The Active Site of a Prototypical “Rigid” Drug Target is Marked by Extensive Conformational Dynamics

Agmatine is the product of the decarboxylation of L-arginine by the enzyme arginine decarboxylase. This amine has been attributed to neurotransmitter functions, anticonvulsant, anti-neurotoxic, and antidepressant in mammals and is a potential therapeutic agent for diseases such as Alzheimer’s, Parkinson’s, and cancer. Agmatinase enzyme hydrolyze agmatine into urea and putrescine, which belong to one of the pathways producing polyamines, essential for cell proliferation. Agmatinase from Escherichia coli (EcAGM) has been widely studied and kinetically characterized, described as highly specific for agmatine. In this study, we analyze the amino acids involved in the high specificity of EcAGM, performing a series of mutations in two loops critical to the active-site entrance. Two structures in different space groups were solved by X-ray crystallography, one at low resolution (3.2 Å), including a guanidine group; and other at high resolution (1.8 Å) which presents urea and agmatine in the active site. These structures made it possible to understand the interface interactions between subunits that allow the hexameric state and postulate a catalytic mechanism according to the Mn2+ and urea/guanidine binding site. Molecular dynamics simulations evaluated the conformational dynamics of EcAGM and residues participating in non-binding interactions. Simulations showed the high dynamics of loops of the active site entrance and evidenced the relevance of Trp68, located in the adjacent subunit, to stabilize the amino group of agmatine by cation-pi interaction. These results allow to have a structural view of the best-kinetic characterized agmatinase in literature up to now.

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The structure of the metallo-β-lactamase VIM-2 in complex with a triazolylthioacetamide inhibitor

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x16016113 ◽

2016 ◽

Vol 72 (11) ◽

pp. 813-819 ◽

Cited By ~ 16

Author(s):

Tony Christopeit ◽

Ke-Wu Yang ◽

Shao-Kang Yang ◽

Hanna-Kirsti S. Leiros

Keyword(s):

Public Health ◽

Crystal Structure ◽

Active Site ◽

Drug Target ◽

Zinc Ions ◽

Clinical Use ◽

Global Public Health ◽

Promising Strategy ◽

Conserved Residue ◽

Substrate Spectrum

The increasing number of pathogens expressing metallo-β-lactamases (MBLs), and in this way achieving resistance to β-lactam antibiotics, is a significant threat to global public health. A promising strategy to treat such resistant pathogens is the co-administration of MBL inhibitors together with β-lactam antibiotics. However, an MBL inhibitor suitable for clinical use has not yet been identified. Verona integron-encoded metallo-β-lactamase 2 (VIM-2) is a widespread MBL with a broad substrate spectrum and hence is an interesting drug target for the treatment of β-lactam-resistant infections. In this study, three triazolylthioacetamides were tested as inhibitors of VIM-2. One of the tested compounds showed clear inhibition of VIM-2, with an IC50of 20 µM. The crystal structure of the inhibitor in complex with VIM-2 was obtained by DMSO-free co-crystallization and was solved at a resolution of 1.50 Å. To our knowledge, this is the first structure of a triazolylthioacetamide inhibitor in complex with an MBL. Analysis of the structure shows that the inhibitor binds to the two zinc ions in the active site of VIM-2 and revealed detailed information on the interactions involved. Furthermore, the crystal structure showed that binding of the inhibitor induced a conformational change of the conserved residue Trp87.

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The Core and Holoenzyme Forms of RNA Polymerase from Mycobacterium smegmatis

Journal of Bacteriology ◽

10.1128/jb.00583-18 ◽

2018 ◽

Vol 201 (4) ◽

Cited By ~ 1

Author(s):

Tomáš Kouba ◽

Jiří Pospíšil ◽

Jarmila Hnilicová ◽

Hana Šanderová ◽

Ivan Barvík ◽

...

Keyword(s):

Electron Microscopy ◽

Rna Polymerase ◽

Conformational Changes ◽

Mycobacterium Smegmatis ◽

Drug Target ◽

Conformational Dynamics ◽

Three Dimensional ◽

Cryo Electron Microscopy ◽

Bacterial Rna ◽

High Degree

ABSTRACT Bacterial RNA polymerase (RNAP) is essential for gene expression and as such is a valid drug target. Hence, it is imperative to know its structure and dynamics. Here, we present two as-yet-unreported forms of Mycobacterium smegmatis RNAP: core and holoenzyme containing σA but no other factors. Each form was detected by cryo-electron microscopy in two major conformations. Comparisons of these structures with known structures of other RNAPs reveal a high degree of conformational flexibility of the mycobacterial enzyme and confirm that region 1.1 of σA is directed into the primary channel of RNAP. Taken together, we describe the conformational changes of unrestrained mycobacterial RNAP. IMPORTANCE We describe here three-dimensional structures of core and holoenzyme forms of mycobacterial RNA polymerase (RNAP) solved by cryo-electron microscopy. These structures fill the thus-far-empty spots in the gallery of the pivotal forms of mycobacterial RNAP and illuminate the extent of conformational dynamics of this enzyme. The presented findings may facilitate future designs of antimycobacterial drugs targeting RNAP.

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Coupling between conformational dynamics and catalytic function at the active site of the lead-dependent ribozyme

RNA ◽

10.1261/rna.067579.118 ◽

2018 ◽

Vol 24 (11) ◽

pp. 1542-1554 ◽

Cited By ~ 2

Author(s):

Neil A. White ◽

Minako Sumita ◽

Victor E. Marquez ◽

Charles G. Hoogstraten

Keyword(s):

Active Site ◽

Conformational Dynamics ◽

Catalytic Function

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Dynamism and Cooperativity Essential for Efficient Catalysis in Aspergillus Niger Monoamine Oxidase

10.26434/chemrxiv.7707857.v1 ◽

2019 ◽

Author(s):

Christian Curado-Carballada ◽

Ferran Feixas ◽

Sílvia Osuna

Keyword(s):

Aspergillus Niger ◽

Monoamine Oxidase ◽

Active Site ◽

Conformational Dynamics ◽

Catalytic Efficiency ◽

Building Blocks ◽

Chiral Amine ◽

Evolutionary Pathway ◽

Accelerated Molecular Dynamics ◽

Open States

Aspergillus niger Monoamine Oxidase (MAO-N) is a homodimeric enzyme responsible for the oxidation of amines into the corresponding imine. Laboratory evolved variants of MAO-N in combination with a non-selective chemical reductant represents a powerful strategy for the deracemisation of chiral amine mixtures and, thus, is of interest for obtaining chiral amine building blocks. MAO-N presents a rich conformational dynamics with a flexible ß-hairpin region that can adopt closed, partially closed and open states. Despite the ß-hairpin conformational dynamics is altered along the laboratory evolutionary pathway of MAO-N, the connection between the ß-hairpin conformational dynamics and active site catalysis still remains unclear. In this work, we use accelerated molecular dynamics to elucidate the potential interplay between the ß-hairpin conformational dynamics and catalytic activity in MAO-N wild type and its evolved D5 variant. Our study reveals a delicate communication between both MAO-N subunits that impacts the active site architecture, and thus its catalytic efficiency. In both MAO-N WT and the laboratory evolved D5 variant, the ß-hairpin conformation in one of the monomers affects the productive binding of the substrate in the active site of the other subunit. However, both MAO-N WT and D5 variants show a quite different behaviour due to the distal mutations introduced experimentally with Directed Evolution.

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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

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.699203 ◽

2021 ◽

Vol 8 ◽

Author(s):

Jeffrey A. Purslow ◽

Jolene N. Thimmesch ◽

Valeria Sivo ◽

Trang T. Nguyen ◽

Balabhadra Khatiwada ◽

...

Keyword(s):

Protein Design ◽

Active Site ◽

Single Point ◽

Conformational Dynamics ◽

Phosphorylation Site ◽

Phosphoryl Group ◽

Single Point Mutation ◽

Phosphotransferase System ◽

Function Relationship ◽

Enzyme I

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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Targeting FGL2, a molecular drug target for glioblastoma, with natural compounds through virtual screening method

Future Medicinal Chemistry ◽

10.4155/fmc-2020-0331 ◽

2021 ◽

Author(s):

Fahad Hassan Shah ◽

Song Ja Kim

Keyword(s):

Virtual Screening ◽

Active Site ◽

Drug Target ◽

Screening Method ◽

Tumor Development ◽

Pharmacokinetic Profile ◽

Natural Compounds ◽

Screening Platform ◽

Proliferation In Vitro

Background: Fibroleukin-2 protein (FGL2) causes redevelopment of brain tumors. Inhibition of these proteins has shown to improve glioblastoma prognosis and treatment efficacy. Aim: The current study gathered recently exploited natural compounds that suppress glioblastoma proliferation in vitro, tested against FGL2 protein. Method: Twenty-five compounds were explored through a virtual screening platform. Results: Three natural compounds (betanine, hesperetin and ovatodiolide) hit the active site of FGL2. Furthermore, the influence of these compounds was also assessed using in silico gene expression, and ADMET tools showed downregulation of some genes, which caused rapid tumor development while possessing a moderate acute toxicity and pharmacokinetic profile. Conclusion: Our study presents three compounds that are good candidates for evaluation in FGL2 mutated glioblastoma animal models.

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