Application of hydrogen/deuterium exchange mass spectrometry to study protein tyrosine phosphatase dynamics, ligand binding, and substrate specificity.
Protein tyrosine phosphatases (PTPs) are signaling enzymes that control a diverse array of cellular processes. Further insight into the specific functional roles of PTPs in cellular signaling requires detailed understanding of the molecular basis for substrate recognition by the PTPs. A central question is how PTPs discriminate between multiple structurally diverse substrates that they encounter in the cell. Although X-ray crystallography is capable of revealing the intimate structural details for molecular interaction, structures of higher order PTP.substrate complexes are often difficult to obtain. Hydrogen/deuterium exchange mass spectrometry (H/DX-MS) is a powerful tool for mapping protein-protein interfaces, as well as identifying conformational and dynamic perturbations in proteins. In addition, H/DX-MS enables analysis of large protein complexes at physiological concentrations and provides insight into the solution behavior of these complexes that can not be gleaned from crystal structures. We have utilized H/DX-MS to probe PTP dynamics, ligand binding, and the structural basis of substrate recognition. In this article, we review general principles of H/DX-MS technology as applied to study protein-protein interactions and dynamics. We also provide protocols for H/DX-MS successfully used in our laboratory to define the molecular basis of ERK2 substrate recognition by MKP3. Many of the aspects that we cover in detail should be applicable to the study of other PTPs with their specific targets.
Zhou
Bo
B
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA.
Zhang
Zhong-Yin
ZY
D041961:Deuterium Exchange Measurement / Q000379:methods*; D054642:Dual Specificity Phosphatase 6; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods*; D019950:Mitogen-Activated Protein Kinase 1 / Q000032:analysis; D025941:Protein Interaction Mapping / Q000379:methods; D017027:Protein Tyrosine Phosphatases / Q000032:analysis / Q000378:metabolism*; D013379:Substrate Specificity
D008024:Ligands; D019950:Mitogen-Activated Protein Kinase 1; D054642:Dual Specificity Phosphatase 6; D017027:Protein Tyrosine Phosphatases