Slow-onset inhibition of fumarylacetoacetate hydrolase by phosphinate mimics of the tetrahedral intermediate: kinetics, crystal structure and pharmacokinetics

FAH (fumarylacetoacetate hydrolase) catalyses the final step of tyrosine catabolism to produce fumarate and acetoacetate. HT1 (hereditary tyrosinaemia type 1) results from deficiency of this enzyme. Previously, we prepared a partial mimic of the putative tetrahedral intermediate in the reaction catalysed by FAH co-crystallized with the enzyme to reveal details of the mechanism [Bateman, Bhanumoorthy, Witte, McClard, Grompe and Timm (2001) J. Biol. Chem. 276, 15284–15291]. We have now successfully synthesized complete mimics CEHPOBA {4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate} and COPHPAA {3-[(3-carboxy-2-oxopropyl)hydroxyphosphinyl]acrylate}, which inhibit FAH in slow-onset tight-binding mode with Ki values of 41 and 12 nM respectively. A high-resolution (1.35 Å; 1 Å=0.1 nm) crystal structure of the FAH·CEHPOBA complex was solved to reveal the affinity determinants for these compounds and to provide further insight into the mechanism of FAH catalysis. These compounds are active in vivo, and CEHPOBA demonstrated a notable dose-dependent increase in SA (succinylacetone; a metabolite seen in patients with HT1) in mouse serum after repeated injections, and, following a single injection (1 μmol/g; intraperitoneal), only a modest regain of FAH enzyme activity was detected in liver protein isolates after 24 h. These potent inhibitors provide a means to chemically phenocopy the metabolic defects of either HT1 or FAH knockout mice and promise future pharmacological utility for hepatocyte transplantation.

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Unique Pharmacological Properties of α-Conotoxin OmIA at α7 nAChRs

Frontiers in Pharmacology ◽

10.3389/fphar.2021.803397 ◽

2021 ◽

Vol 12 ◽

Author(s):

Thao N.T. Ho ◽

Nikita Abraham ◽

Richard J. Lewis

Keyword(s):

Crystal Structure ◽

Prolonged Exposure ◽

Tight Binding ◽

Binding Mode ◽

Pharmacological Properties ◽

Acetylcholine Binding Protein ◽

Step Model ◽

High Affinity Binding Site ◽

Potent Antagonist ◽

Α7 Nachrs

OmIA, isolated from Conus omaria venom, is a potent antagonist at α7 nAChRs. We determined the co-crystal structure of OmIA with Lymnae stagnalis acetylcholine binding protein (Ls-AChBP) that identified His5, Val10 and Asn11 as key determinants for the high potency of OmIA at α7 nAChRs. Remarkably, despite a competitive binding mode observed in the co-crystal structure, OmIA and analogues displayed functional insurmountable antagonism at α7 and α3β4 nAChRs, except OmIA analogues having long side chain at position 10 ([V10Q]OmIA and [V10L]OmIA), which were partial insurmountable antagonist at α7 nAChRs in the presence of type II positive allosteric modulators (PAMs). A “two-state, two-step” model was used to explain these observations, with [V10Q]OmIA and [V10L]OmIA co-existing in a fast reversible/surmountable as well as a tight binding/insurmountable state. OmIA and analogues also showed biphasic-inhibition at α7 nAChRs in the presence of PNU120596, with a preference for the high-affinity binding site following prolonged exposure. The molecular basis of binding and complex pharmacological profile of OmIA at α7 nAChRs presented in here expands on the potential of α-conotoxins to probe the pharmacological properties of nAChRs and may help guide the development novel α7 modulators.

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Structural basis for regiospecific midazolam oxidation by human cytochrome P450 3A4

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1616198114 ◽

2016 ◽

Vol 114 (3) ◽

pp. 486-491 ◽

Cited By ~ 43

Author(s):

Irina F. Sevrioukova ◽

Thomas L. Poulos

Keyword(s):

Crystal Structure ◽

Cytochrome P450 ◽

Active Site ◽

Structural Information ◽

Binding Mode ◽

Structural Basis ◽

Cytochrome P450 3A4 ◽

Human Cytochrome ◽

Cyp3a4 Substrate

Human cytochrome P450 3A4 (CYP3A4) is a major hepatic and intestinal enzyme that oxidizes more than 60% of administered therapeutics. Knowledge of how CYP3A4 adjusts and reshapes the active site to regioselectively oxidize chemically diverse compounds is critical for better understanding structure–function relations in this important enzyme, improving the outcomes for drug metabolism predictions, and developing pharmaceuticals that have a decreased ability to undergo metabolism and cause detrimental drug–drug interactions. However, there is very limited structural information on CYP3A4–substrate interactions available to date. Despite the vast variety of drugs undergoing metabolism, only the sedative midazolam (MDZ) serves as a marker substrate for the in vivo activity assessment because it is preferentially and regioselectively oxidized by CYP3A4. We solved the 2.7 Å crystal structure of the CYP3A4–MDZ complex, where the drug is well defined and oriented suitably for hydroxylation of the C1 atom, the major site of metabolism. This binding mode requires H-bonding to Ser119 and a dramatic conformational switch in the F–G fragment, which transmits to the adjacent D, E, H, and I helices, resulting in a collapse of the active site cavity and MDZ immobilization. In addition to providing insights on the substrate-triggered active site reshaping (an induced fit), the crystal structure explains the accumulated experimental results, identifies possible effector binding sites, and suggests why MDZ is predominantly metabolized by the CYP3A enzyme subfamily.

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Structural studies on the folded domain of the human prion protein bound to the Fab fragment of the antibody POM1

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444912037328 ◽

2012 ◽

Vol 68 (11) ◽

pp. 1501-1512 ◽

Cited By ~ 19

Author(s):

Pravas Kumar Baral ◽

Barbara Wieland ◽

Mridula Swayampakula ◽

Magdalini Polymenidou ◽

Muhammad Hafiz Rahman ◽

...

Keyword(s):

Crystal Structure ◽

Prion Protein ◽

Prion Diseases ◽

Passive Immunization ◽

Antibody Fragment ◽

Binding Mode ◽

Cellular Prion Protein ◽

Fab Fragment ◽

Human Prion Protein

Prion diseases are neurodegenerative diseases characterized by the conversion of the cellular prion protein PrPcinto a pathogenic isoform PrPsc. Passive immunization with antiprion monoclonal antibodies can arrest the progression of prion diseases. Here, the crystal structure of the Fab fragment of an antiprion monoclonal antibody, POM1, in complex with human prion protein (huPrPc) has been determined to 2.4 Å resolution. The prion epitope of POM1 is in close proximity to the epitope recognized by the purportedly therapeutic antibody fragment ICSM18 Fab in complex with huPrPc. POM1 Fab forms a 1:1 complex with huPrPcand the measuredKdof 4.5 × 10−7 Mreveals moderately strong binding between them. Structural comparisons have been made among three prion–antibody complexes: POM1 Fab–huPrPc, ICSM18 Fab–huPrPcand VRQ14 Fab–ovPrPc. The prion epitopes recognized by ICSM18 Fab and VRQ14 Fab are adjacent to a prion glycosylation site, indicating possible steric hindrance and/or an altered binding mode to the glycosylated prion proteinin vivo. However, both of the glycosylation sites on huPrPcare positioned away from the POM1 Fab binding epitope; thus, the binding mode observed in this crystal structure and the binding affinity measured for this antibody are most likely to be the same as those for the native prion proteinin vivo.

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Triglycine-Based Approach for Identifying the Substrate Recognition Site of an Enzyme

Crystals ◽

10.3390/cryst9090444 ◽

2019 ◽

Vol 9 (9) ◽

pp. 444 ◽

Cited By ~ 1

Author(s):

Nam

Keyword(s):

Crystal Structure ◽

Amino Acids ◽

Main Chain ◽

Structural Information ◽

Substrate Recognition ◽

Binding Mode ◽

Recognition Site ◽

Proteinase K ◽

X Ray

Various peptides or non-structural amino acids are recognized by their specific target proteins, and perform a biological role in various pathways in vivo. Understanding the interactions between target protein and peptides (or non-structural amino acids) provides key information on the molecular interactions, which can be potentially translated to the development of novel drugs. However, it is experimentally challenging to determine the crystal structure of protein–peptide complexes. To obtain structural information on the substrate recognition of the peptide-recognizing enzyme, X-ray crystallographic studies were performed using triglycine (Gly-Gly-Gly) as the main-chain of the peptide. The crystal structure of Parengyodontium album Proteinase K in complex with triglcyine was determined at a 1.4 Å resolution. Two different bound conformations of triglycine were observed at the substrate recognition site. The triglycine backbone forms stable interactions with β5-α4 and α5-β6 loops of the main-chain. One of the triglycine-binding conformations was identical to the binding mode of a peptide-based inhibitor from a previously reported crystal structure of Proteinase K. Triglycine has potential application in X-ray crystallography in order to identify the substrate recognition sites in the peptide binding enzymes.

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Triglycine-Based Approach for Identifying the Substrate Recognition Site of an Enzyme

10.20944/preprints201908.0126.v1 ◽

2019 ◽

Author(s):

Ki Hyun Nam

Keyword(s):

Crystal Structure ◽

Amino Acids ◽

Main Chain ◽

Structural Information ◽

Substrate Recognition ◽

Binding Mode ◽

Recognition Site ◽

Proteinase K ◽

X Ray

Various peptides or non-structural amino acids are recognized by their specific target proteins and perform biological role in various pathways in vivo. Understanding the interactions between target protein and peptides (or non-structural amino acids) provides key information on the molecular interactions, which can be potentially translated to the development of novel drugs. However, it is experimentally challenging to determine the crystal structure of protein-peptide complexes. To obtain structural information on substrate recognition of peptide-recognizing enzyme, X-ray crystallographic studies were performed using triglycine (Gly-Gly-Gly) as main-chain of peptide. The crystal structure of Parengyodontium album Proteinase K in complex with triglcyine was determined at 1.4 Å resolution. Two different bound conformations of triglycine were observed at the substrate recognition site. The triglycine backbone forms stable interactions with β5-α4 and α5-β6 loops of main-chain. One of the triglycine-binding conformations was identical with the binding mode of a peptide-based inhibitor from a previously reported crystal structure of Proteinase K. Triglycine has potential application X-ray crystallography to identify substrate recognition sites in peptide binding enzymes.

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Ruthenium(II) Complexes Bearing Thioether-Appended α- Iminopyridine Ligands: Arene Precursors Permit Access to K2-N,N and K3-N,N,S Complexes

10.26434/chemrxiv.9693506.v1 ◽

2019 ◽

Author(s):

Victoria A. Ternes ◽

Hannah A. Morgan ◽

Austin P. Lanquist ◽

Michael P. Murray ◽

Bradley Wile

Keyword(s):

Crystal Structure ◽

Solid State ◽

Strong Field ◽

Active Role ◽

Binding Mode ◽

Phenethyl Alcohol ◽

Ru Complexes

Herein we report the preparation of a series of Ru(II) complexes featuring alpha-iminopyridine ligands bearing thioether functionality (NNSR, where R = Me, CH2Ph, Ph). Metallation using (p cymene)RuCl dimer permits access to (k2-N,N)Ru complexes in which the thioether moiety remains uncoordinated. In the presence of a strong field ligand such as acetonitrile or triphenylphosphine, the p-cymene moiety is displaced, and the ligand adopts a k3-N,N,S binding mode. These complexes are characterized using a combination of solution and solid state methods, including the crystal structure of [(NNSMe)Ru(NCMe)2Cl]Cl. The k2-N,N Ru(II) complexes are shown to serve as efficient precatalysts for the oxidation of sec-phenethyl alcohol at 5 mol% loadings, using a variety of external oxidants and solvents. The complex bearing an S-Ph donor was found to be the most active of those surveyed, suggesting that the thioether donor plays an active role in catalyst speciation for this transformation.

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Spanning the gap: unraveling RSC dynamics in vivo

Current Genetics ◽

10.1007/s00294-020-01144-1 ◽

2021 ◽

Author(s):

Heinz Neumann ◽

Bryan J. Wilkins

Keyword(s):

Cell Cycle ◽

Posttranslational Modifications ◽

Transcriptional Activation ◽

Binding Mode ◽

Binding Modes ◽

Binding Domains ◽

Binding Interface ◽

The Many ◽

And Function

AbstractMultiple reports over the past 2 years have provided the first complete structural analyses for the essential yeast chromatin remodeler, RSC, providing elaborate molecular details for its engagement with the nucleosome. However, there still remain gaps in resolution, particularly within the many RSC subunits that harbor histone binding domains.Solving contacts at these interfaces is crucial because they are regulated by posttranslational modifications that control remodeler binding modes and function. Modifications are dynamic in nature often corresponding to transcriptional activation states and cell cycle stage, highlighting not only a need for enriched spatial resolution but also temporal understanding of remodeler engagement with the nucleosome. Our recent work sheds light on some of those gaps by exploring the binding interface between the RSC catalytic motor protein, Sth1, and the nucleosome, in the living nucleus. Using genetically encoded photo-activatable amino acids incorporated into histones of living yeast we are able to monitor the nucleosomal binding of RSC, emphasizing the regulatory roles of histone modifications in a spatiotemporal manner. We observe that RSC prefers to bind H2B SUMOylated nucleosomes in vivo and interacts with neighboring nucleosomes via H3K14ac. Additionally, we establish that RSC is constitutively bound to the nucleosome and is not ejected during mitotic chromatin compaction but alters its binding mode as it progresses through the cell cycle. Our data offer a renewed perspective on RSC mechanics under true physiological conditions.

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Crystal structures of Ca2+–calmodulin bound to NaV C-terminal regions suggest role for EF-hand domain in binding and inactivation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1818618116 ◽

2019 ◽

Vol 116 (22) ◽

pp. 10763-10772 ◽

Cited By ~ 10

Author(s):

Bernd R. Gardill ◽

Ricardo E. Rivera-Acevedo ◽

Ching-Chieh Tung ◽

Filip Van Petegem

Keyword(s):

Crystal Structure ◽

Binding Sites ◽

Binding Mode ◽

Conformational Switch ◽

Channel Inactivation ◽

Dependent Inactivation ◽

Ef Hand ◽

Inherited Arrhythmia ◽

A Site

Voltage-gated sodium (NaV) and calcium channels (CaV) form targets for calmodulin (CaM), which affects channel inactivation properties. A major interaction site for CaM resides in the C-terminal (CT) region, consisting of an IQ domain downstream of an EF-hand domain. We present a crystal structure of fully Ca2+-occupied CaM, bound to the CT of NaV1.5. The structure shows that the C-terminal lobe binds to a site ∼90° rotated relative to a previous site reported for an apoCaM complex with the NaV1.5 CT and for ternary complexes containing fibroblast growth factor homologous factors (FHF). We show that the binding of FHFs forces the EF-hand domain in a conformation that does not allow binding of the Ca2+-occupied C-lobe of CaM. These observations highlight the central role of the EF-hand domain in modulating the binding mode of CaM. The binding sites for Ca2+-free and Ca2+-occupied CaM contain targets for mutations linked to long-QT syndrome, a type of inherited arrhythmia. The related NaV1.4 channel has been shown to undergo Ca2+-dependent inactivation (CDI) akin to CaVs. We present a crystal structure of Ca2+/CaM bound to the NaV1.4 IQ domain, which shows a binding mode that would clash with the EF-hand domain. We postulate the relative reorientation of the EF-hand domain and the IQ domain as a possible conformational switch that underlies CDI.

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Turnover of total proteins and ornithine aminotransferase during liver regeneration in rats

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1980.238.1.e46 ◽

1980 ◽

Vol 238 (1) ◽

pp. E46-E52

Author(s):

S. L. Augustine ◽

R. W. Swick

Keyword(s):

Amino Acids ◽

Liver Regeneration ◽

Protein Degradation ◽

Partial Hepatectomy ◽

Free Amino ◽

Liver Protein ◽

Ornithine Aminotransferase ◽

Fractional Rate

The recovery of approximately 40% of the total liver protein during the first day after partial hepatectomy was shown to be due to the near cessation of protein breakdown rather than to an increase in protein synthesis. The decrease in degradation of total protein was less if rats were adrenalectomized or protein-depleted prior to partial hepatectomy. The effect of these treatments originally suggested that changes in free amino acid levels in liver might be related to the rate of protein degradation. However, no correlation was found between levels of total free amino acids and rates of breakdown. Measurements of individual amino acids during liver regeneration suggested that levels of free methionine and phenylalanine, amino acids that have been found to lower rates of protein degradation in vitro, are not correlated with rates of breakdown in vivo. The difference between the fractional rate of ornithine aminotransferase degradation (0.68/day and 0.28/day in sham-hepatectomized and partially hepatectomized rats, respectively) was sufficient to account for the higher level of this protein 3 days after surgery in the latter group.

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