Simulations of the regulatory ACT domain of human phenylalanine hydroxylase (PAH) unveil its mechanism of phenylalanine binding
- Yunhui Ge‡,
- Elias Borne§,
- Shannon Stewart§,
- Michael R. Hansen§,
- Emilia C. Arturo§,¶,
- Eileen K. Jaffe§ and
- Vincent A. Voelz‡1
- From the ‡Department of Chemistry, Temple University, Philadelphia, Pennsylvania 19122,
- §Fox Chase Cancer Center, Temple University Health System, Philadelphia, Pennsylvania 19111, and
- ¶Drexel University College of Medicine, Philadelphia, Pennsylvania 19129
- ↵1 To whom correspondence should be addressed. Tel.: 215-204-1973; Fax: 215-204-1532; E-mail: voelz{at}temple.edu.
-
Edited by Norma M. Allewell
Abstract
Phenylalanine hydroxylase (PAH) regulates phenylalanine (Phe) levels in mammals to prevent neurotoxicity resulting from high Phe concentrations as observed in genetic disorders leading to hyperphenylalaninemia and phenylketonuria. PAH senses elevated Phe concentrations by transient allosteric Phe binding to a protein–protein interface between ACT domains of different subunits in a PAH tetramer. This interface is present in an activated PAH (A-PAH) tetramer and absent in a resting-state PAH (RS-PAH) tetramer. To investigate this allosteric sensing mechanism, here we used the GROMACS molecular dynamics simulation suite on the Folding@home computing platform to perform extensive molecular simulations and Markov state model (MSM) analysis of Phe binding to ACT domain dimers. These simulations strongly implicated a conformational selection mechanism for Phe association with ACT domain dimers and revealed protein motions that act as a gating mechanism for Phe binding. The MSMs also illuminate a highly mobile hairpin loop, consistent with experimental findings also presented here that the PAH variant L72W does not shift the PAH structural equilibrium toward the activated state. Finally, simulations of ACT domain monomers are presented, in which spontaneous transitions between resting-state and activated conformations are observed, also consistent with a mechanism of conformational selection. These mechanistic details provide detailed insight into the regulation of PAH activation and provide testable hypotheses for the development of new allosteric effectors to correct structural and functional defects in PAH.
- molecular dynamics
- ligand-binding protein
- kinetics
- allosteric regulation
- conformational change
- binding pathways
- ligand binding kinetics
- Markov state models
- phenylketonuria
- phenylalanine hydroxylase
- molecular simulation
- conformational selection
Footnotes
-
This work was supported by National Institutes of Health Grants 1R01GM123296-01 (to V. A. V.), 1S10OD020095-01, 1R01NS100081 (to E. K. J.), and P30CA006927 (to Fox Chase Cancer Center) and in part by the National Science Foundation through Major Research Instrumentation Grant CNS-09-58854. The in vitro studies were also supported by the National PKU Alliance and BioMarin Pharmaceuticals. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
-
This article was selected as one of our Editors' Picks.
-
This article contains supporting methods, Figs. S1–S27, Tables S1–S9, and Movie S1.
- Received July 16, 2018.
- Revision received September 17, 2018.
- © 2018 Ge et al.
Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
