Functional cross-talk between allosteric effects of activating and inhibiting ligands underlies PKM2 regulation

Jens Kleinjung,Dimitrios Anastasiou,Vesela Encheva,Stephen R Martin,Franca Fraternali,Alina Theisen,Perdita E Barran,Louise Fets,Laura Masino,Ambrosius P Snijders,Paul C Driscoll,Jamie A Macpherson

doi:10.7554/elife.45068

Jens Kleinjung, Dimitrios Anastasiou + Show 10 more

Open Access

https://doi.org/10.7554/elife.45068

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Abstract

Several enzymes can simultaneously interact with multiple intracellular metabolites, however, how the allosteric effects of distinct ligands are integrated to coordinately control enzymatic activity remains poorly understood. We addressed this question using, as a model system, the glycolytic enzyme pyruvate kinase M2 (PKM2). We show that the PKM2 activator fructose 1,6-bisphosphate (FBP) alone promotes tetramerisation and increases PKM2 activity, but addition of the inhibitor L-phenylalanine (Phe) prevents maximal activation of FBP-bound PKM2 tetramers. We developed a method, AlloHubMat, that uses eigenvalue decomposition of mutual information derived from molecular dynamics trajectories to identify residues that mediate FBP-induced allostery. Experimental mutagenesis of these residues identified PKM2 variants in which activation by FBP remains intact but cannot be attenuated by Phe. Our findings reveal residues involved in FBP-induced allostery that enable the integration of allosteric input from Phe and provide a paradigm for the coordinate regulation of enzymatic activity by simultaneous allosteric inputs.

Highlights

Allostery refers to the regulation of protein function resulting from the binding of an effector to a site distal to the protein’s functional centre and is a crucial mechanism for the control of multiple physiological processes (Shen et al, 2016; Nussinov and Tsai, 2013)
Using a novel computational framework to predict residues implicated in allosteric signal transmission from molecular dynamics (MD) simulations, we identify a network of pyruvate kinase M2 (PKM2) residues that mediates allosteric activation by FBP
We investigated whether Phe impedes FBP–induced activation of PKM2 by perturbing the oligomeric state of the protein, using nano-electrospray ionisation and ion mobility (IM) mass spectrometry (MS) (Pacholarz et al, 2017; Beveridge et al, 2016)

Summary

Introduction

Allostery refers to the regulation of protein function resulting from the binding of an effector to a site distal to the protein’s functional centre (the active site in the case of enzymes) and is a crucial mechanism for the control of multiple physiological processes (Shen et al, 2016; Nussinov and Tsai, 2013). The functional response of enzymes to allosteric ligands occurs on the ns - ms time scale, preceding other important regulatory mechanisms such as changes in gene expression or signalling-induced post-translational modifications (PTMs) (Gerosa and Sauer, 2011), thereby enabling fast cellular responses to various stimuli. Despite the demonstrated importance of protein allostery, the investigation of underlying mechanisms remains a challenge for conventional structural approaches and necessitates multi-disciplinary strategies.

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