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

Crystals ◽  
2019 ◽  
Vol 9 (9) ◽  
pp. 444 ◽  
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.

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

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.

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

2016 ◽  
Vol 114 (3) ◽  
pp. 486-491 ◽  
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.

Crystal structure of theAcinetobacter baumanniiouter membrane protein Omp33

2018 ◽  
Vol 74 (9) ◽  
pp. 852-860 ◽  
Author(s):  
Javier Abellón-Ruiz ◽  
Michael Zahn ◽  
Arnaud Baslé ◽  
Bert van den Berg
Keyword(s):  
Crystal Structure ◽  
Structural Information ◽  
Cell Envelope ◽  
Resistance Mechanisms ◽  
Low Permeability ◽  
Extracellular Loop ◽  
X Ray ◽  
Gated Channel ◽  
Delivery Vehicles

Acinetobacter baumanniiis becoming a major threat to human health due to its multidrug resistance. This is owing in a large part to the low permeability of its outer membrane (OM), which prevents high internal antibiotic concentrations and makes antibiotic-resistance mechanisms more effective. To exploit OM channels as potential delivery vehicles for future antibiotics, structural information is required. One abundant OM protein inA. baumanniiis Omp33. This protein has been reported to be important for thein vivofitness and virulence ofA. baumannii, but its structure is not known. Here, the X-ray crystal structure of Omp33 is reported at a resolution of 2.1 Å. Omp33 has a 14-β-stranded barrel without stable extracellular loop constrictions. Instead, an extended and unusual periplasmic turn connecting β-strands 2 and 3 is present, which folds into the pore lumen and completely blocks the aqueous channel. The Omp33 structure helps in understanding howA. baumanniiOM proteins contribute to the low permeability of the cell envelope of this bacterium and suggests that Omp33 might function as a gated channel.

Effects of dietary alteration of bicarbonate and magnesium on rat bone

1986 ◽  
Vol 250 (2) ◽  
pp. F302-F307 ◽  
Author(s):  
J. M. Burnell ◽  
C. Liu ◽  
A. G. Miller ◽  
E. Teubner
Keyword(s):  
Crystal Structure ◽  
Bone Formation ◽  
X Ray Diffraction ◽  
X Ray ◽  
Growing Rats ◽  
Final Composition ◽  
Rat Bone

To study the effects of bicarbonate and magnesium on bone, mild acidosis and/or hypermagnesemia were produced in growing rats by feeding ammonium chloride and/or magnesium sulfate. Bone composition, quantitative histomorphometry, and mineral x-ray diffraction (XRD) characteristics were measured after 6 wk of treatment. The results demonstrated that both acidosis (decreased HCO3) and hypermagnesemia inhibited periosteal bone formation, and, when combined, results were summative; and the previously observed in vitro role of HCO3- and Mg2+ as inhibitors of crystal growth were confirmed in vivo. XRD measurements demonstrated that decreased plasma HCO3 resulted in larger crystals and increased Mg resulted in smaller crystals. However, the combined XRD effects of acidosis and hypermagnesemia resembled acidosis alone. It is postulated that the final composition and crystal structure of bone are strongly influenced by HCO3- and Mg2+, and the effects are mediated by the combined influence on both osteoblastic bone formation and the growth of hydroxyapatite.

scholarly journals The Rosetta all-atom energy function for macromolecular modeling and design

2017 ◽  
Author(s):  
Rebecca F. Alford ◽  
Andrew Leaver-Fay ◽  
Jeliazko R. Jeliazkov ◽  
Matthew J. O'Meara ◽  
Frank P. DiMaio ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acids ◽  
Energy Function ◽  
Crystal Structure Data ◽  
Structural Data ◽  
X Ray ◽  
The Past ◽  
Biomolecular Modeling ◽  
Macromolecular Modeling ◽  
Atom Energy

AbstractOver the past decade, the Rosetta biomolecular modeling suite has informed diverse biological questions and engineering challenges ranging from interpretation of low-resolution structural data to design of nanomaterials, protein therapeutics, and vaccines. Central to Rosetta’s success is the energy function: amodel parameterized from small molecule and X-ray crystal structure data used to approximate the energy associated with each biomolecule conformation. This paper describes the mathematical models and physical concepts that underlie the latest Rosetta energy function, beta_nov15. Applying these concepts,we explain how to use Rosetta energies to identify and analyze the features of biomolecular models.Finally, we discuss the latest advances in the energy function that extend capabilities from soluble proteins to also include membrane proteins, peptides containing non-canonical amino acids, carbohydrates, nucleic acids, and other macromolecules.

Abstract 304: Crystal Structure Of Fak/mef2 Complex Reveals The Role Of Fak In The Regulation Of Mef2 Transcription Factor.

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Alisson C Cardoso ◽  
Ana H Pereira ◽  
Andre L Ambrosio ◽  
Silvio R Consonni ◽  
Sandra M Dias ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Mechanical Stress ◽  
Focal Adhesion ◽  
Molecular Mechanisms ◽  
Structural Information ◽  
Mechanistic Model ◽  
Focal Adhesions ◽  
Saxs Data ◽  
X Ray ◽  
Gene Assay

Members of MEF2 (Myocyte Enhancer Factor 2) family of transcription factors are major regulators of cardiac development and homeostasis. Their functions are regulated at several levels, including the association with a variety of protein partners. We have previously shown that FAK (Focal Adhesion Kinase) regulates the stretch-induced activation of MEF2 in cardiomyocytes. But, the molecular mechanisms, involved in this process, are unclear. Here, we integrated biochemical, imaging and structural analyses to characterize a novel interaction between MEF2 and FAK. An association between MEF2 and FAK was detected by co-immunoprecipitation in the extracts of stretched cardiomyocytes (10%, 60Hz, 2 hours). MEF2 and FAK staining were co-localized in the nuclei of stretched cells. Pull down assays indicated that the Focal Adhesion Targeting (FAT) domain is sufficient to confer FAK interaction with MEF2. Gene reporter assays indicated that the interaction with FAK enhances the MEF2C transcriptional activity in cultured cardiomyocytes. Also, we present a 2.9-Å X-ray crystal structure for the FAK_FAT domain bound to MEF2C (1-95), comprised by the MADS box/MEF2 domain. The structural information, when used in combination with biochemical studies, small-angle X-ray scattering (SAXS) data and reporter gene assay, lead to a mechanistic model describing how FAK binds to MEF2C and stimulates its transcription function in cardiomyocytes. We further validated this model by showing that the binding of FAK to MEF2C is essential for the hypertrophy of cardiomyocyte in response to mechanical stress. Our results present FAK as a new positive regulator of MEF2, implicated in the fine control of the signal transduction between focal adhesions and the nucleus of cardiac myocytes during mechanical stress.

scholarly journals Crystal structure of a pyridoxal 5′-phosphate-dependent aspartate racemase derived from the bivalve mollusc Scapharca broughtonii

2017 ◽  
Vol 73 (12) ◽  
pp. 651-656 ◽  
Author(s):  
Taichi Mizobuchi ◽  
Risako Nonaka ◽  
Motoki Yoshimura ◽  
Katsumasa Abe ◽  
Shouji Takahashi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Active Site ◽  
Metal Binding ◽  
Bivalve Mollusc ◽  
Active Site Residues ◽  
X Ray ◽  
Aspartate Racemase ◽  
Scapharca Broughtonii

Aspartate racemase (AspR) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that is responsible for D-aspartate biosynthesis in vivo. To the best of our knowledge, this is the first study to report an X-ray crystal structure of a PLP-dependent AspR, which was resolved at 1.90 Å resolution. The AspR derived from the bivalve mollusc Scapharca broughtonii (SbAspR) is a type II PLP-dependent enzyme that is similar to serine racemase (SR) in that SbAspR catalyzes both racemization and dehydration. Structural comparison of SbAspR and SR shows a similar arrangement of the active-site residues and nucleotide-binding site, but a different orientation of the metal-binding site. Superposition of the structures of SbAspR and of rat SR bound to the inhibitor malonate reveals that Arg140 recognizes the β-carboxyl group of the substrate aspartate in SbAspR. It is hypothesized that the aromatic proline interaction between the domains, which favours the closed form of SbAspR, influences the arrangement of Arg140 at the active site.

scholarly journals Crystal Structure of the Kinase Domain of MerTK in Complex with AZD7762 Provides Clues for Structure-Based Drug Development

2020 ◽  
Vol 21 (21) ◽  
pp. 7878
Author(s):  
Tae Hyun Park ◽  
Seung-Hyun Bae ◽  
Seoung Min Bong ◽  
Seong Eon Ryu ◽  
Hyonchol Jang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Cancer Cell ◽  
Structural Information ◽  
Antiplatelet Agents ◽  
Binding Mode ◽  
Kinase Domain ◽  
Chemical Library ◽  
Time Resolved ◽  
Cancer Cell Death

Aberrant tyrosine-protein kinase Mer (MerTK) expression triggers prosurvival signaling and contributes to cell survival, invasive motility, and chemoresistance in many kinds of cancers. In addition, recent reports suggested that MerTK could be a primary target for abnormal platelet aggregation. Consequently, MerTK inhibitors may promote cancer cell death, sensitize cells to chemotherapy, and act as new antiplatelet agents. We screened an inhouse chemical library to discover novel small-molecule MerTK inhibitors, and identified AZD7762, which is known as a checkpoint-kinase (Chk) inhibitor. The inhibition of MerTK by AZD7762 was validated using an in vitro homogeneous time-resolved fluorescence (HTRF) assay and through monitoring the decrease in phosphorylated MerTK in two lung cancer cell lines. We also determined the crystal structure of the MerTK:AZD7762 complex and revealed the binding mode of AZD7762 to MerTK. Structural information from the MerTK:AZD7762 complex and its comparison with other MerTK:inhibitor structures gave us new insights for optimizing the development of inhibitors targeting MerTK.

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