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1.
J Mol Biol ; 429(15): 2360-2372, 2017 07 21.
Article in English | MEDLINE | ID: mdl-28625849

ABSTRACT

Drug-like molecules targeting allosteric sites in proteins are of great therapeutic interest; however, identification of potential sites is not trivial. A straightforward approach to identify hidden allosteric sites is demonstrated in protein tyrosine phosphatases (PTP) by creation of single alanine mutations in the catalytic acid loop of PTP1B and VHR. This approach relies on the reciprocal interactions between an allosteric site and its coupled orthosteric site. The resulting NMR chemical shift perturbations (CSPs) of each mutant reveal clusters of distal residues affected by acid loop mutation. In PTP1B and VHR, two new allosteric clusters were identified in each enzyme. Mutations in these allosteric clusters altered phosphatase activity with changes in kcat/KM ranging from 30% to nearly 100-fold. This work outlines a simple method for identification of new allosteric sites in PTP, and given the basis of this method in thermodynamics, it is expected to be generally useful in other systems.


Subject(s)
Allosteric Site , Dual Specificity Phosphatase 3/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 1/chemistry , Amino Acid Substitution , DNA Mutational Analysis , Dual Specificity Phosphatase 3/genetics , Dual Specificity Phosphatase 3/metabolism , Kinetics , Magnetic Resonance Spectroscopy , Models, Molecular , Mutagenesis, Site-Directed , Mutant Proteins/chemistry , Mutant Proteins/genetics , Protein Conformation , Protein Tyrosine Phosphatase, Non-Receptor Type 1/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism
2.
Biochem Mol Biol Educ ; 45(5): 403-410, 2017 Sep.
Article in English | MEDLINE | ID: mdl-28294503

ABSTRACT

Here, we present a 10-week project-oriented laboratory module designed to provide a course-based undergraduate research experience in biochemistry that emphasizes the importance of biomolecular structure and dynamics in enzyme function. This module explores the impact of mutagenesis on an important active site loop for a biomedically-relevant human enzyme, protein tyrosine phosphatase 1B (PTP1B). Over the course of the semester students guide their own mutant of PTP1B from conception to characterization in a cost-effective manner and gain exposure to fundamental techniques in biochemistry, including site-directed DNA mutagenesis, bacterial recombinant protein expression, affinity column purification, protein quantitation, SDS-PAGE, and enzyme kinetics. This project-based approach allows an instructor to simulate a research setting and prepare students for productive research beyond the classroom. Potential modifications to expand or contract this module are also provided. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(5):403-410, 2017.


Subject(s)
Biochemistry/education , Laboratories , Protein Tyrosine Phosphatase, Non-Receptor Type 1/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism , Research/education , Humans , Protein Conformation , Protein Tyrosine Phosphatase, Non-Receptor Type 1/isolation & purification , Students
3.
Biochemistry ; 56(1): 96-106, 2017 Jan 10.
Article in English | MEDLINE | ID: mdl-27959494

ABSTRACT

Protein tyrosine phosphatase 1B (PTP1B) is a known regulator of the insulin and leptin signaling pathways and is an active target for the design of inhibitors for the treatment of type II diabetes and obesity. Recently, cichoric acid (CHA) and chlorogenic acid (CGA) were predicted by docking methods to be allosteric inhibitors that bind distal to the active site. However, using a combination of steady-state inhibition kinetics, solution nuclear magnetic resonance experiments, and molecular dynamics simulations, we show that CHA is a competitive inhibitor that binds in the active site of PTP1B. CGA, while a noncompetitive inhibitor, binds in the second aryl phosphate binding site, rather than the predicted benzfuran binding pocket. The molecular dynamics simulations of the apo enzyme and cysteine-phosphoryl intermediate states with and without bound CGA suggest CGA binding inhibits PTP1B by altering hydrogen bonding patterns at the active site. This study provides a mechanistic understanding of the allosteric inhibition of PTP1B.


Subject(s)
Caffeic Acids/pharmacology , Chlorogenic Acid/pharmacology , Enzyme Inhibitors/pharmacology , Protein Tyrosine Phosphatase, Non-Receptor Type 1/antagonists & inhibitors , Succinates/pharmacology , Algorithms , Allosteric Regulation , Binding Sites , Binding, Competitive , Caffeic Acids/chemistry , Caffeic Acids/metabolism , Catalytic Domain , Chlorogenic Acid/chemistry , Chlorogenic Acid/metabolism , Enzyme Inhibitors/metabolism , Humans , Hydrogen Bonding , Kinetics , Magnetic Resonance Spectroscopy , Molecular Dynamics Simulation , Protein Binding , Protein Domains , Protein Tyrosine Phosphatase, Non-Receptor Type 1/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism , Succinates/chemistry , Succinates/metabolism
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