Hydrophobic interactions in the trypsin active center. The sensitivity of the hydrophobic binding site to side chain modifications in competitive inhibitors of the amidinium type

It has been known for some time that penicillin exerts its antibiotic action by inhibiting the synthesis of the cell wall mucopeptide polymer in sensitive bacteria. Richmond and I, seeking an explanation for this specificity, suggested that there was a structural similarity between penicillin and N -acetylmuramic acid, one of the components in the polymer, and that penicillin might inhibit one of the enzymes involved in the synthesis of the polymer because of this similarity (Collins & Richmond 1962). It is therefore very interesting to hear that phenoxymethyl-penicillin has a single binding site in the lysozyme molecule, which is also the binding site for α -benzyl- N -acetylmuramic acid (the site number 5, in Dr Phillips’s notation). With the data from the 6 Å resolution picture of these enzyme complexes obtained by Dr Johnson, it is not possible to determine which groups are involved in interactions between lysozyme and the bound molecules of penicillin and benzy- N -acetylmuramic acid. Examination of the lysozyme model in the region where these molecules are bound shows some groups which may be involved in hydrophobic interactions, but few polar groups. This weakens the possibility that the similarity suggested by Richmond and me underlies the interaction between the molecules in these complexes, since we found the maximum similarity to lie in the disposition of polar groups in penicillin and in N -acetylmuramic acid. Briefly, we drew an analogy between the carboxyl groups, between the ring nitrogen atom in penicillin and the ether oxygen atom in the lactyl side-chain of N -acetylmuramic acid, and between the carbonyl groups in the amide links in the two molecules.

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Crystal Structure of Tetrameric Homoisocitrate Dehydrogenase from an Extreme Thermophile, Thermus thermophilus: Involvement of Hydrophobic Dimer-Dimer Interaction in Extremely High Thermotolerance

Journal of Bacteriology ◽

10.1128/jb.187.19.6779-6788.2005 ◽

2005 ◽

Vol 187 (19) ◽

pp. 6779-6788 ◽

Cited By ~ 16

Author(s):

Junichi Miyazaki ◽

Kuniko Asada ◽

Shinya Fushinobu ◽

Tomohisa Kuzuyama ◽

Makoto Nishiyama

Keyword(s):

Crystal Structure ◽

Binding Site ◽

Thermal Inactivation ◽

Hydrophobic Interactions ◽

Substrate Binding ◽

Thermus Thermophilus ◽

Lysine Biosynthesis ◽

Side Chain ◽

Substrate Binding Site ◽

Tetramer Formation

ABSTRACT The crystal structure of homoisocitrate dehydrogenase involved in lysine biosynthesis from Thermus thermophilus (TtHICDH) was determined at 1.85-Å resolution. Arg85, which was shown to be a determinant for substrate specificity in our previous study, is positioned close to the putative substrate binding site and interacts with Glu122. Glu122 is highly conserved in the equivalent position in the primary sequence of ICDH and archaeal 3-isopropylmalate dehydrogenase (IPMDH) but interacts with main- and side-chain atoms in the same domain in those paralogs. In addition, a conserved Tyr residue (Tyr125 in TtHICDH) which extends its side chain toward a substrate and thus has a catalytic function in the related β-decarboxylating dehydrogenases, is flipped out of the substrate-binding site. These results suggest the possibility that the conformation of the region containing Glu122-Tyr125 is changed upon substrate binding in TtHICDH. The crystal structure of TtHICDH also reveals that the arm region is involved in tetramer formation via hydrophobic interactions and might be responsible for the high thermotolerance. Mutation of Val135, located in the dimer-dimer interface and involved in the hydrophobic interaction, to Met alters the enzyme to a dimer (probably due to steric perturbation) and markedly decreases the thermal inactivation temperature. Both the crystal structure and the mutation analysis indicate that tetramer formation is involved in the extremely high thermotolerance of TtHICDH.

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Binding affinity and stereoselectivity of local anesthetics in single batrachotoxin-activated Na+ channels.

The Journal of General Physiology ◽

10.1085/jgp.96.5.1105 ◽

1990 ◽

Vol 96 (5) ◽

pp. 1105-1127 ◽

Cited By ~ 31

Author(s):

G K Wang

Keyword(s):

Binding Site ◽

Binding Affinity ◽

Local Anesthetics ◽

Dwell Time ◽

Hydrophobic Interactions ◽

Na Channels ◽

Structural Determinants ◽

Planar Bilayers ◽

Binding Domains ◽

Hydrophobic Binding

Several local anesthetics (LA) have been previously shown to block muscle batrachotoxin (BTX)-activated Na+ channels in planar bilayers. The mean dwell time of different LA drugs, however, varies widely, from less than 10 ms to longer than several seconds. In this study, we have examined the structural determinants that govern the dwell time, the binding affinity, and the stereoselectivity of LA drugs using cocaine and bupivacaine homologues, RAC compounds, and their available stereoisomers. Our results from the structure-activity experiments reveal that (a) there are two apparent hydrophobic binding domains present in the LA binding site; one interacts with the aromatic moiety of the LA drugs, and the other interacts with the alkyl group attached to the tertiary amine of the LA drugs; (b) the LA mean dwell time and the binding affinity are largely determined by the hydrophobic interactions; (c) the LA binding site is highly stereoselective, with a difference in KD values over 50- and 6-fold for (+/-) cocaine and (+/-) bupivacaine, respectively; (d) the cocaine stereoselectivity is comparable among muscle, brain, and heart BTX-activated Na+ channels; and finally and most unexpectedly, (e) the stereoselectivity of LA drugs in BTX-activated Na+ channels appears greatly different from that reported in normal Na+ channels. Possible explanations for this difference are discussed.

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Kinetic Study of the Effect of Heparin on the Amidase Activity of Trypsin, Plasmin and Urokinase

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657362 ◽

1983 ◽

Vol 49 (03) ◽

pp. 199-203 ◽

Cited By ~ 10

Author(s):

V M Yomtova ◽

N A Stambolieva ◽

B M Blagoev

Keyword(s):

Kinetic Study ◽

Active Center ◽

Simultaneous Presence ◽

Amidase Activity ◽

Competitive Inhibitors ◽

Uncompetitive Inhibitor

SummaryIt was found that the effect of heparin on the amidase activity of urokinase (E C 3.4.21.31), plasmin (E C 3.4.21.7) and trypsin (E C 3.4.21.4) depended on the substrate used. No effect of heparin on the amidase activity of urokinase and trypsin was observed when Pyro Glu-Gly-Arg-p-nitroanilide (S-2444) and α-N-acetyl-L-lysine-p-nitroanilide (ALNA) were used as substrates. Heparin acted as a uncompetitive inhibitor of trypsin (Ki = 1.2×10-6 M), plasmin (Ki = 4.9×10-6 M) and urokinase (Ki = l.0×10-7 M) when Bz-Phe-Val-Arg-p-nitroanilide (S-2160), H-D-Val-Leu-Lys-p-nitroanilide (S-2251) and plasminogen, respectively, were used as substrates. These results, as well as the data obtained by studying the effect of the simultaneous presence of heparin and competitive inhibitors suggest that although heparin is not bound at the active center of these enzymes, it may influence the effectivity of catalysis.

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Schistosomal Sulfotransferase Interaction with Oxamniquine Involves Hybrid Mechanism of Induced-fit and Conformational Selection

Current Computer - Aided Drug Design ◽

10.2174/1573409915666190708103132 ◽

2020 ◽

Vol 16 (4) ◽

pp. 451-459 ◽

Cited By ~ 1

Author(s):

Fortunatus C. Ezebuo ◽

Ikemefuna C. Uzochukwu

Keyword(s):

Molecular Dynamics ◽

Amino Acid ◽

Binding Energy ◽

Binding Site ◽

Conformational Dynamics ◽

Side Chain ◽

Amino Acid Residues ◽

Conformational Selection ◽

Hybrid Mechanism ◽

Dynamics Simulations

Background: Sulfotransferase family comprises key enzymes involved in drug metabolism. Oxamniquine is a pro-drug converted into its active form by schistosomal sulfotransferase. The conformational dynamics of side-chain amino acid residues at the binding site of schistosomal sulfotransferase towards activation of oxamniquine has not received attention. Objective: The study investigated the conformational dynamics of binding site residues in free and oxamniquine bound schistosomal sulfotransferase systems and their contribution to the mechanism of oxamniquine activation by schistosomal sulfotransferase using molecular dynamics simulations and binding energy calculations. Methods: Schistosomal sulfotransferase was obtained from Protein Data Bank and both the free and oxamniquine bound forms were subjected to molecular dynamics simulations using GROMACS-4.5.5 after modeling it’s missing amino acid residues with SWISS-MODEL. Amino acid residues at its binding site for oxamniquine was determined and used for Principal Component Analysis and calculations of side-chain dihedrals. In addition, binding energy of the oxamniquine bound system was calculated using g_MMPBSA. Results: The results showed that binding site amino acid residues in free and oxamniquine bound sulfotransferase sampled different conformational space involving several rotameric states. Importantly, Phe45, Ile145 and Leu241 generated newly induced conformations, whereas Phe41 exhibited shift in equilibrium of its conformational distribution. In addition, the result showed binding energy of -130.091 ± 8.800 KJ/mol and Phe45 contributed -9.8576 KJ/mol. Conclusion: The results showed that schistosomal sulfotransferase binds oxamniquine by relying on hybrid mechanism of induced fit and conformational selection models. The findings offer new insight into sulfotransferase engineering and design of new drugs that target sulfotransferase.

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Mechanism of SARS-CoV-2 polymerase stalling by remdesivir

Nature Communications ◽

10.1038/s41467-020-20542-0 ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Goran Kokic ◽

Hauke S. Hillen ◽

Dimitry Tegunov ◽

Christian Dienemann ◽

Florian Seitz ◽

...

Keyword(s):

Electron Microscopy ◽

Rna Polymerase ◽

Binding Site ◽

Molecular Mechanism ◽

Active Center ◽

Rna Synthesis ◽

Active Form ◽

Nucleoside Triphosphate ◽

Rna Dependent Rna Polymerase ◽

Cryo Electron Microscopy

AbstractRemdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analog and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3ʹ-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3ʹ-nucleotide of the RNA product is matched and located with the template base in the active center, and this may impair proofreading by the viral 3ʹ-exonuclease. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.

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Mitigating the Adverse Effects of Polychlorinated Biphenyl Derivatives on Estrogenic Activity via Molecular Modification Techniques

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph18094999 ◽

2021 ◽

Vol 18 (9) ◽

pp. 4999

Author(s):

Wei He ◽

Wenhui Zhang ◽

Zhenhua Chu ◽

Yu Li

Keyword(s):

Amino Acid ◽

Binding Site ◽

Hydrophobic Interaction ◽

Oestrogen Receptor ◽

Polychlorinated Biphenyl ◽

Hydrophobic Interactions ◽

Estrogenic Activity ◽

Average Distance ◽

Amino Acid Residues ◽

Hydrophobic Amino Acid

The aim of this paper is to explore the mechanism of the change in oestrogenic activity of PCBs molecules before and after modification by designing new PCBs derivatives in combination with molecular docking techniques through the constructed model of oestrogenic activity of PCBs molecules. We found that the weakened hydrophobic interaction between the hydrophobic amino acid residues and hydrophobic substituents at the binding site of PCB derivatives and human oestrogen receptor alpha (hERα) was the main reason for the weakened binding force and reduced anti-oestrogenic activity. It was consistent with the information that the hydrophobic field displayed by the 3D contour maps in the constructed oestrogen activity CoMSIA model was one of the main influencing force fields. The hydrophobic interaction between PCB derivatives and oestrogen-active receptors was negatively correlated with the average distance between hydrophobic substituents and hydrophobic amino acid residues at the hERα-binding site, and positively correlated with the number of hydrophobic amino acid residues. In other words, the smaller the average distance between the hydrophobic amino acid residues at the binding sites between the two and the more the number of them, and the stronger the oestrogen activity expression degree of PCBS derivative molecules. Therefore, hydrophobic interactions between PCB derivatives and the oestrogen receptor can be reduced by altering the microenvironmental conditions in humans. This reduces the ability of PCB derivatives to bind to the oestrogen receptor and can effectively modulate the risk of residual PCB derivatives to produce oestrogenic activity in humans.

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Pontocerebellar hypoplasia due to bi-allelic variants in MINPP1

European Journal of Human Genetics ◽

10.1038/s41431-020-00749-x ◽

2020 ◽

Author(s):

Bart Appelhof ◽

Matias Wagner ◽

Julia Hoefele ◽

Anja Heinze ◽

Timo Roser ◽

...

Keyword(s):

Autosomal Recessive ◽

Hydrophobic Interactions ◽

Inositol Phosphates ◽

Side Chain ◽

Globular Domain ◽

Pontocerebellar Hypoplasia ◽

Disease Mechanism ◽

Nonsense Mediated Decay ◽

Inositol Phosphatase ◽

Induced Apoptosis

Abstract Pontocerebellar hypoplasia (PCH) describes a group of rare heterogeneous neurodegenerative diseases with prenatal onset. Here we describe eight children with PCH from four unrelated families harboring the homozygous MINPP1 (NM_004897.4) variants; c.75_94del, p.(Leu27Argfs*39), c.851 C > A, p.(Ala284Asp), c.1210 C > T, p.(Arg404*), and c.992 T > G, p.(Ile331Ser). The homozygous p.(Leu27Argfs*39) change is predicted to result in a complete absence of MINPP1. The p.(Arg404*) would likely lead to a nonsense mediated decay, or alternatively, a loss of several secondary structure elements impairing protein folding. The missense p.(Ala284Asp) affects a buried, hydrophobic residue within the globular domain. The introduction of aspartic acid is energetically highly unfavorable and therefore predicted to cause a significant reduction in protein stability. The missense p.(Ile331Ser) affects the tight hydrophobic interactions of the isoleucine by the disruption of the polar side chain of serine, destabilizing the structure of MINPP1. The overlap of the above-mentioned genotypes and phenotypes is highly improbable by chance. MINPP1 is the only enzyme that hydrolyses inositol phosphates in the endoplasmic reticulum lumen and several studies support its role in stress induced apoptosis. The pathomechanism explaining the disease mechanism remains unknown, however several others genes of the inositol phosphatase metabolism (e.g., INPP5K, FIG4, INPP5E, ITPR1) are correlated with phenotypes of neurodevelopmental disorders. Taken together, we present MINPP1 as a novel autosomal recessive pontocerebellar hypoplasia gene.

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