An allosteric pocket for inhibition of bacterial Enzyme I identified by NMR-based fragment screening

Nguyen, Thanh Tuan; Venditti, Vincenzo

doi:10.1016/j.yjsbx.2020.100034

Cited by 7 publications

(5 citation statements)

References 44 publications

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“…178 STD NMR protocols and theoretical modeling have been extensively developed and tested for screening projects aimed at identifying ligands for protein drug targets. 177,[181][182][183] More recently, several STD NMR applications to the investigation of NP adsorption have appeared in the literature. For example, STD NMR has been used to screen libraries of small molecules against NP targets, 184,185 to determine the affinities of NP-ligand adducts, 184,186 and to investigate the forces driving small molecule adsorption.…”

Section: Saturation Transfer Methodsmentioning

confidence: 99%

Solution NMR methods for structural and thermodynamic investigation of nanoparticle adsorption equilibria

Sedinkin

Venditti

2022

Nanoscale Adv.

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Section: Saturation Transfer Methodsmentioning

confidence: 99%

Solution NMR methods for structural and thermodynamic investigation of nanoparticle adsorption equilibria

Sedinkin

Venditti

2022

Nanoscale Adv.

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“…Considering the reasons outlined in the introductory paragraphs, the trend over the past few years has been to identify PCs that bind to alternative sites of proteins rather than competing with the known ligand [ 20 ]. This can be experimentally achieved by, e.g., the use of molecular probes like in the multiple solvent crystal structure (MSCS) X-ray diffraction technique [ 108 , 109 , 110 ] or fragment-based NMR [ 111 , 112 ]. Alternative binding sites can also be discovered using bioinformatics tools mapping the surface/volume of a protein structure, allowing the search to be expanded to modeled proteins.…”

Section: Pocket Prediction Toolsmentioning

confidence: 99%

Pharmacological Chaperones and Protein Conformational Diseases: Approaches of Computational Structural Biology

Grasso

Galderisi

Santucci

et al. 2023

IJMS

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Whenever a protein fails to fold into its native structure, a profound detrimental effect is likely to occur, and a disease is often developed. Protein conformational disorders arise when proteins adopt abnormal conformations due to a pathological gene variant that turns into gain/loss of function or improper localization/degradation. Pharmacological chaperones are small molecules restoring the correct folding of a protein suitable for treating conformational diseases. Small molecules like these bind poorly folded proteins similarly to physiological chaperones, bridging non-covalent interactions (hydrogen bonds, electrostatic interactions, and van der Waals contacts) loosened or lost due to mutations. Pharmacological chaperone development involves, among other things, structural biology investigation of the target protein and its misfolding and refolding. Such research can take advantage of computational methods at many stages. Here, we present an up-to-date review of the computational structural biology tools and approaches regarding protein stability evaluation, binding pocket discovery and druggability, drug repurposing, and virtual ligand screening. The tools are presented as organized in an ideal workflow oriented at pharmacological chaperones’ rational design, also with the treatment of rare diseases in mind.

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“…The phosphorylation state of EI dictates the phosphorylation state of all other downstream components of the PTS ( Deutscher et al, 2014 ) and malfunction of EI has been linked to reduced growth-rate and attenuated virulence in several bacterial species ( Edelstein et al, 1999 ; Jones et al, 2000 ; Lau et al, 2001 ; Kok et al, 2003 ). Given its central role in controlling bacterial metabolism, EI has been proposed as a target for antimicrobial design ( Kok et al, 2003 ; Huang et al, 2013 ; Nguyen and Venditti, 2020 ) or for metabolic engineering efforts aimed at developing more efficient systems for microbial production of chemicals from biomass feedstocks ( Doucette et al, 2011 ; Venditti et al, 2013 ).…”

Section: Introductionmentioning

confidence: 99%

“…Coupling NMR relaxation experiments with Small Angle X-ray Scattering, we showed that binding of PEP results in further quenching of μs-ms dynamics at the EIC catalytic loops that triggers the open-to-close interdomain rearrangement and activates EI for catalysis ( Venditti et al, 2015b ). Finally, by combining NMR with Molecular Dynamics (MD) simulations, we noticed that residual conformational heterogeneity at the EIC active site in the activated enzyme-substrate complex determines the enzymatic turnover ( Dotas et al, 2020 ) and that perturbing conformational dynamics at the active site loops is an effective strategy to inhibit the phosphoryl-transfer reaction ( Nguyen and Venditti 2020 ).…”

Section: Introductionmentioning

confidence: 99%

A Single Point Mutation Controls the Rate of Interconversion Between the g+ and g− Rotamers of the Histidine 189 χ2 Angle That Activates Bacterial Enzyme I for Catalysis

Purslow

Thimmesch

Sivo

et al. 2021

Front. Mol. Biosci.

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Enzyme I (EI) of the bacterial phosphotransferase system (PTS) is a master regulator of bacterial metabolism and a promising target for development of a new class of broad-spectrum antibiotics. The catalytic activity of EI is mediated by several intradomain, interdomain, and intersubunit conformational equilibria. Therefore, in addition to its relevance as a drug target, EI is also a good model for investigating the dynamics/function relationship in multidomain, oligomeric proteins. Here, we use solution NMR and protein design to investigate how the conformational dynamics occurring within the N-terminal domain (EIN) affect the activity of EI. We show that the rotameric g+-to-g− transition of the active site residue His189 χ2 angle is decoupled from the state A-to-state B transition that describes a ∼90° rigid-body rearrangement of the EIN subdomains upon transition of the full-length enzyme to its catalytically competent closed form. In addition, we engineered EIN constructs with modulated conformational dynamics by hybridizing EIN from mesophilic and thermophilic species, and used these chimeras to assess the effect of increased or decreased active site flexibility on the enzymatic activity of EI. Our results indicate that the rate of the autophosphorylation reaction catalyzed by EI is independent from the kinetics of the g+-to-g− rotameric transition that exposes the phosphorylation site on EIN to the incoming phosphoryl group. In addition, our work provides an example of how engineering of hybrid mesophilic/thermophilic chimeras can assist investigations of the dynamics/function relationship in proteins, therefore opening new possibilities in biophysics.

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An allosteric pocket for inhibition of bacterial Enzyme I identified by NMR-based fragment screening

Abstract: Graphical abstract

Cited by 7 publications

References 44 publications

Solution NMR methods for structural and thermodynamic investigation of nanoparticle adsorption equilibria

Solution NMR methods for structural and thermodynamic investigation of nanoparticle adsorption equilibria

Pharmacological Chaperones and Protein Conformational Diseases: Approaches of Computational Structural Biology

A Single Point Mutation Controls the Rate of Interconversion Between the g+ and g− Rotamers of the Histidine 189 χ2 Angle That Activates Bacterial Enzyme I for Catalysis

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