A salt bridge of the C-terminal carboxyl group regulates PHPT1 substrate affinity and catalytic activity.

PHPT1 is a histidine phosphatase that modulates signaling in eukaryotes through its catalytic activity. Here, we present an analysis of the structure and dynamics of PHPT1 through a combination of solution NMR, molecular dynamics, and biochemical experiments. We identify a salt bridge formed between the R78 guanidinium moiety and the C-terminal carboxyl group on Y125 that is critical for ligand binding. Disruption of the salt bridge by appending a glycine residue at the C-terminus (G126) leads to a decrease in catalytic activity and binding affinity for the pseudo substrate, para-nitrophenylphosphate (pNPP), as well as the active site inhibitor, phenylphosphonic acid (PPA). We show through NMR chemical shift, 15N relaxation measurements, and analysis of molecular dynamics trajectories, that removal of this salt bridge results in an active site that is altered both structurally and dynamically thereby significantly impacting enzymatic function and confirming the importance of this electrostatic interaction.

Leveraging Reciprocity to Identify and Characterize Unknown Allosteric Sites in Protein Tyrosine Phosphatases.

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.

Exploring protein structure and dynamics through a project-oriented biochemistry laboratory module.

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.

Characterization of Protein Tyrosine Phosphatase 1B Inhibition by Chlorogenic Acid and Cichoric Acid.

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.

Elucidating concepts in drug design through taste with natural and artificial sweeteners.

Fundamental concepts in biochemistry important for drug design often lack connection to the macroscopic world and can be difficult for students to grasp, particularly those in introductory science courses at the high school and college level. Educational research has shown that multisensory teaching facilitates learning, but teaching at the high school and college level is almost exclusively limited to the visual and auditory senses. This approach neglects the lifetime of experience our students bring to the classroom in the form of taste perception and makes our teaching less supportive of those with sensory impairment. In this article, we outline a novel guided-inquiry activity that utilizes taste perception for a series of natural and artificial sweetener solutions to introduce the concepts of substrate affinity and selectivity in the context of drug design. The findings from this study demonstrate clear gains in student knowledge, as well as an increase in enthusiasm for the fields of biochemistry and drug design. © 2016 by The International Union of Biochemistry and Molecular Biology, 44(6):550-554, 2016.

Nanometer propagation of millisecond motions in V-type allostery.

Imidazole glycerol phosphate synthase (IGPS) is a V-type allosteric enzyme, which is catalytically inactive for glutamine hydrolysis until the allosteric effector, N'-[(5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) binds 30 Å away. In the apo state, NMR relaxation dispersion experiments indicate the absence of millisecond (ms) timescale motions. Binding of the PRFAR to form the active ternary complex is endothermic with a large positive entropy change. In addition, there is a protein wide enhancement of conformational motions in the ternary complex, which connect the two active sites. NMR chemical shift changes and acrylamide quenching experiments suggest that little in the way of structural changes accompany these motions. The data indicate that enzyme activation in the ternary complex is primarily due to an enhancement of ms motions that allows formation of a population of enzymatically active conformers.

Monitoring molecular interactions by NMR.

The ability of proteins to interact with small molecules or other proteins is essential in all aspects of biology. In many cases these interactions cause detectable changes in NMR chemical shifts, lineshapes, and relaxation rates and therefore provide a means by which to study these biologically important phenomena. Here we review the theory upon which this analysis is based, provide several illustrative examples, and highlight potential problems in the study of binding interactions by solution NMR.

1H, 15N and 13C resonance assignment of imidazole glycerol phosphate (IGP) synthase protein HisF from Thermotoga maritima.

HisF comprises one half of the heterodimeric protein complex imidazole glycerol phosphate (IGP) synthase responsible for the fifth step of histidine biosynthesis. Here we report backbone and side chain assignments necessary for characterization of protein dynamics involved in the allosteric mechanism of IGP synthase.