Structure and analysis of nucleoside diphosphate kinase fromBorrelia burgdorferiprepared in a transition-state complex with ADP and vanadate moieties

Nucleoside diphosphate kinases (NDKs) are implicated in a wide variety of cellular functions owing to their enzymatic conversion of NDP to NTP. NDK fromBorrelia burgdorferi(BbNDK) was selected for functional and structural analysis to determine whether its activity is required for infection and to assess its potential for therapeutic inhibition. The Seattle Structural Genomics Center for Infectious Diseases (SSGCID) expressed recombinantBbNDK protein. The protein was crystallized and structures were solved of both the apoenzyme and a liganded form with ADP and vanadate ligands. This provided two structures and allowed the elucidation of changes between the apo and ligand-bound enzymes. Infectivity studies withndktransposon mutants demonstrated that NDK function was important for establishing a robust infection in mice, and provided a rationale for therapeutic targeting ofBbNDK. The protein structure was compared with other NDK structures found in the Protein Data Bank and was found to have similar primary, secondary, tertiary and quaternary structures, with conserved residues acting as the catalytic pocket, primarily using His132 as the phosphohistidine-transfer residue. Vanadate and ADP complexes model the transition state of this phosphoryl-transfer reaction, demonstrating that the pocket closes when bound to ADP, while allowing the addition or removal of a γ-phosphate. This analysis provides a framework for the design of potential therapeutics targetingBbNDK inhibition.

Download Full-text

Faculty Opinions recommendation of Anionic charge is prioritized over geometry in aluminum and magnesium fluoride transition state analogs of phosphoryl transfer enzymes.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1126855.583957 ◽

2008 ◽

Author(s):

Arieh Warshel

Keyword(s):

Transition State ◽

Magnesium Fluoride ◽

Phosphoryl Transfer ◽

Transition State Analogs ◽

Anionic Charge

Download Full-text

Crystal structure of Ssu72, an essential eukaryotic phosphatase specific for the C-terminal domain of RNA polymerase II, in complex with a transition state analogue

Biochemical Journal ◽

10.1042/bj20101471 ◽

2011 ◽

Vol 434 (3) ◽

pp. 435-444 ◽

Cited By ~ 27

Author(s):

Yong Zhang ◽

Mengmeng Zhang ◽

Yan Zhang

Keyword(s):

Rna Polymerase ◽

Transition State ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Complex Structure ◽

Protein Tyrosine Phosphatases ◽

Cysteine Residue ◽

Phosphoryl Transfer ◽

Terminal Domain ◽

Deep Groove

Reversible phosphorylation of the CTD (C-terminal domain) of the eukaryotic RNA polymerase II largest subunit represents a critical regulatory mechanism during the transcription cycle and mRNA processing. Ssu72 is an essential phosphatase conserved in eukaryotes that dephosphorylates phosphorylated Ser5 of the CTD heptapeptide. Its function is implicated in transcription initiation, elongation and termination, as well as RNA processing. In the present paper we report the high resolution X-ray crystal structures of Drosophila melanogaster Ssu72 phosphatase in the apo form and in complex with an inhibitor mimicking the transition state of phosphoryl transfer. Ssu72 facilitates dephosphorylation of the substrate through a phosphoryl-enzyme intermediate, as visualized in the complex structure of Ssu72 with the oxo-anion compound inhibitor vanadate at a 2.35 Å (1 Å=0.1 nm) resolution. The structure resembles the transition state of the phosphoryl transfer with vanadate exhibiting a trigonal bi-pyramidal geometry covalently bonded to the nucleophilic cysteine residue. Interestingly, the incorporation of oxo-anion compounds greatly stabilizes a flexible loop containing the general acid, as detected by an increase of melting temperature of Ssu72 detected by differential scanning fluorimetry. The Ssu72 structure exhibits a core fold with a similar topology to that of LMWPTPs [low-molecular-mass PTPs (protein tyrosine phosphatases)], but with an insertion of a unique ‘cap’ domain to shelter the active site from the solvent with a deep groove in between where the CTD substrates bind. Mutagenesis studies in this groove established the functional roles of five residues (Met17, Pro46, Asp51, Tyr77 and Met85) that are essential specifically for substrate recognition.

Download Full-text

ChemInform Abstract: Phosphoryl Transfer to Anionic Oxygen Nucleophiles. Nature of the Transition State and Electrostatic Repulsion.

ChemInform ◽

10.1002/chin.199001262 ◽

1990 ◽

Vol 21 (1) ◽

Author(s):

D. HERSCHLAG ◽

W. P. JENCKS

Keyword(s):

Transition State ◽

Phosphoryl Transfer ◽

Electrostatic Repulsion

Download Full-text

Transition-state stabilization and molecular recognition: acceleration of phosphoryl-transfer reactions by an artificial receptor

Journal of the American Chemical Society ◽

10.1021/ja00182a017 ◽

1990 ◽

Vol 112 (26) ◽

pp. 9586-9590 ◽

Cited By ~ 37

Author(s):

Paolo Tecilla ◽

Suk Kyu Chang ◽

Andrew D. Hamilton

Keyword(s):

Molecular Recognition ◽

Transition State ◽

Transfer Reactions ◽

Phosphoryl Transfer ◽

Transition State Stabilization ◽

Artificial Receptor

Download Full-text

Alkali metal ion catalysis in nucleophilic displacement by ethoxide ion on p-nitrophenyl phenylphosphonate: Evidence for multiple metal ion catalysis

Canadian Journal of Chemistry ◽

10.1139/v02-202 ◽

2003 ◽

Vol 81 (1) ◽

pp. 53-63 ◽

Cited By ~ 37

Author(s):

Erwin Buncel ◽

Ruby Nagelkerke ◽

Gregory RJ Thatcher

Keyword(s):

Alkali Metal ◽

Transition State ◽

Metal Ions ◽

Metal Ion ◽

Alkali Metal Ions ◽

Phosphoryl Transfer ◽

Alkali Metal Ion ◽

Nucleophilic Displacement ◽

Displacement Reactions ◽

Metal Ion Catalysis

In continuation of our studies of alkali metal ion catalysis and inhibition at carbon, phosphorus, and sulfur centers, the role of alkali metal ions in nucleophilic displacement reactions of p-nitrophenyl phenylphosphonate (PNPP) has been examined. All alkali metal ions studied acted as catalysts. Alkali metal ions added as inert salts increased the rate while decreased rate resulted on M+ complexation with 18-crown-6 ether. Kinetic analysis indicated the interaction of possibly three potassium ions, four sodium ions, and five lithium ions in the transition state of the reactions of ethoxide with PNPP. Pre-association of the anionic substrate with two metals ions in the ground state gave the best fit to the experimental data of the sodium system. Thus, the study gives evidence of the role of several metal ions in nucleophilic displacement reactions of ethoxide with anionic PNPP, both in the ground state and in the transition state. Molecular modeling of the anionic transition state implies that the size of the monovalent cation and the steric requirement of the pentacoordinate transition state are the primary limitations on the number of cations that can be brought to bear to stabilize the transition state and catalyze nucleophilic substitution at phosphorus. The bearing of the present work on metal ion catalysis in enzyme systems is discussed, in particular enzymes that catalyze phosphoryl transfer, which often employ multiple metal ions. Our results, both kinetic and modeling, reveal the importance of electrostatic stabilization of the transition state for phosphoryl transfer that may be effected by multiple cations, either monovalent metal ions or amino acid residues. The more such cations can be brought into contact with the anionic transition state, the greater the catalysis observed.Key words: alkali metal ion catalysis, nucleophilic displacement at phosphorus, multiple metal ion catalysis, phosphoryl transfer.

Download Full-text

Identification of a novel selective and potent inhibitor of glycogen synthase kinase-3

AJP Cell Physiology ◽

10.1152/ajpcell.00061.2019 ◽

2019 ◽

Vol 317 (6) ◽

pp. C1289-C1303 ◽

Cited By ~ 5

Author(s):

Mahboubeh S. Noori ◽

Pooja M. Bhatt ◽

Maria C. Courreges ◽

Davoud Ghazanfari ◽

Chaz Cuckler ◽

...

Keyword(s):

Design Space ◽

Potent Inhibitor ◽

Glycogen Synthase ◽

Selective Inhibition ◽

Glycogen Synthase Kinase ◽

Glycogen Synthase Kinase 3 ◽

Cellular Functions ◽

Starting Point ◽

Potential Therapeutics

Glycogen synthase kinase-3 (GSK-3) is a multitasking protein kinase that regulates numerous critical cellular functions. Not surprisingly, elevated GSK-3 activity has been implicated in a host of diseases including pathological inflammation, diabetes, cancer, arthritis, asthma, bipolar disorder, and Alzheimer’s. Therefore, reagents that inhibit GSK-3 activity provide a means to investigate the role of GSK-3 in cellular physiology and pathophysiology and could become valuable therapeutics. Finding a potent inhibitor of GSK-3 that can selectively target this kinase, among over 500 protein kinases in the human genome, is a significant challenge. Thus there remains a critical need for the identification of selective inhibitors of GSK-3. In this work, we introduce a novel small organic compound, namely COB-187, which exhibits potent and highly selective inhibition of GSK-3. Specifically, this study 1) utilized a molecular screen of 414 kinase assays, representing 404 unique kinases, to reveal that COB-187 is a highly potent and selective inhibitor of GSK-3; 2) utilized a cellular assay to reveal that COB-187 decreases the phosphorylation of canonical GSK-3 substrates indicating that COB-187 inhibits cellular GSK-3 activity; and 3) reveals that a close isomer of COB-187 is also a selective and potent inhibitor of GSK-3. Taken together, these results demonstrate that we have discovered a region of chemical design space that contains novel GSK-3 inhibitors. These inhibitors will help to elucidate the intricate function of GSK-3 and can serve as a starting point for the development of potential therapeutics for diseases that involve aberrant GSK-3 activity.

Download Full-text