Allosteric inhibition of VIM metallo-β-lactamases by a camelid nanobody

2013 ◽  
Vol 450 (3) ◽  
pp. 477-486 ◽  
Author(s):  
Jean S. Sohier ◽  
Clémentine Laurent ◽  
Andy Chevigné ◽  
Els Pardon ◽  
Vasundara Srinivasan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Active Site ◽  
Substrate Inhibition ◽  
Inhibition Mechanism ◽  
Bacterial Strains ◽  
Single Domain Antibody ◽  
Alanine Scan ◽  
Hydrophobic Amino Acids ◽  
Micromolar Range

MβL (metallo-β-lactamase) enzymes are usually produced by multi-resistant Gram-negative bacterial strains and have spread worldwide. An approach on the basis of phage display was used to select single-domain antibody fragments (VHHs, also called nanobodies) that would inhibit the clinically relevant VIM (Verona integron-encoded MβL)-4 MβL. Out of more than 50 selected nanobodies, only the NbVIM_38 nanobody inhibited VIM-4. The paratope, inhibition mechanism and epitope of the NbVIM_38 nanobody were then characterized. An alanine scan of the NbVIM_38 paratope showed that its binding was driven by hydrophobic amino acids. The inhibitory concentration was in the micromolar range for all β-lactams tested. In addition, the inhibition was found to follow a mixed hyperbolic profile with a predominantly uncompetitive component. Moreover, substrate inhibition was recorded only after nanobody binding. These kinetic data are indicative of a binding site that is distant from the active site. This finding was confirmed by epitope mapping analysis that was performed using peptides, and which identified two stretches of amino acids in the L6 loop and at the end of the α2 helix. Because this binding site is distant from the active site and alters both the substrate binding and catalytic properties of VIM-4, this nanobody can be considered as an allosteric inhibitor.

Structural Basis for the Inhibition Mechanism of Ecotin against Neutrophil Elastase by Targeting the Active Site and Secondary Binding Site

Biochemistry ◽  
2020 ◽  
Vol 59 (30) ◽  
pp. 2788-2795
Author(s):  
Chacko Jobichen ◽  
Mahalakshmi Tirumuru Prabhakar ◽  
Su Ning Loh ◽  
J. Sivaraman
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Neutrophil Elastase ◽  
Structural Basis ◽  
Inhibition Mechanism

scholarly journals Structural basis of HIV inhibition by translocation-defective RT inhibitor 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA)

2016 ◽  
Vol 113 (33) ◽  
pp. 9274-9279 ◽  
Author(s):  
Zhe Li Salie ◽  
Karen A. Kirby ◽  
Eleftherios Michailidis ◽  
Bruno Marchand ◽  
Kamalendra Singh ◽  
...  
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Complex Structure ◽  
Primer Binding Site ◽  
Structural Basis ◽  
Inhibition Mechanism ◽  
Catalytic Complex ◽  
Hiv Reverse Transcriptase ◽  
A Chain ◽  
Ordered Water

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.

scholarly journals Amino acids from both N-terminal hydrophobic regions of the Lassa virus envelope glycoprotein GP-2 are critical for pH-dependent membrane fusion and infectivity

2007 ◽  
Vol 88 (8) ◽  
pp. 2320-2328 ◽  
Author(s):  
Christian Klewitz ◽  
Hans-Dieter Klenk ◽  
Jan ter Meulen
Keyword(s):  
Amino Acids ◽  
Structural Changes ◽  
Point Mutations ◽  
Lassa Virus ◽  
Virus Envelope ◽  
Glycoprotein Precursor ◽  
Alanine Scan ◽  
N Terminus ◽  
Hydrophobic Amino Acids ◽  
Ph Dependent

Lassa virus glycoprotein 2 (LASV GP-2) belongs to the class I fusion protein family. Its N terminus contains two stretches of highly conserved hydrophobic amino acids (residues 260–266 and 276–298) that have been proposed as N-terminal or internal fusion peptide segments (N-FPS, I-FPS) by analogy with similar sequences of other viral glycoproteins or based on experimental data obtained with synthetic peptides, respectively. By using a pH-dependent, recombinant LASV glycoprotein mediated cell–cell fusion assay and a retroviral pseudotype infectivity assay, an alanine scan of all hydrophobic amino acids within both proposed FPSs was performed. Fusogenicity and infectivity were correlated, both requiring correct processing of the glycoprotein precursor. Most point mutations in either FPS accounted for reduced or abolished fusion or infection, respectively. Some mutations also had an effect on pre-fusion steps of virus entry, possibly by inducing structural changes in the glycoprotein. The data demonstrate that several amino acids from both hydrophobic regions of the N terminus, some of which (W264, G277, Y278 and L280) are 100 % conserved in all arenaviruses, are involved in fusogenicity and infectivity of LASV GP-2.

scholarly journals Probing the specificity of cysteine proteinases at subsites remote from the active site: analysis of P4, P3, P2′ and P3′ variations in extended substrates

2000 ◽  
Vol 347 (1) ◽  
pp. 123-129 ◽  
Author(s):  
Fernada C. Vieira PORTARO ◽  
Ana Beatriz F. SANTOS ◽  
Maria Helena S. CEZARI ◽  
Maria Aparecida JULIANO ◽  
Luiz JULIANO ◽  
...  
Keyword(s):  
Amino Acids ◽  
Significant Influence ◽  
Active Site ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Cysteine Proteinases ◽  
Aminobenzoic Acid ◽  
Site Analysis ◽  
Hydrophobic Amino Acids ◽  
Human Cathepsin

We have determined the kinetic parameters for the hydrolysis by papain, cathepsin B and cathepsin L of internally quenched fluorescent peptides derived from the lead peptides Abz-AAFRSAQ-EDDnp [in which Abz and EDDnp stand for o-aminobenzoic acid and N-(2,4-dinitrophenyl)ethylenediamine respectively], to map the specificity of S4 and S3 subsites, and Abz-AFRSAAQ-EDDnp, to identify the specificity of S2ʹ and S3ʹ. Abz and EDDnp were the fluorescent quencher pair. These two series of peptides were cleaved at the Arg-Ser bond and systematic modifications at P4, P3, P2ʹ and P3ʹ were made. The S4 to S2ʹ subsites had a significant influence on the hydrolytic efficiencies of the three enzymes. Only papain activity was observed to be dependent on S3ʹ, indicating that its binding site is larger than those of cathepsins B and L. Hydrophobic amino acids were accepted at S4, S3, S2ʹ and S3ʹ of the three enzymes. The best substrates for cathepsins L and B had Trp and Asn at P2ʹ respectively; variations at this position were less accepted by these enzymes. The best substrates for papain were peptides containing Trp, Tyr or Asn at P3ʹ. Basic residues at P3 and P4 were well accepted by cathepsin L and papain. We also explored the susceptibility of substrates Abz-AFRSXAQ-EDDnp, modified at P2ʹ (X), to human cathepsin B mutants from which one or two occluding loop contacts had been removed. The modifications at His111 (H111A) and His110 (H110A) of cathepsin B led to an increase in kcat values of one or two orders of magnitude. The hydrolytic efficiencies of these cathepsin B mutants became closer to those of papain or cathepsin L.

scholarly journals Peptide Mimics of the Ribosomal P Stalk Inhibit the Activity of Ricin A Chain by Preventing Ribosome Binding

Toxins ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 371 ◽  
Author(s):  
Xiao-Ping Li ◽  
Jennifer Kahn ◽  
Nilgun Tumer
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Active Site ◽  
Ribosome Binding Site ◽  
Ricin A Chain ◽  
Ribosome Binding ◽  
P Proteins ◽  
A Chain ◽  
Ribosomal P ◽  
Ricin A

Ricin A chain (RTA) depurinates the sarcin/ricin loop (SRL) by interacting with the C-termini of the ribosomal P stalk. The ribosome interaction site and the active site are located on opposite faces of RTA. The interaction with P proteins allows RTA to depurinate the SRL on the ribosome at physiological pH with an extremely high activity by orienting the active site towards the SRL. Therefore, if an inhibitor disrupts RTA–ribosome interaction by binding to the ribosome binding site of RTA, it should inhibit the depurination activity. To test this model, we synthesized peptides mimicking the last 3 to 11 amino acids of P proteins and examined their interaction with wild-type RTA and ribosome binding mutants by Biacore. We measured the inhibitory activity of these peptides on RTA-mediated depurination of yeast and rat liver ribosomes. We found that the peptides interacted with the ribosome binding site of RTA and inhibited depurination activity by disrupting RTA–ribosome interactions. The shortest peptide that could interact with RTA and inhibit its activity was four amino acids in length. RTA activity was inhibited by disrupting its interaction with the P stalk without targeting the active site, establishing the ribosome binding site as a new target for inhibitor discovery.

scholarly journals Distribution of Charged and Hydrophobic Amino Acids on the Surfaces of Two Types of Beta-Fructosidase from Thermotoga Maritima

2020 ◽  
Vol 2 (1) ◽  
pp. 4
Author(s):  
Farkhat Sakibaev ◽  
Marina Holyavka ◽  
Victoria Koroleva ◽  
Valeriy Artyukhov
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Active Center ◽  
Active Site ◽  
Thermotoga Maritima ◽  
Significant Loss ◽  
Hydrophobic Residues ◽  
Charged Amino Acids ◽  
Hydrophobic Amino Acids

Thermotoga maritima beta-fructosidases are enzymes that release beta-D-fructose from sucrose, raffinose, and fructan polymers such as inulin. The surfaces of beta-fructosidases 1UYP and 1W2T from Thermotoga maritima were studied in this work. It was showed that amino acids are not distributed equally on the surfaces of the enzymes. Several clusters of charged and hydrophobic residues were detected at pH 7.0. Such clusters were detected by calculation of the distances between them. It was determined that on surfaces of beta-fructosidases PDB ID: 1UYP and PDB ID: 1W2T, 96% and 95% of charged amino acids and also 50% and 42% of hydrophobic amino acids form clusters, respectively. Six clusters of charged amino acids on the surface of beta-fructosidase 1UYP and five clusters on the surface of beta-fructosidase 1W2T were detected. The composition of such clusters is presented. Both types of beta-fructosidase have three clusters of hydrophobic amino acids on their surface. These facts should be considered when choosing immobilization conditions. It was shown that a charged matrix is more promising for the immobilization of beta-fructosidases 1UYP and 1W2T from Thermotoga maritima due to the possibility of binding without any significant loss of activity due to their overlapping active center. Hydrophobic carriers are less promising due to the probable active site overlap. Such binding may have a loss of enzyme activity as a result.

scholarly journals Exploring the action of new FimH inhibitors against CTX– 15 enzyme by enzoinformatics approach: A plausible arsenal against drug-resistant uropathogenic bacterial strains

2021 ◽  
Vol 20 (11) ◽  
pp. 2363-2370
Author(s):  
Amir Saeed ◽  
Khalid Alshaghdali ◽  
Mohd Saeed ◽  
Mousa Alreshidi
Keyword(s):  
Amino Acids ◽  
Molecular Docking ◽  
Active Site ◽  
Treatment Options ◽  
Positive Control ◽  
Drug Resistant ◽  
Amino Acid Residues ◽  
Bacterial Strains ◽  
Docking Approach ◽  
Uropathogenic Bacteria

Purpose: To explore the potency of FimH inhibitors against CTX-M β-lactamase enzyme type 15, in view of the increasing prevalence of CTX-M 15 in uropathogenic strains which has reduced the treatment options to minimal.Method: FimH inhibitors were targeted against CTXM-15 by a molecular docking approach. Thereafter, the best ligand-target confirmation was selected and analyzed using LIGPLOT+ Version v.2.1. The hydrophobic and hydrogen bonding among the catalytic site amino acids of CTXM-15 and the FimH inhibitors were analyzed and 3-D structures were converted into 2-D images by LIGPLOT algorithm.Results: Out of all the FimH inhibitors tested, 3′-chloro-4′- (α-D-mannopyranosyloxy) biphenyl-4- carbonitrile, para-biphenyl-2-methyl-3′-methylamidemannoside, para-biphenyl-2-methyl-3′,5′dimethylamide-α-D-mannoside, and thiazolylamino mannoside exhibited better interaction with the CTX-M15 active site than the positive control avibactam. Moreover, in CTX-M 15, the amino acid residues, Ser70, Tyr105, Ser130, Asn132, Thr216, Thr235, Gly236, and Ser237 were commonly interacting with these FimH inhibitors as well as avibactam.Conclusion: The predicted findings suggest that these FimH inhibitors could be explored as potential CTX-M 15 inhibitors to cope-up with resistance issues of uropathogenic bacteria in the form of an alternate strategy.

scholarly journals Interaction of lecithin:cholesterol acyltransferase with lipid surfaces and apolipoprotein A-I-derived peptides

2018 ◽  
Vol 59 (4) ◽  
pp. 670-683 ◽  
Author(s):  
Marco G. Casteleijn ◽  
Petteri Parkkila ◽  
Tapani Viitala ◽  
Artturi Koivuniemi
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Lipid Binding ◽  
Unesterified Cholesterol ◽  
Transfer Free Energy ◽  
Apolipoprotein A ◽  
Hydrophobic Amino Acids ◽  
Lipid Surface ◽  
Membrane Anchoring

LCAT is an enzyme responsible for the formation of cholesteryl esters from unesterified cholesterol (UC) and phospholipid (PL) molecules in HDL particles. However, it is poorly understood how LCAT interacts with lipoproteins and how apoA-I activates it. Here we have studied the interactions between LCAT and lipids through molecular simulations. In addition, we studied the binding of LCAT to apoA-I-derived peptides, and their effect on LCAT lipid association-utilizing experiments. Results show that LCAT anchors itself to lipoprotein surfaces by utilizing nonpolar amino acids located in the membrane-binding domain and the active site tunnel opening. Meanwhile, the membrane-anchoring hydrophobic amino acids attract cholesterol molecules next to them. The results also highlight the role of the lid-loop in the lipid binding and conformation of LCAT with respect to the lipid surface. The apoA-I-derived peptides from the LCAT-activating region bind to LCAT and promote its lipid surface interactions, although some of these peptides do not bind lipids individually. The transfer free-energy of PL from the lipid bilayer into the active site is consistent with the activation energy of LCAT. Furthermore, the entry of UC molecules into the active site becomes highly favorable by the acylation of SER181.

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

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.

13C nuclear magnetic resonance study of cyclodipeptides containing 13C enriched hydrophobic amino acids

1980 ◽  
Vol 45 (2) ◽  
pp. 482-490 ◽  
Author(s):  
Jaroslav Vičar ◽  
François Piriou ◽  
Pierre Fromageot ◽  
Karel Bláha ◽  
Serge Fermandjian
Keyword(s):  
Amino Acids ◽  
Nuclear Magnetic Resonance ◽  
Nuclear Magnetic Resonance Study ◽  
Coupling Constants ◽  
Side Chain ◽  
Amino Acid Residues ◽  
Magnetic Resonance Study ◽  
Side Chain Conformation ◽  
13C Nuclear Magnetic Resonance ◽  
Hydrophobic Amino Acids

The diastereoisomeric pairs of cyclodipeptides cis- and trans-cyclo(Ala-Ala), cyclo(Ala-Phe), cyclo(Val-Val) and cyclo(Leu-Leu) containing 85% 13C enriched amino-acid residues were synthesized and their 13C-13C coupling constants were measured. The combination of 13C-13C and 1H-1H coupling constants enabled to estimate unequivocally the side chain conformation of the valine and leucine residues.

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