Elucidation of the coenzyme binding mode of further B12-dependent enzymes using a base-off analogue of coenzyme B12

Glutamate mutase catalyses an unusual isomerization involving free-radical intermediates that are generated by homolysis of the cobalt–carbon bond of the coenzyme adenosylcobalamin (coenzyme B12). A variety of techniques have been used to examine the interaction between the protein and adenosylcobalamin, and between the protein and the products of coenzyme homolysis, cob(II)alamin and 5′-deoxyadenosine. These include equilibrium gel filtration, isothermal titration calorimetry, and resonance Raman, UV-visible and EPR spectroscopies. The thermodynamics of adenosylcobalamin binding to the protein have been examined and appear to be entirely entropy-driven, with ∆S = 109 Jċmol-1ċK-1. The cobalt–carbon bond stretching frequency is unchanged upon coenzyme binding to the protein, arguing against a ground-state destabilization of the cobalt–carbon bond of adenosylcobalamin by the protein. However, reconstitution of the enzyme with cob(II)alamin and 5′-deoxyadenosine, the two stable intermediates formed subsequent to homolysis, results in the blue-shifting of two of the bands comprising the UV-visible spectrum of the corrin ring. The most plausible interpretation of this result is that an interaction between the protein, 5′-deoxyadenosine and cob(II)alamin introduces a distortion into the ring corrin that perturbs its electronic properties.

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Ethanolamine Ammonia-Lyase Has a “Base-On” Binding Mode for Coenzyme B12

Archives of Biochemistry and Biophysics ◽

10.1006/abbi.1999.1382 ◽

1999 ◽

Vol 370 (1) ◽

pp. 138-141 ◽

Cited By ~ 35

Author(s):

Andreas Abend ◽

Vahe Bandarian ◽

Rainer Nitsche ◽

Erhard Stupperich ◽

Janos Rétey ◽

...

Keyword(s):

Binding Mode ◽

Coenzyme B12

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Unique coenzyme binding mode of hyperthermophilic archaealsn-glycerol-1-phosphate dehydrogenase fromPyrobaculum calidifontis

Proteins Structure Function and Bioinformatics ◽

10.1002/prot.25161 ◽

2016 ◽

Vol 84 (12) ◽

pp. 1786-1796 ◽

Cited By ~ 1

Author(s):

Junji Hayashi ◽

Kaori Yamamoto ◽

Kazunari Yoneda ◽

Toshihisa Ohshima ◽

Haruhiko Sakuraba

Keyword(s):

Binding Mode ◽

Coenzyme Binding

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Characterization of coenzyme binding and selectivity determinants in Mycobacterium tuberculosis flavoprotein reductase A: analysis of Arg199 and Arg200 mutants at the NADP(H) 2′-phosphate binding site

Biochemical Journal ◽

10.1042/bj20080466 ◽

2008 ◽

Vol 417 (1) ◽

pp. 103-114 ◽

Cited By ~ 7

Author(s):

Muna Sabri ◽

Adrian J. Dunford ◽

Kirsty J. McLean ◽

Rajasekhar Neeli ◽

Nigel S. Scrutton ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Substrate Inhibition ◽

Reduction Rate ◽

Binding Mode ◽

Phosphate Binding ◽

Ferricyanide Reduction ◽

Coenzyme Specificity ◽

Coenzyme Binding ◽

Kd Values ◽

Adrenodoxin Reductase

Mycobacterium tuberculosis FprA (flavoprotein reductase A) is an NAD(P)H- and FAD-binding reductase that is structurally/evolutionarily related to adrenodoxin reductase. Structural analysis implicates Arg199 and Arg200 in interactions with the NADP(H) 2′-phosphate group. R199A, R200A and R199A/R200A mutants were characterized to explore the roles of these basic residues. All mutations abolished neutral FAD semiquinone stabilization in the NADPH-reduced enzyme, owing to weakened NADPH affinity. Instead, FAD hydroquinone was formed in all mutants, and each displayed substantially enhanced autooxidation rates (20–40-fold) compared with NADPH-reduced WT (wild-type) FprA. Steady-state ferricyanide reduction studies revealed diminished NADPH affinity (higher Km values), but lower NADH Km values. Despite a lowered kcat, the R199A/R200A mutant exhibited a 200-fold coenzyme specificity switch towards NADH, although substrate inhibition was observed at high NADH concentrations (Ki=250 μM). Stopped-flow FAD reduction studies confirmed substantially increased NADPH Kd values, although the limiting flavin reduction rate constant was similar in all mutants. The R199A mutation abolished electron transfer between hydroquinone FprA and NADP+, while this reaction progressed (via an FADH2-NADP+ charge-transfer intermediate) for R200A FprA, albeit more slowly (klim=58.1 s−1 compared with >300 s−1) than in WT. All mutations caused positive shifts in FAD potential (∼40–65 mV). Binding of an NADPH analogue (tetrahydro-NADP) induced negative shifts in potential (∼30–40 mV) only for variants with the R200A mutation, indicating distinctive effects of Arg199/Arg200 on coenzyme binding mode and FAD potential. Collectively, these data reveal important roles for the phylogenetically conserved arginines in controlling FprA FAD environment, thermodynamics, coenzyme selectivity and reactivity.

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Mobile loop dynamics in adenosyltransferase control binding and reactivity of coenzyme B12

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2007332117 ◽

2020 ◽

Vol 117 (48) ◽

pp. 30412-30422

Author(s):

Romila Mascarenhas ◽

Markus Ruetz ◽

Liam McDevitt ◽

Markos Koutmos ◽

Ruma Banerjee

Keyword(s):

Mycobacterium Tuberculosis ◽

Active Sites ◽

Solvent Accessibility ◽

Binding Mode ◽

Coenzyme B12 ◽

Coordination Geometry ◽

Loop Dynamics ◽

N Terminus ◽

Bond Homolysis ◽

Side Pocket

Cobalamin is a complex organometallic cofactor that is processed and targeted via a network of chaperones to its dependent enzymes. AdoCbl (5′-deoxyadenosylcobalamin) is synthesized from cob(II)alamin in a reductive adenosylation reaction catalyzed by adenosyltransferase (ATR), which also serves as an escort, delivering AdoCbl to methylmalonyl-CoA mutase (MCM). The mechanism by which ATR signals that its cofactor cargo is ready (AdoCbl) or not [cob(II)alamin] for transfer to MCM, is not known. In this study, we have obtained crystallographic snapshots that reveal ligand-induced ordering of the N terminus ofMycobacterium tuberculosisATR, which organizes a dynamic cobalamin binding site and exerts exquisite control over coordination geometry, reactivity, and solvent accessibility. Cob(II)alamin binds with its dimethylbenzimidazole tail splayed into a side pocket and its corrin ring buried. The cosubstrate, ATP, enforces a four-coordinate cob(II)alamin geometry, facilitating the unfavorable reduction to cob(I)alamin. The binding mode for AdoCbl is notably different from that of cob(II)alamin, with the dimethylbenzimidazole tail tucked under the corrin ring, displacing the N terminus of ATR, which is disordered. In this solvent-exposed conformation, AdoCbl undergoes facile transfer to MCM. The importance of the tail in cofactor handover from ATR to MCM is revealed by the failure of 5′-deoxyadenosylcobinamide, lacking the tail, to transfer. In the absence of MCM, ATR induces a sacrificial cobalt–carbon bond homolysis reaction in an unusual reversal of the heterolytic chemistry that was deployed to make the same bond. The data support an important role for the dimethylbenzimidazole tail in moving the cobalamin cofactor between active sites.

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The –SH Protection Method for Determining Accurate Kd Values for Enzyme-Coenzyme Complexes of NAD+-Dependent Glutamate Dehydrogenase and Engineered Mutants: Evidence for Nonproductive NADPH Complexes

Enzyme Research ◽

10.4061/2010/951472 ◽

2010 ◽

Vol 2010 ◽

pp. 1-5 ◽

Cited By ~ 1

Author(s):

Joanna Griffin ◽

Paul C. Engel

Keyword(s):

Glutamate Dehydrogenase ◽

Binding Mode ◽

Apparent Contradiction ◽

Double Mutation ◽

Coenzyme Binding ◽

Protection Method ◽

Kd Values ◽

Activity Measurements ◽

Nonproductive Binding

Inactivation rates have been measured for clostridial glutamate dehydrogenase and several engineered mutants at various DTNB concentrations. Analysis of rate constants allowed determination of Kd for each non-covalent enzyme-DTNB complex and the rate constant for reaction to form the inactive enzyme-thionitrobenzoate adduct. Both parameters are sensitive to the mutations F238S, P262S, the double mutation F238S/P262S, and D263K, all in the coenzyme binding site. Study of the effects of NAD+, NADH and NADPH at various concentrations in protecting against inactivation by 200 μM DTNB allowed determination of Kd values for binding of these coenzymes to each protein, yielding surprising results. The mutations were originally devised to lessen discrimination against the disfavoured coenzyme NADP(H), and activity measurements showed this was achieved. However, the Kd determinations indicated that, although Kd values for NAD+ and NADH were increased considerably, Kd for NADPH was increased even more than for NADH, so that discrimination against binding of NADPH was not decreased. This apparent contradiction can only be explained if NADPH has a nonproductive binding mode that is not weakened by the mutations, and a catalytically productive mode that, though strengthened, is masked by the nonproductive binding. Awareness of the latter is important in planning further mutagenesis.

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Experimental validation of the predicted TGR5 binding mode model

Zeitschrift für Gastroenterologie ◽

10.1055/s-0034-1397135 ◽

2015 ◽

Vol 53 (01) ◽

Author(s):

L Spomer ◽

CGW Gertzen ◽

D Häussinger ◽

H Gohlke ◽

V Keitel

Keyword(s):

Experimental Validation ◽

Binding Mode

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Binding Modes of Ligands Using Enhanced Sampling (BLUES): Rapid Decorrelation of Ligand Binding Modes Using Nonequilibrium Candidate Monte Carlo

10.26434/chemrxiv.5406907.v2 ◽

2017 ◽

Author(s):

Samuel Gill ◽

Nathan M. Lim ◽

Patrick Grinaway ◽

Ariën S. Rustenburg ◽

Josh Fass ◽

...

Keyword(s):

Monte Carlo ◽

Ligand Binding ◽

Binding Mode ◽

Free Energy Calculations ◽

Dynamics Simulation ◽

Binding Modes ◽

Enhanced Sampling ◽

Recent Success ◽

Multiple Binding ◽

Proper Sampling

<div>Accurately predicting protein-ligand binding is a major goal in computational chemistry, but even the prediction of ligand binding modes in proteins poses major challenges. Here, we focus on solving the binding mode prediction problem for rigid fragments. That is, we focus on computing the dominant placement, conformation, and orientations of a relatively rigid, fragment-like ligand in a receptor, and the populations of the multiple binding modes which may be relevant. This problem is important in its own right, but is even more timely given the recent success of alchemical free energy calculations. Alchemical calculations are increasingly used to predict binding free energies of ligands to receptors. However, the accuracy of these calculations is dependent on proper sampling of the relevant ligand binding modes. Unfortunately, ligand binding modes may often be uncertain, hard to predict, and/or slow to interconvert on simulation timescales, so proper sampling with current techniques can require prohibitively long simulations. We need new methods which dramatically improve sampling of ligand binding modes. Here, we develop and apply a nonequilibrium candidate Monte Carlo (NCMC) method to improve sampling of ligand binding modes.</div><div><br></div><div>In this technique the ligand is rotated and subsequently allowed to relax in its new position through alchemical perturbation before accepting or rejecting the rotation and relaxation as a nonequilibrium Monte Carlo move. When applied to a T4 lysozyme model binding system, this NCMC method shows over two orders of magnitude improvement in binding mode sampling efficiency compared to a brute force molecular dynamics simulation. This is a first step towards applying this methodology to pharmaceutically relevant binding of fragments and, eventually, drug-like molecules. We are making this approach available via our new Binding Modes of Ligands using Enhanced Sampling (BLUES) package which is freely available on GitHub.</div>

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Binding Modes of Ligands Using Enhanced Sampling (BLUES): Rapid Decorrelation of Ligand Binding Modes Using Nonequilibrium Candidate Monte Carlo

10.26434/chemrxiv.5406907 ◽

2018 ◽

Author(s):

Samuel Gill ◽

Nathan M. Lim ◽

Patrick Grinaway ◽

Ariën S. Rustenburg ◽

Josh Fass ◽

...

Keyword(s):

Monte Carlo ◽

Ligand Binding ◽

Binding Mode ◽

Free Energy Calculations ◽

Dynamics Simulation ◽

Binding Modes ◽

Enhanced Sampling ◽

Recent Success ◽

Multiple Binding ◽

Proper Sampling

<div>Accurately predicting protein-ligand binding is a major goal in computational chemistry, but even the prediction of ligand binding modes in proteins poses major challenges. Here, we focus on solving the binding mode prediction problem for rigid fragments. That is, we focus on computing the dominant placement, conformation, and orientations of a relatively rigid, fragment-like ligand in a receptor, and the populations of the multiple binding modes which may be relevant. This problem is important in its own right, but is even more timely given the recent success of alchemical free energy calculations. Alchemical calculations are increasingly used to predict binding free energies of ligands to receptors. However, the accuracy of these calculations is dependent on proper sampling of the relevant ligand binding modes. Unfortunately, ligand binding modes may often be uncertain, hard to predict, and/or slow to interconvert on simulation timescales, so proper sampling with current techniques can require prohibitively long simulations. We need new methods which dramatically improve sampling of ligand binding modes. Here, we develop and apply a nonequilibrium candidate Monte Carlo (NCMC) method to improve sampling of ligand binding modes.</div><div><br></div><div>In this technique the ligand is rotated and subsequently allowed to relax in its new position through alchemical perturbation before accepting or rejecting the rotation and relaxation as a nonequilibrium Monte Carlo move. When applied to a T4 lysozyme model binding system, this NCMC method shows over two orders of magnitude improvement in binding mode sampling efficiency compared to a brute force molecular dynamics simulation. This is a first step towards applying this methodology to pharmaceutically relevant binding of fragments and, eventually, drug-like molecules. We are making this approach available via our new Binding Modes of Ligands using Enhanced Sampling (BLUES) package which is freely available on GitHub.</div>

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D3R Grand Challenge 4: Ligand Similarity and MM-GBSA-Based Pose Prediction and Affinity Ranking for BACE-1 Inhibitors

10.26434/chemrxiv.8259074.v1 ◽

2019 ◽

Author(s):

Sukanya Sasmal ◽

Léa El Khoury ◽

David Mobley

Keyword(s):

Binding Mode ◽

Energy Estimates ◽

Poor Correlation ◽

Grand Challenge ◽

Pose Prediction ◽

Design Data ◽

Data Resource ◽

Affinity Ranking ◽

Ligand Similarity ◽

Bace 1

The Drug Design Data Resource (D3R) Grand Challenges present an opportunity to assess, in the context of a blind predictive challenge, the accuracy and the limits of tools and methodologies designed to help guide pharmaceutical drug discovery projects. Here, we report the results of our participation in the D3R Grand Challenge 4, which focused on predicting the binding poses and affinity ranking for compounds targeting the beta-amyloid precursor protein (BACE-1). Our ligand similarity-based protocol using HYBRID (OpenEye Scientific Software) successfully identified poses close to the native binding mode for most of the ligands with less than 2 A RMSD accuracy. Furthermore, we compared the performance of our HYBRID-based approach to that of AutoDock Vina and Dock 6 and found that HYBRID performed better here for pose prediction. We also conducted end-point free energy estimates on protein-ligand complexes using molecular mechanics combined with generalized Born surface area method (MM-GBSA). We found that the binding affinity ranking based on MM-GBSA scores have poor correlation with the experimental values. Finally, the main lessons from our participation in D3R Grand Challenge 4 suggest that: i) the generation of the macrocycles conformers is a key step for successful pose prediction, ii) the protonation states of the BACE-1 binding site should be treated carefully, iii) the MM-GBSA method could not discriminate well between different predicted binding poses, and iv) the MM-GBSA method does not perform well at predicting protein-ligand binding affinities here.

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