1976 ◽  
Author(s):  
Clorinda Lori ◽  
Alessandra Pasquo ◽  
Roberta Montanari ◽  
Davide Capelli ◽  
Valerio Consalvi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Partial Agonist ◽  
Salt Bridge ◽  
Van Der Waals Interactions ◽  
Structural Basis ◽  
Wild Type ◽  
Partial Lipodystrophy ◽  
X Ray ◽  
Familial Partial Lipodystrophy

The peroxisome proliferator-activated receptors (PPARs) are transcription factors that regulate glucose and lipid metabolism. The role of PPARs in several chronic diseases such as type 2 diabetes, obesity and atherosclerosis is well known and, for this reason, they are the targets of antidiabetic and hypolipidaemic drugs. In the last decade, some rare mutations in human PPARγ that might be associated with partial lipodystrophy, dyslipidaemia, insulin resistance and colon cancer have emerged. In particular, the F360L mutant of PPARγ (PPARγ2 residue 388), which is associated with familial partial lipodystrophy, significantly decreases basal transcriptional activity and impairs stimulation by synthetic ligands. To date, the structural reason for this defective behaviour is unclear. Therefore, the crystal structure of PPARγ F360L together with the partial agonist LT175 has been solved and the mutant has been characterized by circular-dichroism spectroscopy (CD) in order to compare its thermal stability with that of the wild-type receptor. The X-ray analysis showed that the mutation induces dramatic conformational changes in the C-terminal part of the receptor ligand-binding domain (LBD) owing to the loss of van der Waals interactions made by the Phe360 residue in the wild type and an important salt bridge made by Arg357, with consequent rearrangement of loop 11/12 and the activation function helix 12 (H12). The increased mobility of H12 makes the binding of co-activators in the hydrophobic cleft less efficient, thereby markedly lowering the transactivation activity. The spectroscopic analysis in solution and molecular-dynamics (MD) simulations provided results which were in agreement and consistent with the mutant conformational changes observed by X-ray analysis. Moreover, to evaluate the importance of the salt bridge made by Arg357, the crystal structure of the PPARγ R357A mutant in complex with the agonist rosiglitazone has been solved.

scholarly journals Impaired Peroxisome Proliferator-Activated Receptor γ Function through Mutation of a Conserved Salt Bridge (R425C) in Familial Partial Lipodystrophy

2007 ◽  
Vol 21 (5) ◽  
pp. 1049-1065 ◽  
Author(s):  
Ellen H. Jeninga ◽  
Olivier van Beekum ◽  
Aalt D. J. van Dijk ◽  
Nicole Hamers ◽  
Brenda I. Hendriks-Stegeman ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Transcriptional Activity ◽  
Salt Bridge ◽  
Dominant Negative ◽  
Heterozygous Mutation ◽  
Peroxisome Proliferator ◽  
Wild Type ◽  
Peroxisome Proliferator Activated Receptor ◽  
Partial Lipodystrophy ◽  
Familial Partial Lipodystrophy

Abstract The nuclear receptor peroxisome proliferator-activated receptor (PPAR) γ plays a key role in the regulation of glucose and lipid metabolism in adipocytes by regulating their differentiation, maintenance, and function. A heterozygous mutation in the PPARG gene, which changes an arginine residue at position 425 into a cysteine (R425C), has been reported in a patient with familial partial lipodystrophy subtype 3 (FPLD3). The strong conservation of arginine 425 among nuclear receptors that heterodimerize with retinoic acid X receptor prompted us to investigate the functional consequences of the R425C mutation on PPARγ function. Here we show that this mutant displayed strongly reduced transcriptional activity compared with wild-type PPARγ, irrespective of cell type, promoter context, or ligand, whereas transrepression of nuclear factor-κB activity remained largely intact. Our data indicate that the reduced transcriptional activity of PPARγ R425C is not caused by impaired corepressor release, but due to reduced dimerization with retinoic acid X receptor α in combination with reduced ligand binding and subsequent coactivator binding. As a consequence of these molecular defects, the R425C mutant was less effective in inducing adipocyte differentiation. PPARγ R425C did not inhibit its wild-type counterpart in a dominant-negative manner, suggesting a haploinsufficiency mechanism in at least some FPLD3 patients. Using molecular dynamics simulations, substitution of R425 with cysteine is predicted to cause the formation of an alternative salt bridge. This structural change provides a likely explanation of how mutation of a single conserved residue in a patient with FPLD3 can disrupt the function of the adipogenic transcription factor PPARγ on multiple levels.

scholarly journals Domain structure of human complement C4b extends with increasing NaCl concentration: implications for its regulatory mechanism

2016 ◽  
Vol 473 (23) ◽  
pp. 4473-4491 ◽  
Author(s):  
Ka Wai Fung ◽  
David W. Wright ◽  
Jayesh Gor ◽  
Marcus J. Swann ◽  
Stephen J. Perkins
Keyword(s):  
Crystal Structure ◽  
Domain Structure ◽  
Conformational Changes ◽  
Analytical Ultracentrifugation ◽  
Sedimentation Coefficient ◽  
Salt Bridge ◽  
X Ray ◽  
X Ray Scattering ◽  
Nacl Concentration ◽  
Dual Polarisation Interferometry

During the activation of complement C4 to C4b, the exposure of its thioester domain (TED) is crucial for the attachment of C4b to activator surfaces. In the C4b crystal structure, TED forms an Arg104–Glu1032 salt bridge to tether its neighbouring macroglobulin (MG1) domain. Here, we examined the C4b domain structure to test whether this salt bridge affects its conformation. Dual polarisation interferometry of C4b immobilised at a sensor surface showed that the maximum thickness of C4b increased by 0.46 nm with an increase in NaCl concentration from 50 to 175 mM NaCl. Analytical ultracentrifugation showed that the sedimentation coefficient s20,w of monomeric C4b of 8.41 S in 50 mM NaCl buffer decreased to 7.98 S in 137 mM NaCl buffer, indicating that C4b became more extended. Small angle X-ray scattering reported similar RG values of 4.89–4.90 nm for C4b in 137–250 mM NaCl. Atomistic scattering modelling of the C4b conformation showed that TED and the MG1 domain were separated by 4.7 nm in 137–250 mM NaCl and this is greater than that of 4.0 nm in the C4b crystal structure. Our data reveal that in low NaCl concentrations, both at surfaces and in solution, C4b forms compact TED–MG1 structures. In solution, physiologically relevant NaCl concentrations lead to the separation of the TED and MG1 domain, making C4b less capable of binding to its complement regulators. These conformational changes are similar to those seen previously for complement C3b, confirming the importance of this salt bridge for regulating both C4b and C3b.

scholarly journals Crystal structure of pimecrolimus Form B, C43H68ClNO11

2021 ◽  
Vol 36 (1) ◽  
pp. 35-42
Author(s):  
Shivang Bhaskar ◽  
Joseph T. Golab ◽  
James A. Kaduk ◽  
Amy M. Gindhart ◽  
Thomas N. Blanton
Keyword(s):  
Crystal Structure ◽  
Powder Diffraction ◽  
Density Functional ◽  
Van Der Waals Interactions ◽  
Powder Diffraction File ◽  
X Ray ◽  
Weak Intermolecular Interactions ◽  
Significant Difference ◽  
Atomic Coordinates ◽  
Solid State Conformation

The crystal structure of pimecrolimus Form B has been solved and refined using synchrotron X-ray powder diffraction data and optimized using density functional techniques. Pimecrolimus crystallizes in the space group P21 (#4) with a = 15.28864(7), b = 13.31111(4), c = 10.95529(5) Å, β = 96.1542(3)°, V = 2216.649(9) Å3, and Z = 2. Although there are an intramolecular six-ring hydrogen bond and some larger chain and ring patterns, the crystal structure is dominated by van der Waals interactions. There is a significant difference between the conformation of the Rietveld-refined and the DFT-optimized structures in one portion of the macrocyclic ring. Although weak, intermolecular interactions are apparently important in determining the solid-state conformation. The powder pattern is included in the Powder Diffraction File™ (PDF®) as entry 00-066-1619. This study provides the atomic coordinates to be added to the PDF entry.

scholarly journals Structural Basis for Imipenem Inhibition of Class C β-Lactamases

2002 ◽  
Vol 46 (12) ◽  
pp. 3978-3980 ◽  
Author(s):  
Beth M. Beadle ◽  
Brian K. Shoichet
Keyword(s):  
Crystal Structure ◽  
Carbonyl Oxygen ◽  
Structural Basis ◽  
Enzyme Complex ◽  
X Ray ◽  
Class A ◽  
Lactam Carbonyl ◽  
Class C ◽  
Expected Position

ABSTRACT To determine how imipenem inhibits the class C β-lactamase AmpC, the X-ray crystal structure of the acyl-enzyme complex was determined to a resolution of 1.80 Å. In the complex, the lactam carbonyl oxygen of imipenem has flipped by approximately 180° compared to its expected position; the electrophilic acyl center is thus displaced from the point of hydrolytic attack. This conformation resembles that of imipenem bound to the class A enzyme TEM-1 but is different from that of moxalactam bound to AmpC.

scholarly journals Structural Analysis of The OXA-48 Carbapenemase Bound to A “Poor” Carbapenem Substrate, Doripenem

Antibiotics ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 145 ◽  
Author(s):  
Krisztina M. Papp-Wallace ◽  
Vijay Kumar ◽  
Elise T. Zeiser ◽  
Scott A. Becka ◽  
Focco van den Akker
Keyword(s):  
Public Health ◽  
Crystal Structure ◽  
Salt Bridge ◽  
Hydrolytic Activity ◽  
Van Der Waals Interactions ◽  
Side Chain ◽  
Carbapenem Resistant ◽  
Mechanistic Basis ◽  
Further Development ◽  
Carbapenem Resistant Enterobacteriaceae

Carbapenem-resistant Enterobacteriaceae are a significant threat to public health, and a major resistance determinant that promotes this phenotype is the production of the OXA-48 carbapenemase. The activity of OXA-48 towards carbapenems is a puzzling phenotype as its hydrolytic activity against doripenem is non-detectable. To probe the mechanistic basis for this observation, we determined the 1.5 Å resolution crystal structure of the deacylation deficient K73A variant of OXA-48 in complex with doripenem. Doripenem is observed in the Δ1R and Δ1S tautomeric states covalently attached to the catalytic S70 residue. Likely due to positioning of residue Y211, the carboxylate moiety of doripenem is making fewer hydrogen bonding/salt-bridge interactions with R250 compared to previously determined carbapenem OXA structures. Moreover, the hydroxyethyl side chain of doripenem is making van der Waals interactions with a key V120 residue, which likely affects the deacylation rate of doripenem. We hypothesize that positions V120 and Y211 play important roles in the carbapenemase profile of OXA-48. Herein, we provide insights for the further development of the carbapenem class of antibiotics that could render them less effective to hydrolysis by or even inhibit OXA carbapenemases.

Co-Crystal Structures of 7-Azaindole Inhibitors of Wild-Type and T315I Imatinib-Resistant Mutant Forms of the BCR-ABL Tyrosine Kinase.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1018-1018
Author(s):  
Hal A. Lewis ◽  
Fred Zhang ◽  
Richard Romero ◽  
Pierre-Yves Bounaud ◽  
Mark E. Wilson ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Tyrosine Kinase ◽  
Crystal Structures ◽  
Kinase Domain ◽  
Hinge Region ◽  
Wild Type ◽  
X Ray ◽  
Abl Kinase ◽  
Imatinib Resistant ◽  
Mutant Forms

Abstract Chronic myelogenous leukemia (CML) arises from uncontrolled cell growth driven by a constitutively active BCR-ABL fusion protein tyrosine kinase, which is the product of the pathognomonic Philadelphia chromosomal translocation. Imatinib mesylate (Gleevec) is a BCR-ABL inhibitor used as a first line treatment of CML. Although imatinib is highly effective in chronic phase CML, in advanced disease patients frequently relapse due to the emergence of drug resistance. Approximately two-thirds of resistance is caused by point mutations in the BCR-ABL kinase domain, which give rise to active mutant forms of the enzyme that are insensitive to Gleevec. The T315I mutation represents one of the most common causes of resistance, is resistant to the second generation BCR-ABL inhibitors dasatinib and nilotinib, and represents an important and challenging target for discovery of next generation targeted CML treatments. We have applied X-ray crystallographic screening of our FAST™ fragment library and structure-guided hit-to-lead optimization to identify potent inhibitors of both wild-type and T315I mutant BCR-ABL. These efforts yielded a 7-azaindole compound series that exhibits binding to and inhibition of both wild-type and T315I BCR-ABL. Methods: Wild-type (with Y393F) and T315I Abl kinase domain protein were expressed in E. coli and purified to homogeneity. These proteins were crystallized in the presence of a reference inhibitor followed by addition of the 7-azaindole series compounds soaked into the preformed crystals to displace the reference compound, giving the desired co-crystal. X-ray diffraction data were recorded at the company’s proprietary synchrotron beamline SGX-CAT at the Advanced Photon Source. Three-dimensional enzyme-inhibitor co-crystal structures were determined by molecular replacement and refined to permit modeling of bound ligand. Results: Both wild-type and T315I Abl structures revealed enzyme in the active conformation with inhibitors bound to the kinase hinge region. The crystal structure of 2-amino-5-[3-(1-ethyl-1H-pyrazol-5-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl]-N,N-dimethylbenzamide in complex with T315I, illustrates the typical binding mode which is independent of the 315 residue, and therefore accounts for the compound inhibiting the T315I mutant form of BCR-ABL (see figure). The inhibitor binds to the hinge region of ABL utilizing hydrogen bonding to backbone carbonyl of Glu316 and NH of Met318, with the pyrazole ring stacking in a lipophilic pocket between Phe382 and Tyr253. In addition, the benzamide carbonyl participates in a hydrogen bond interactioin with the backbone-NH of Glu249 of the p-loop. Conclusions: X-ray crystallographic fragment screening and co-crystal structure studies have been successfully employed in discovery/optimization of 7-azaindole series compounds, yielding potent, selective inhibitors of both wild-type and imatinib-resistant forms of BCR-ABL. Figure Figure

scholarly journals Crystal structure of the DNA sequence d(CGTGAATTCACG)2with DAPI

2017 ◽  
Vol 73 (9) ◽  
pp. 500-504 ◽  
Author(s):  
Hristina I. Sbirkova-Dimitrova ◽  
Boris Shivachev
Keyword(s):  
Crystal Structure ◽  
Crystal Packing ◽  
Minor Groove ◽  
Van Der Waals Interactions ◽  
Water Molecules ◽  
X Ray Diffraction ◽  
Base Pairs ◽  
X Ray ◽  
Dna Molecule ◽  
Dna Fragment

The structure of 4′,6-diamidine-2-phenylindole (DAPI) bound to the synthetic B-DNA oligonucleotide d(CGTGAATTCACG) has been solved in space groupP212121by single-crystal X-ray diffraction at a resolution of 2.2 Å. The structure is nearly isomorphous to that of the previously reported crystal structure of the oligonucleotide d(CGTGAATTCACG) alone. The adjustments in crystal packing between the native DNA molecule and the DNA–DAPI complex are described. DAPI lies in the narrow minor groove near the centre of the B-DNA fragment, positioned over the A–T base pairs. It is bound to the DNA by hydrogen-bonding and van der Waals interactions. Comparison of the two structures (with and without ligand) shows that DAPI inserts into the minor groove, displacing the ordered spine waters. Indeed, as DAPI is hydrophobic it confers this behaviour on the DNA and thus restricts the presence of water molecules.

scholarly journals Crystal structure of EndoS, an immunomodulatory endoglycosidase specific for human IgG antibodies

2014 ◽  
Vol 70 (a1) ◽  
pp. C256-C256
Author(s):  
Beatriz Trastoy
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Igg Antibodies ◽  
Human Pathogens ◽  
X Ray ◽  
Effector Functions ◽  
Encounter Complex ◽  
Key Enzyme

In order to evade host immune mechanisms, many bacteria secrete immunomodulatory enzymes. Streptococcus pyogenes, one of the most common human pathogens, secretes a large endoglycosidase, EndoS, which removes carbohydrates in a highly specific manner from IgG antibodies. This modification renders antibodies incapable of eliciting host effector functions through either complement or Fc γ receptors, providing the bacteria with a survival advantage. On account of this antibody-specific modifying activity, EndoS is being developed as a promising injectable therapeutic for autoimmune diseases that rely on autoantibodies. Additionally, EndoS is a key enzyme used in the chemoenzymatic synthesis of homogenously glycosylated antibodies with tailored Fc γ receptor-mediated effector functions. Despite the tremendous utility of this enzyme, the molecular basis of EndoS specificity for, and processing of, IgG antibodies has remained poorly understood. Here, we report the X-ray crystal structure of EndoS and provide a model of its encounter complex with its substrate, the IgG1 Fc domain. We show that EndoS is composed of five distinct protein domains, including glycosidase, leucine-rich repeat, hybrid Ig, carbohydrate binding module, and three-helix bundle domains, arranged in a distinctive V-shaped conformation. Our data suggest that the substrate enters the concave interior of the enzyme structure, is held in place by the carbohydrate binding module, and that concerted conformational changes in both enzyme and substrate are required for subsequent antibody deglycosylation. The EndoS structure presented here provides a framework from which novel endoglycosidases could be engineered for additional clinical and biotechnological applications.

scholarly journals Einkristallzüchtung und Röntgenstrukturanalyse des polymeren 1:1-Komplexes zwischen CuCl2 und Pyridazin / Crystal Growth in Gels and X-Ray Characterization of the Polymeric 1:1-Complex of CuCl2 with Pyridazine

1990 ◽  
Vol 45 (2) ◽  
pp. 199-202 ◽  
Author(s):  
Th. Fetzer ◽  
A. Lentz ◽  
T. Debaerdemaeker ◽  
O. Abou-El-Wafa
Keyword(s):  
Crystal Structure ◽  
Thermal Decomposition ◽  
Crystal Growth ◽  
Single Crystals ◽  
Crystal Structure Analysis ◽  
Van Der Waals Interactions ◽  
Monoclinic Space Group ◽  
Crystal Data ◽  
X Ray

Single crystals of Cu(pdz)Cl2 were grown by using gel methods with tetramethoxysilane as the gel-forming reagent. Thermal decomposition is interpreted. Crystal data for the complex : monoclinic, space group C2/c with a = 938.3(3) pm, b = 1242.9(3) pm, c = 675.7(4) pm, β = 128.48(5)°. A crystal structure analysis reveals CuCl2 molecules connected by pyridazine in the form of zigzag-chains. Between these chains there are only Van-der-Waals-Interactions.

scholarly journals X-Ray Crystal Structure of the Multidomain Endoglucanase Cel9G from Clostridium cellulolyticum Complexed with Natural and Synthetic Cello-Oligosaccharides

2003 ◽  
Vol 185 (14) ◽  
pp. 4127-4135 ◽  
Author(s):  
David Mandelman ◽  
Anne Belaich ◽  
J. P. Belaich ◽  
Nushin Aghajari ◽  
Hugues Driguez ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Catalytic Domain ◽  
Cellulose Degradation ◽  
Cellulase Activity ◽  
Structural Basis ◽  
Crystalline Cellulose ◽  
X Ray ◽  
Clostridium Cellulolyticum ◽  
Active Site Cleft ◽  
Cellulose Binding

ABSTRACT Complete cellulose degradation is the first step in the use of biomass as a source of renewable energy. To this end, the engineering of novel cellulase activity, the activity responsible for the hydrolysis of the β-1,4-glycosidic bonds in cellulose, is a topic of great interest. The high-resolution X-ray crystal structure of a multidomain endoglucanase from Clostridium cellulolyticum has been determined at a 1.6-Å resolution. The endoglucanase, Cel9G, is comprised of a family 9 catalytic domain attached to a family IIIc cellulose-binding domain. The two domains together form a flat platform onto which crystalline cellulose is suggested to bind and be fed into the active-site cleft for endolytic hydrolysis. To further dissect the structural basis of cellulose binding and hydrolysis, the structures of Cel9G in the presence of cellobiose, cellotriose, and a DP-10 thio-oligosaccharide inhibitor were resolved at resolutions of 1.7, 1.8, and 1.9 Å, respectively.

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