Structural Basis for the Active Site Inhibition Mechanism of Human Kidney-Type Glutaminase (KGA)

4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is the most potent nucleoside analog inhibitor of HIV reverse transcriptase (RT). It retains a 3′-OH yet acts as a chain-terminating agent by diminishing translocation from the pretranslocation nucleotide-binding site (N site) to the posttranslocation primer-binding site (P site). Also, facile misincorporation of EFdA-monophosphate (MP) results in difficult-to-extend mismatched primers. To understand the high potency and unusual inhibition mechanism of EFdA, we solved RT crystal structures (resolutions from 2.4 to 2.9 Å) that include inhibition intermediates (i) before inhibitor incorporation (catalytic complex, RT/DNA/EFdA-triphosphate), (ii) after incorporation of EFdA-MP followed by dT-MP (RT/DNAEFdA-MPP•dT-MPN), or (iii) after incorporation of two EFdA-MPs (RT/DNAEFdA-MPP•EFdA-MPN); (iv) the latter was also solved with EFdA-MP mismatched at the N site (RT/DNAEFdA-MPP•EFdA-MP*N). We report that the inhibition mechanism and potency of EFdA stem from interactions of its 4′-ethynyl at a previously unexploited conserved hydrophobic pocket in the polymerase active site. The high resolution of the catalytic complex structure revealed a network of ordered water molecules at the polymerase active site that stabilize enzyme interactions with nucleotide and DNA substrates. Finally, decreased translocation results from favorable interactions of primer-terminating EFdA-MP at the pretranslocation site and unfavorable posttranslocation interactions that lead to observed localized primer distortions.

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Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

Biochemical Journal ◽

10.1042/bcj20190399 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3333-3353 ◽

Cited By ~ 3

Author(s):

Malti Yadav ◽

Kamalendu Pal ◽

Udayaditya Sen

Keyword(s):

Active Site ◽

Metal Binding ◽

Metal Ion ◽

Triosephosphate Isomerase ◽

Catalytic Mechanism ◽

Degradation Mechanism ◽

Structural Basis ◽

Conserved Residues ◽

Phosphodiester Bond ◽

Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

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Theoretical Studies of the Acid-Base Equilibria in a Model Active Site of the Human 20S Proteasome

10.26434/chemrxiv.13388753.v1 ◽

2020 ◽

Author(s):

Jon Uranga ◽

Lukas Hasecke ◽

Jonny Proppe ◽

Jan Fingerhut ◽

Ricardo A. Mata

Keyword(s):

Active Site ◽

Boronic Acid ◽

Computational Study ◽

Ubiquitin Proteasome System ◽

Bayesian Optimization ◽

Inhibition Mechanism ◽

20S Proteasome ◽

Acid Base ◽

Ubiquitin Proteasome ◽

Infection Cycles

The 20S Proteasome is a macromolecule responsible for the chemical step in the ubiquitin-proteasome system of degrading unnecessary and unused proteins of the cell. It plays a central role both in the rapid growth of cancer cells as well as in viral infection cycles. Herein, we present a computational study of the acid-base equilibria in an active site of the human proteasome, an aspect which is often neglected despite the crucial role protons play in the catalysis. As example substrates, we take the inhibition by epoxy and boronic acid containing warheads. We have combined cluster quantum mechanical calculations, replica exchange molecular dynamics and Bayesian optimization of non-bonded potential terms in the inhibitors. In relation to the latter, we propose an easily scalable approach to the reevaluation of non-bonded potentials making use of QM/MM dynamics information. Our results show that coupled acid-base equilibria need to be considered when modeling the inhibition mechanism. The coupling between a neighboring lysine and the reacting threonine is not affected by the presence of the inhibitor.

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Discovery of natural product ovalicin sensitive type 1 methionine aminopeptidases: molecular and structural basis

Biochemical Journal ◽

10.1042/bcj20180874 ◽

2019 ◽

Vol 476 (6) ◽

pp. 991-1003 ◽

Cited By ~ 1

Author(s):

Vijaykumar Pillalamarri ◽

Tarun Arya ◽

Neshatul Haque ◽

Sandeep Chowdary Bala ◽

Anil Kumar Marapaka ◽

...

Keyword(s):

Natural Product ◽

Active Site ◽

Covalent Modification ◽

Structural Basis ◽

Wild Type ◽

Methionine Aminopeptidases ◽

Synthetic Derivative ◽

Dynamics Simulations

Abstract Natural product ovalicin and its synthetic derivative TNP-470 have been extensively studied for their antiangiogenic property, and the later reached phase 3 clinical trials. They covalently modify the conserved histidine in Type 2 methionine aminopeptidases (MetAPs) at nanomolar concentrations. Even though a similar mechanism is possible in Type 1 human MetAP, it is inhibited only at millimolar concentration. In this study, we have discovered two Type 1 wild-type MetAPs (Streptococcus pneumoniae and Enterococcus faecalis) that are inhibited at low micromolar to nanomolar concentrations and established the molecular mechanism. F309 in the active site of Type 1 human MetAP (HsMetAP1b) seems to be the key to the resistance, while newly identified ovalicin sensitive Type 1 MetAPs have a methionine or isoleucine at this position. Type 2 human MetAP (HsMetAP2) also has isoleucine (I338) in the analogous position. Ovalicin inhibited F309M and F309I mutants of human MetAP1b at low micromolar concentration. Molecular dynamics simulations suggest that ovalicin is not stably placed in the active site of wild-type MetAP1b before the covalent modification. In the case of F309M mutant and human Type 2 MetAP, molecule spends more time in the active site providing time for covalent modification.

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Structural Basis for Dual-Inhibition Mechanism of a Non-Classical Kazal-Type Serine Protease Inhibitor from Horseshoe Crab in Complex with Subtilisin

PLoS ONE ◽

10.1371/journal.pone.0018838 ◽

2011 ◽

Vol 6 (4) ◽

pp. e18838 ◽

Cited By ~ 10

Author(s):

Rajesh T. Shenoy ◽

Saravanan Thangamani ◽

Adrian Velazquez-Campoy ◽

Bow Ho ◽

Jeak Ling Ding ◽

...

Keyword(s):

Protease Inhibitor ◽

Serine Protease ◽

Horseshoe Crab ◽

Serine Protease Inhibitor ◽

Structural Basis ◽

Inhibition Mechanism ◽

Dual Inhibition

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Structural basis for an atypical active site of anl-aspartate/glutamate-specific racemase fromEscherichia coli

FEBS Letters ◽

10.1016/j.febslet.2015.11.003 ◽

2015 ◽

Vol 589 (24PartB) ◽

pp. 3842-3847 ◽

Cited By ~ 10

Author(s):

Jae-Woo Ahn ◽

Jeong Ho Chang ◽

Kyung-Jin Kim

Keyword(s):

Active Site ◽

Structural Basis ◽

Fromescherichia Coli

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Structure of undecaprenyl pyrophosphate synthase from Acinetobacter baumannii

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x18012931 ◽

2018 ◽

Vol 74 (12) ◽

pp. 765-769 ◽

Cited By ~ 3

Author(s):

Tzu-Ping Ko ◽

Chi-Hung Huang ◽

Shu-Jung Lai ◽

Yeh Chen

Keyword(s):

Acinetobacter Baumannii ◽

Active Site ◽

Multidrug Resistant ◽

Structural Basis ◽

X Ray Diffraction ◽

X Ray ◽

Peptidoglycan Synthesis ◽

C Terminus ◽

Dimeric Protein ◽

Β Sheet

Undecaprenyl pyrophosphate (UPP) is an important carrier of the oligosaccharide component in peptidoglycan synthesis. Inhibition of UPP synthase (UPPS) may be an effective strategy in combating the pathogen Acinetobacter baumannii, which has evolved to be multidrug-resistant. Here, A. baumannii UPPS (AbUPPS) was cloned, expressed, purified and crystallized, and its structure was determined by X-ray diffraction. Each chain of the dimeric protein folds into a central β-sheet with several surrounding α-helices, including one at the C-terminus. In the active site, two molecules of citrate interact with the side chains of the catalytic aspartate and serine. These observations may provide a structural basis for inhibitor design against AbUPPS.

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Molecular mechanism of uncompetitive inhibition of human placental and germ-cell alkaline phosphatase

Biochemical Journal ◽

10.1042/bj2860023 ◽

1992 ◽

Vol 286 (1) ◽

pp. 23-30 ◽

Cited By ~ 55

Author(s):

M F Hoylaerts ◽

T Manes ◽

J L Millán

Keyword(s):

Germ Cell ◽

Active Site ◽

Molecular Model ◽

Inhibition Mechanism ◽

Side Group ◽

Alkaline Phosphatases ◽

Uncompetitive Inhibition ◽

Guanidinium Group ◽

Hydrolysis Of ◽

Site Binding

Placental (PLAP) and germ-cell (GCAP) alkaline phosphatases are inhibited uncompetitively by L-Leu and L-Phe. Whereas L-Phe inhibits PLAP and GCAP to the same extent, L-Leu inhibits GCAP 17-fold more strongly than it does PLAP. This difference has been attributed [Hummer & Millán (1991) Biochem. J 274, 91-95] to a Glu----Gly substitution at position 429 in GCAP. The D-Phe and D-Leu enantiomorphs are also inhibitory through an uncompetitive mechanism but with greatly decreased efficiencies. Replacement of the active-site residue Arg-166 by Ala-166 changes the inhibition mechanism of the resulting PLAP mutant to a more complex mixed-type inhibition, with decreased affinities for L-Leu and L-Phe. The uncompetitive mechanism is restored on the simultaneous introduction of Gly-429 in the Ala-166 mutant, but the inhibitions of [Ala166,Gly429]PLAP and even [Lys166,Gly429]PLAP by L-Leu and L-Phe are considerably decreased compared with that of [Gly429]PLAP. These findings point to the importance of Arg-166 during inhibition. Active-site binding of L-Leu requires the presence of covalently bound phosphate in the active-site pocket, and the inhibition of PLAP by L-Leu is pH-sensitive, gradually disappearing when the pH is decreased from 10.5 to 7.5. Our data are compatible with the following molecular model for the uncompetitive inhibition of PLAP and GCAP by L-Phe and L-Leu: after binding of a phosphorylated substrate to the active site, the guanidinium group of Arg-166 (normally involved in positioning phosphate) is redirected to the carboxy group of L-Leu (or L-Phe), thus stabilizing the inhibitor in the active site. Therefore leucinamide and leucinol are weaker inhibitors of [Gly429]PLAP than is L-Leu. During this Arg-166-regulated event, the amino acid side group is positioned in the loop containing Glu-429 or Gly-429, leading to further stabilization. Replacement of Glu-429 by Gly-429 eliminates steric constraints experienced by the bulky L-Leu side group during its positioning and also increases the active-site accessibility for the inhibitor, providing the basis for the 17-fold difference in inhibition efficiency between PLAP and GCAP. Finally, the inhibitor's unprotonated amino group co-ordinates with the active-site Zn2+ ion 1, interfering with the hydrolysis of the phosphoenzyme intermediate, a phenomenon that determines the uncompetitive nature of the inhibition.

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Cryo-EM Structures of Prestin and the Molecular Basis of Outer Hair Cell Electromotility

10.1101/2021.08.06.455374 ◽

2021 ◽

Author(s):

Navid Bavi ◽

Michael D Clark ◽

Gustavo F Contreras ◽

Rong Shen ◽

Bharat Reddy ◽

...

Keyword(s):

Outer Hair Cell ◽

Binding Pocket ◽

Structural Features ◽

Outer Hair Cells ◽

Anion Binding ◽

Structural Basis ◽

Inhibition Mechanism ◽

Core Domain ◽

The Core ◽

Voltage Dependent

The voltage-dependent motor protein, Prestin (SLC26A5) is responsible for the electromotive behavior of outer hair cells (OHCs). Here, we determined the structure of dolphin Prestin in complex with Cl- and the inhibitor Salicylate using single particle cryo-electron microscopy. These structures establish the specific structural features of mammalian Prestin and reveal small but significant differences with the transporter members of the SLC26 family of membrane proteins. Comparison with SLC26A9 point to conformational differences in the special relationship between the core and gate domains. Importantly, we highlight substantial alterations to the hydrophobic footprint of Prestin as it relates to the membrane, which point to a potential influence of Prestin on its surrounding lipid. The structure of Prestin bound to the inhibitor Salicylate confirms the nature of the anion binding pocket, formed by TM3 and TM10 in the Core domain and a set of anion coordinating residues which include Q97, F101, F137, S398 and R399. The presence of a well-defined density for Salycilate points to an inhibition mechanism based on competition for the anion-binding pocket of Prestin. These observations illuminate the structural basis of Prestin electromotility, a key component in the mammalian cochlear amplifier.

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