Mutation of outer-shell residues modulates metal ion co-ordination strength in a metalloenzyme

The metal ion co-ordination sites of many metalloproteins have been characterized by a variety of spectroscopic techniques and small-molecule model systems, revealing many important insights into the structural determinants of metal ion co-ordination. However, our understanding of this fundamentally and practically important phenomenon remains frustratingly simplistic; in many proteins it is essentially impossible to predict metal ion specificity and the effects of remote ‘outer-shell’ residues on metal ion co-ordination strength are also poorly defined. This is exemplified by our inability to explain why metalloenzymes with identical metal ion co-ordination spheres, such as the closely related orthologues of bacterial PTE (phosphotriesterase) from Agrobacterium radiobacter and Pseudomonas diminuta, display different metal ion specificity and co-ordination strength. In the present study, we present a series of PTE variants that all possess identical metal ion co-ordination spheres, yet display large differences in their metal ion co-ordination strength. Using measurement of the rates of metal ion dissociation from the active site alongside analysis of structural data obtained through X-ray crystallography, we show that ‘outer-shell’ residues provide essential support for the metal ion ligands, in effect buttressing them in their optimal orientation. Remote mutations appear to modulate metal ion interactions by increasing or decreasing the stabilizing effects of these networks. The present study therefore provides a description of how the greater protein fold can be modified to ‘tune’ the strength of metal ion co-ordination and metal ion specificity, as well as reinforcing the concept of proteins as ensembles of conformational states with unique structures and biochemical properties.

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

Scientific Reports ◽

10.1038/s41598-021-93419-x ◽

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.

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Structural Basis for Receptor Recognition by Lujo Virus

10.1101/329482 ◽

2018 ◽

Author(s):

Hadas Cohen-Dvashi ◽

Itay Kilimnik ◽

Ron Diskin

Keyword(s):

Metal Ion ◽

New World ◽

Cell Attachment ◽

Evolutionary Relationship ◽

Structural Data ◽

Structural Basis ◽

Human Populations ◽

X Ray Crystallography ◽

Transferrin Receptor 1 ◽

Receptor Recognition

AbstractLujo virus (LUJV) has emerged as a novel and highly fatal human pathogen. Despite its membership among the Arenaviridae, LUJV does not classify with the known Old and New World groups of that viral family. Likewise, LUJV was recently found to use neuropilin-2 (NRP2) as a cellular receptor instead of the canonical α-dystroglycan (α-DG) or transferrin receptor 1 (TfR1) utilized by Old World (OW) and New World (NW) arenaviruses, respectively. The emergence of a deadly new pathogen into human populations using an unprecedented entry route raises many questions regarding the mechanism of cell recognition and the risk that Arenaviruses are further diversifying their infection strategies. To provide the basis for combating LUJV in particular, and to increase our general understanding of the molecular changes that accompany an evolutionary switch to a new receptor for Arenaviruses, we used X-ray crystallography to reveal how the GP1 receptor-binding domain of LUJV (LUJVGP1) recognizes NRP2. Our structural data imply that LUJV is evolutionary closer to OW than to NW arenaviruses. Structural analysis supported by experimental validation further suggests that NRP2 recognition is metal ion dependent and that the complete NRP2 binding is formed in the context of the trimeric spike. Taken together, our data provide the mechanism for the cell attachment step of LUJV, the evolutionary relationship between the GP1 domain of this novel pathogen and other arenaviruses, and indispensable information for combating LUJV.

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The structure of the Mn 4 Ca 2+ cluster of photosystem II and its protein environment as revealed by X-ray crystallography

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2007.2208 ◽

2007 ◽

Vol 363 (1494) ◽

pp. 1129-1138 ◽

Cited By ~ 45

Author(s):

James Barber ◽

James W Murray

Keyword(s):

Fine Structure ◽

Photosystem Ii ◽

Water Splitting ◽

Metal Ion ◽

Spectroscopic Techniques ◽

Precise Position ◽

X Ray ◽

X Ray Crystallography ◽

X Ray Absorption ◽

Protein Environment

The location, structure and protein environment of the Mn 4 Ca 2+ cluster, which catalyses the light-driven, water-splitting reaction of photosystem II, has been revealed by X-ray crystallography. However, owing to the low resolutions of the crystal structures reported to date, and the possibility of radiation damage at the catalytic centre, the precise position of each metal ion remains unknown. To some extent, these problems have been overcome by applying spectroscopic techniques like extended X-ray absorption fine structure. Taking into account the most recent results obtained with these two X-ray-based techniques, we have attempted to refine models of the structure of the Mn 4 Ca 2+ cluster and its protein environment.

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Versatile Colorimetric Chemosensor Based on Bis(rhodamine) B Hydrazone for Rapid Detection of Multi-Analytes (Fe3+, Bi3+, Cu2+, and Hg2+) in Aqueous Media

Australian Journal of Chemistry ◽

10.1071/ch20046 ◽

2020 ◽

Vol 73 (11) ◽

pp. 1112

Author(s):

Tamer El Malah ◽

Hany F. Nour

Keyword(s):

Rhodamine B ◽

Metal Ion ◽

Chemical Structure ◽

Aqueous Media ◽

Spectroscopic Techniques ◽

Ion Interactions ◽

Colour Changes ◽

Metal Ion Interactions ◽

Job's Method ◽

Naked Eye Detection

A new bis(rhodamine) B hydrazone L, bearing two rhodamine chromophores, has been designed and synthesised in 70% yield. The chemical structure of the new ligand L was characterised using different spectroscopic techniques. The receptor L was utilised for rapid ‘naked eye’ detection of Fe3+, Bi3+, Cu2+, and Hg2+ in THF–H2O (1:1, v/v) by displaying significant colour changes. The mechanism of detection of metal ions was based on the ring-opening of the spirolactam rings of L upon recognition. UV/Vis spectrometry was employed to investigate L–metal ion interactions in THF–H2O (1:1, v/v). The stoichiometries of the complexes were determined using Job’s method, which revealed 1:2L–metal ion stoichiometries in the case of L–(Fe3+/Bi3+/Hg2+) complexes, while a 1:1L–metal ion stoichiometry was recognised for the L–Cu2+ complex. 1H NMR spectroscopy was applied to study the L–metal ion interactions in DMSO-d6 and results are presented.

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Interpretation of spectroscopic data using molecular simulations for the secondary active transporter BetP

The Journal of General Physiology ◽

10.1085/jgp.201812111 ◽

2019 ◽

Vol 151 (3) ◽

pp. 381-394 ◽

Cited By ~ 2

Author(s):

Vanessa Leone ◽

Izabela Waclawska ◽

Katharina Kossmann ◽

Caroline Koshy ◽

Monika Sharma ◽

...

Keyword(s):

Spectroscopic Data ◽

Double Resonance ◽

Structural Data ◽

Spin Labels ◽

Conformational Ensemble ◽

Spectroscopic Techniques ◽

Coupled Transport ◽

X Ray Crystallography ◽

Simulation Techniques ◽

Double Electron Electron Resonance

Mechanistic understanding of dynamic membrane proteins such as transporters, receptors, and channels requires accurate depictions of conformational ensembles, and the manner in which they interchange as a function of environmental factors including substrates, lipids, and inhibitors. Spectroscopic techniques such as electron spin resonance (ESR) pulsed electron–electron double resonance (PELDOR), also known as double electron–electron resonance (DEER), provide a complement to atomistic structures obtained from x-ray crystallography or cryo-EM, since spectroscopic data reflect an ensemble and can be measured in more native solvents, unperturbed by a crystal lattice. However, attempts to interpret DEER data are frequently stymied by discrepancies with the structural data, which may arise due to differences in conditions, the dynamics of the protein, or the flexibility of the attached paramagnetic spin labels. Recently, molecular simulation techniques such as EBMetaD have been developed that create a conformational ensemble matching an experimental distance distribution while applying the minimal possible bias. Moreover, it has been proposed that the work required during an EBMetaD simulation to match an experimentally determined distribution could be used as a metric with which to assign conformational states to a given measurement. Here, we demonstrate the application of this concept for a sodium-coupled transport protein, BetP. Because the probe, protein, and lipid bilayer are all represented in atomic detail, the different contributions to the work, such as the extent of protein backbone movements, can be separated. This work therefore illustrates how ranking simulations based on EBMetaD can help to bridge the gap between structural and biophysical data and thereby enhance our understanding of membrane protein conformational mechanisms.

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4-vinylpyridine derivatives: Protonation, methylation and silver(I) coordination chemistry

Journal of Chemical Research ◽

10.1177/1747519821989659 ◽

2021 ◽

pp. 174751982198965

Author(s):

Guoqi Zhang

Keyword(s):

Mass Spectrometry ◽

Schiff Base ◽

Coordination Chemistry ◽

Elemental Analysis ◽

Silver Complex ◽

Spectroscopic Techniques ◽

X Ray ◽

X Ray Crystallography ◽

Pyridine Moiety ◽

New Compounds

( E)-4-[2-(Pyridin-4-yl)vinyl]benzaldehyde, containing both a 4-vinylpyridine and an aldehyde functionality, is utilized to develop new, highly conjugated chalcone compounds and a bis-Schiff base azine compound. The chalcone-containing compounds are further explored for their protonation, methylation and silver(I) coordination chemistry using the pyridine moiety. In parallel, a cyano-containing analogue, ( E)-4-[2-(pyridin-4-yl)vinyl]benzonitrile is also synthesized and studied for its silver(I) coordination chemistry. These new compounds are fully characterized by mass spectrometry, elemental analysis and spectroscopic techniques. The methylated product of ( E)-1-(9-anthryl)-3-{4-[2-(pyridin-4-yl)vinyl]phenyl}prop-2-en-1-one and a silver complex of ( E)-4-[2-(pyridin-4-yl)vinyl]benzonitrile are structurally determined by X-ray crystallography.

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.005134 ◽

2018 ◽

Vol 293 (47) ◽

pp. 18296-18308 ◽

Cited By ~ 10

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.

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