scholarly journals A novel inhibitor of complement C5 provides structural insights into activation

2019 ◽  
Author(s):  
Martin P. Reichhardt ◽  
Steven Johnson ◽  
Terence Tang ◽  
Thomas Morgan ◽  
Nchimunya Tebeka ◽  
...  
Keyword(s):  
Function Analysis ◽  
Structural Data ◽  
The Novel ◽  
Tick Saliva ◽  
X Ray Crystallography ◽  
Inhibitory Function ◽  
Binding Interface ◽  
Rhipicephalus Pulchellus ◽  
Direct Binding ◽  
Immune Defences

AbstractThe complement system is a crucial part of innate immune defences against invading pathogens. The blood-meal of the tick Rhipicephalus pulchellus lasts for days, and the tick must therefore rely on inhibitors to counter complement activation. We have identified a novel class of inhibitors from tick saliva, the CirpT family, and generated detailed structural data revealing their mechanism of action. We show direct binding of a CirpT to complement C5 and have determined the structure of the C5-CirpT complex by cryo-electron microscopy. This reveals an interaction with the peripheral macro globulin domain 4 (C5_MG4) of C5. To achieve higher resolution detail, the structure of the C5_MG4-CirpT complex was solved by X-ray crystallography (at 2.7 Å). We thus present the novel fold of the CirpT protein family, and provide detailed mechanistic insights into its inhibitory function. Analysis of the binding interface reveals a novel mechanism of C5 inhibition, and provides information to expand our biological understanding of the activation of C5, and thus the terminal complement pathway.

scholarly journals An inhibitor of complement C5 provides structural insights into activation

2019 ◽  
Vol 117 (1) ◽  
pp. 362-370 ◽  
Author(s):  
Martin P. Reichhardt ◽  
Steven Johnson ◽  
Terence Tang ◽  
Thomas Morgan ◽  
Nchimunya Tebeka ◽  
...  
Keyword(s):  
Function Analysis ◽  
Structural Data ◽  
Tick Saliva ◽  
X Ray Crystallography ◽  
Inhibitory Function ◽  
Binding Interface ◽  
Rhipicephalus Pulchellus ◽  
Direct Binding ◽  
The Complement System ◽  
Crucial Part

The complement system is a crucial part of innate immune defenses against invading pathogens. The blood-meal of the tickRhipicephalus pulchelluslasts for days, and the tick must therefore rely on inhibitors to counter complement activation. We have identified a class of inhibitors from tick saliva, the CirpT family, and generated detailed structural data revealing their mechanism of action. We show direct binding of a CirpT to complement C5 and have determined the structure of the C5–CirpT complex by cryoelectron microscopy. This reveals an interaction with the peripheral macro globulin domain 4 (C5_MG4) of C5. To achieve higher resolution detail, the structure of the C5_MG4–CirpT complex was solved by X-ray crystallography (at 2.7 Å). We thus present the fold of the CirpT protein family, and provide detailed mechanistic insights into its inhibitory function. Analysis of the binding interface reveals a mechanism of C5 inhibition, and provides information to expand our biological understanding of the activation of C5, and thus the terminal complement pathway.

Axial Ligand Effects in Sterically Strained Porphyrins: A Crystallographic Study of Five- and Six-coordinated Metal Complexes of 2,3,7,8,12,13,17,18-Octaethyl-5,10,15,20-tetranitroporphyrin

1998 ◽  
Vol 02 (02) ◽  
pp. 107-121 ◽  
Author(s):  
MATHIAS O. SENGE
Keyword(s):  
High Spin ◽  
Mutual Influence ◽  
Structural Data ◽  
Axial Ligand ◽  
The Novel ◽  
X Ray Crystallography ◽  
Axial Ligands ◽  
Metal Derivatives ◽  
Ligand Orientation ◽  
Derivatives Of

Porphyrins with steric hindrance at the periphery are known to exhibit severely non-planar macrocycle conformations. Among other dodecasubstituted porphyrins, the title compound has been studied widely and shows a typical saddle-distorted macrocycle. The specific conformation of the porphyrin leads to the formation of distinct cavities on both sides of the macrocycle. Compared to planar porphyrins this should increase steric interactions between the macrocycle and axial ligands in five- and six-coordinated metal derivatives. In order to study the influence of different axial ligands on the conformation (or vice versa) a variety of five- and six-coordinated metal derivatives of 2,3,7,8,12,13,17,18-octaethyl-5,10,15,20-tetranitroporphyrin (oetnp) were prepared and their conformation investigated by X-ray crystallography. Structural data for Zn II oetnp (L) where L is imidazole, 1-methylimidazole, 2-methylimidazole, 4-methylimidazole and 3,5-lutidine clearly indicated that a mutual influence exists between axial ligands and non-planar porphyrins. An asymmetric macrocycle distortion and axial ligand orientation was found in the sterically hindered 2- and 4-methylimidazole derivatives. The presence of four electron-withdrawing nitro-groups led to the formation of the novel polymeric porphyrins ( Zn II oetnp )n and ( Co II oetnp )n where polymer formation was achieved via the utilization of nitro oxygen atoms as axial ligands to the metal centre of neighbouring porphyrins. The structure of the six-coordinated Ni II oetnp ( pyr )2 (where pyr is pyridine) yielded first structural data on neutral, non-planar, high-spin Ni ( II ) porphyrins. A comparison with the respective low-spin Ni II oetnp clearly showed that a change to the high-spin form leads to a less non-planar macrocycle conformation further proving that spin state changes can have a profound effect on the conformation in distorted porphyrins.

scholarly journals Identification of V6.51L as a selectivity hotspot in stereoselective A2B adenosine receptor antagonist recognition

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Xuesong Wang ◽  
Willem Jespers ◽  
Rubén Prieto-Díaz ◽  
Maria Majellaro ◽  
Adriaan P. IJzerman ◽  
...  
Keyword(s):  
Radioligand Binding ◽  
Structural Data ◽  
Binding Mode ◽  
Selective Recognition ◽  
Chiral Hplc ◽  
Binding Assays ◽  
Structural Determinants ◽  
X Ray Crystallography ◽  
Adenosine Receptor Antagonist ◽  
Energy Perturbation

AbstractThe four adenosine receptors (ARs) A1AR, A2AAR, A2BAR, and A3AR are G protein-coupled receptors (GPCRs) for which an exceptional amount of experimental and structural data is available. Still, limited success has been achieved in getting new chemical modulators on the market. As such, there is a clear interest in the design of novel selective chemical entities for this family of receptors. In this work, we investigate the selective recognition of ISAM-140, a recently reported A2BAR reference antagonist. A combination of semipreparative chiral HPLC, circular dichroism and X-ray crystallography was used to separate and unequivocally assign the configuration of each enantiomer. Subsequently affinity evaluation for both A2A and A2B receptors demonstrate the stereospecific and selective recognition of (S)-ISAM140 to the A2BAR. The molecular modeling suggested that the structural determinants of this selectivity profile would be residue V2506.51 in A2BAR, which is a leucine in all other ARs including the closely related A2AAR. This was herein confirmed by radioligand binding assays and rigorous free energy perturbation (FEP) calculations performed on the L249V6.51 mutant A2AAR receptor. Taken together, this study provides further insights in the binding mode of these A2BAR antagonists, paving the way for future ligand optimization.

scholarly journals Endo-fucoidan hydrolases from glycoside hydrolase family 107 (GH107) display structural and mechanistic similarities to α-l-fucosidases from GH29

2018 ◽  
Vol 293 (47) ◽  
pp. 18296-18308 ◽  
Author(s):  
Chelsea Vickers ◽  
Feng Liu ◽  
Kento Abe ◽  
Orly Salama-Alber ◽  
Meredith Jenkins ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Biological Activities ◽  
Bacterial Species ◽  
Structural Data ◽  
Side Chain ◽  
Glycoside Hydrolase Family ◽  
X Ray Crystallography ◽  
Catalytic Mechanisms ◽  
Thickening Agents ◽  
Hydrolase Family

Fucoidans are chemically complex and highly heterogeneous sulfated marine fucans from brown macro algae. Possessing a variety of physicochemical and biological activities, fucoidans are used as gelling and thickening agents in the food industry and have anticoagulant, antiviral, antitumor, antibacterial, and immune activities. Although fucoidan-depolymerizing enzymes have been identified, the molecular basis of their activity on these chemically complex polysaccharides remains largely uninvestigated. In this study, we focused on three glycoside hydrolase family 107 (GH107) enzymes: MfFcnA and two newly identified members, P5AFcnA and P19DFcnA, from a bacterial species of the genus Psychromonas. Using carbohydrate-PAGE, we show that P5AFcnA and P19DFcnA are active on fucoidans that differ from those depolymerized by MfFcnA, revealing differential substrate specificity within the GH107 family. Using a combination of X-ray crystallography and NMR analyses, we further show that GH107 family enzymes share features of their structures and catalytic mechanisms with GH29 α-l-fucosidases. However, we found that GH107 enzymes have the distinction of utilizing a histidine side chain as the proposed acid/base catalyst in its retaining mechanism. Further interpretation of the structural data indicated that the active-site architectures within this family are highly variable, likely reflecting the specificity of GH107 enzymes for different fucoidan substructures. Together, these findings begin to illuminate the molecular details underpinning the biological processing of fucoidans.

scholarly journals Molecular mechanism of ATP versus GTP selectivity of adenylate kinase

2018 ◽  
Vol 115 (12) ◽  
pp. 3012-3017 ◽  
Author(s):  
Per Rogne ◽  
Marie Rosselin ◽  
Christin Grundström ◽  
Christian Hedberg ◽  
Uwe H. Sauer ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
Adenylate Kinase ◽  
Function Analysis ◽  
Substrate Selectivity ◽  
Cellular Functions ◽  
X Ray ◽  
Precise Control ◽  
X Ray Crystallography ◽  
The Family ◽  
Atp Analogs

Enzymatic substrate selectivity is critical for the precise control of metabolic pathways. In cases where chemically related substrates are present inside cells, robust mechanisms of substrate selectivity are required. Here, we report the mechanism utilized for catalytic ATP versus GTP selectivity during adenylate kinase (Adk) -mediated phosphorylation of AMP. Using NMR spectroscopy we found that while Adk adopts a catalytically competent and closed structural state in complex with ATP, the enzyme is arrested in a catalytically inhibited and open state in complex with GTP. X-ray crystallography experiments revealed that the interaction interfaces supporting ATP and GTP recognition, in part, are mediated by coinciding residues. The mechanism provides an atomic view on how the cellular GTP pool is protected from Adk turnover, which is important because GTP has many specialized cellular functions. In further support of this mechanism, a structure–function analysis enabled by synthesis of ATP analogs suggests that a hydrogen bond between the adenine moiety and the backbone of the enzyme is vital for ATP selectivity. The importance of the hydrogen bond for substrate selectivity is likely general given the conservation of its location and orientation across the family of eukaryotic protein kinases.

scholarly journals A Structural Overview of Vascular Endothelial Growth Factors Pharmacological Ligands: From Macromolecules to Designed Peptidomimetics

Molecules ◽  
2021 ◽  
Vol 26 (22) ◽  
pp. 6759
Author(s):  
Xiaoqing Ye ◽  
Jean-François Gaucher ◽  
Michel Vidal ◽  
Sylvain Broussy
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Unnatural Amino Acids ◽  
Structural Data ◽  
Age Related Macular Degeneration ◽  
Vascular Endothelial ◽  
Binding Modes ◽  
Therapeutic Applications ◽  
X Ray Crystallography ◽  
Age Related

The vascular endothelial growth factor (VEGF) family of cytokines plays a key role in vasculogenesis, angiogenesis, and lymphangiogenesis. VEGF-A is the main member of this family, alongside placental growth factor (PlGF), VEGF-B/C/D in mammals, and VEGF-E/F in other organisms. To study the activities of these growth factors under physiological and pathological conditions, resulting in therapeutic applications in cancer and age-related macular degeneration, blocking ligands have been developed. These have mostly been large biomolecules like antibodies. Ligands with high affinities, at least in the nanomolar range, and accurate structural data from X-ray crystallography and NMR spectroscopy have been described. They constitute the main focus of this overview, which evidences similarities and differences in their binding modes. For VEGF-A ligands, and to a limited extent also for PlGF, a transition is now observed towards developing smaller ligands like nanobodies and peptides. These include unnatural amino acids and chemical modifications for designed and improved properties, such as serum stability and greater affinity. However, this review also highlights the scarcity of such small molecular entities and the striking lack of small organic molecule ligands. It also shows the gap between the rather large array of ligands targeting VEGF-A and the general absence of ligands binding other VEGF members, besides some antibodies. Future developments in these directions are expected in the upcoming years, and the study of these growth factors and their promising therapeutic applications will be welcomed.

scholarly journals A Novel Monovalent Cation-Stabilized Fold Mediates an Aptamer-Protein Complex

2014 ◽  
Vol 70 (a1) ◽  
pp. C1164-C1164
Author(s):  
Camille Padlan ◽  
Vladimir Malashkevich ◽  
Steve Almo ◽  
Matthew Levy ◽  
Michael Brenowitz ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Binding Free Energy ◽  
Monovalent Cation ◽  
Rna Aptamers ◽  
Diagnostic Tools ◽  
Surprising Result ◽  
Chemical Mapping ◽  
X Ray Crystallography ◽  
Binding Interface ◽  
Shape Mapping

RNA aptamers are structured single-stranded oligonucleotides selected to bind tightly and specifically to a broad spectrum of biomolecular targets. The structural stability and diverse functionality of aptamers have enabled their use as diagnostic tools, inhibitors and potential therapeutic agents. However, since very few attempts at solving atomic structures of protein-aptamer complexes have succeeded, surprisingly little is known about how aptamers specifically bind to selected regions on the surface of proteins and cells. We show that aptamers can be effectively minimized for structural analysis using chemical mapping to experimentally define the secondary structure and identify tertiary contacts within the RNA and with the target protein. Ribonuclease and SHAPE mapping were used to determine the correct predicted secondary structure of a high affinity aptamer (Lys1) selected against lysozyme (KD ~ 30 nM). A deletion variant, minE (KD ~ 20 nM), was engineered to delete a long, apparently unstructured region. The lysozyme-minE complex was determined by x-ray crystallography at a 2.0 Å resolution, yielding a seventh RNA aptamer-protein structure. Solution hydroxyl-radical footprinting confirms the binding interface observed in the crystal. Although the minE aptamer interacts with a positively charged face of lysozyme, the electrostatic contribution to the binding free energy is minimal. The minE aptamer was found to inhibit the function of lysozyme in the standard cell-wall hydrolysis assay – a surprising result since the aptamer binding site is quite far from the catalytic site, and no structural differences between free lysozyme and that in complex with the aptamer could be detected. The long term goal of this study is to develop a systematic approach to aptamer minimization and use solved structures to probe the mechanisms by which RNA aptamers bind their targets and regulate catalytic activity and/or cellular function.

scholarly journals Functional CBS modules make IMPDHs octameric

2014 ◽  
Vol 70 (a1) ◽  
pp. C1283-C1283
Author(s):  
Gilles Labesse ◽  
Thomas Alexandre ◽  
Laurène Vaupré ◽  
Isabelle Salard-Arnaud ◽  
Joséphine Lai Kee Him ◽  
...  
Keyword(s):  
Analytical Ultracentrifugation ◽  
Quaternary Structure ◽  
Catalytic Domain ◽  
Structural Data ◽  
Site Directed Mutagenesis ◽  
X Ray Crystallography ◽  
Binding Pockets ◽  
Essential Enzyme ◽  
Cbs Domains

Inosine-5'-monophosphate dehydrogenase (1, 2) (IMPDH) is a major target for antiviral, antiparasitic, antileukemic and immunosuppressive therapies. It is an ubiquitous and essential enzyme for the biosynthesis of guanosine nucleotides. Up to now, IMPDHs have been reported as tetrameric enzymes harbouring a catalytic domain and a tandem of cystathionine-ß-synthase (CBS) modules. The latter had no precise function assigned despite their nearly absolute conservation among IMPDHs and consistent indication of their importance in vivo. The aim of our study was to provide evidence for the role of the CBS modules on the quaternary structure and on the regulation of IMPDHs. A multidisciplinary approach involving enzymology, site-directed mutagenesis, analytical ultracentrifugation, X-ray crystallography, SAXS, cryo-electron microscopy and molecular modelling allowed us to demonstrate that the Pseudomonas aeruginosa IMPDH is functionally active as an octamer and allosterically regulated by MgATP via each CBS module. Revisiting deposited structural data, we found this newly discovered octameric organization conserved in other IMPDH structures. Meanwhile, we demonstrated that the human IMPDH1 formed two distinct octamers that can pile up into isolated fibres in the presence of MgATP while its pathogenic mutant D226N, localised into the CBS domains, appeared to form massively aggregating fibres. The dramatic impact of this mutation could explain the severe retinopathy adRP10. Our data (3) revealed for the first time that IMPDH has an octameric architecture modulated by MgATP binding to the CBS modules, inducing large structural rearrangements. Thus, the regulatory CBS modules in IMPDHs are functional and they can either modulate catalysis or/and macromolecular assembly. Targeting the conserved effector binding pockets identified within the CBS modules might be promising to develop antibacterial compounds or drugs to counter retinopathy onset.

scholarly journals How Do Molecular Dynamics Data Complement Static Structural Data of GPCRs

2020 ◽  
Vol 21 (16) ◽  
pp. 5933 ◽  
Author(s):  
Mariona Torrens-Fontanals ◽  
Tomasz Maciej Stepniewski ◽  
David Aranda-García ◽  
Adrián Morales-Pastor ◽  
Brian Medel-Lacruz ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Structural Data ◽  
Md Simulations ◽  
Full Potential ◽  
Missing Information ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
G Protein Coupled ◽  
Gpcr Signal ◽  
Important Drug

G protein-coupled receptors (GPCRs) are implicated in nearly every physiological process in the human body and therefore represent an important drug targeting class. Advances in X-ray crystallography and cryo-electron microscopy (cryo-EM) have provided multiple static structures of GPCRs in complex with various signaling partners. However, GPCR functionality is largely determined by their flexibility and ability to transition between distinct structural conformations. Due to this dynamic nature, a static snapshot does not fully explain the complexity of GPCR signal transduction. Molecular dynamics (MD) simulations offer the opportunity to simulate the structural motions of biological processes at atomic resolution. Thus, this technique can incorporate the missing information on protein flexibility into experimentally solved structures. Here, we review the contribution of MD simulations to complement static structural data and to improve our understanding of GPCR physiology and pharmacology, as well as the challenges that still need to be overcome to reach the full potential of this technique.

scholarly journals Assignment of the Ferriheme Resonances of the Low-Spin Complexes of Nitrophorins 1 and 4 by1H and13C NMR Spectroscopy:  Comparison to Structural Data Obtained from X-ray Crystallography

2007 ◽  
Vol 46 (6) ◽  
pp. 2041-2056 ◽  
Author(s):  
Tatiana Kh. Shokhireva ◽  
Andrzej Weichsel ◽  
Kevin M. Smith ◽  
Robert E. Berry ◽  
Nikolai V. Shokhirev ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Structural Data ◽  
X Ray ◽  
X Ray Crystallography
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