Structures of PGAM5 Provide Insight into Active Site Plasticity and Multimeric Assembly

Apirat Chaikuad,Panagis Filippakopoulos,Sean R Marcsisin,Sarah Picaud,Martin Schröder,Shiori Sekine,Hidenori Ichijo,John R Engen,Kohsuke Takeda,Stefan Knapp

doi:10.1016/j.str.2017.05.020

Abstract

SummaryPGAM5 is a mitochondrial membrane protein that functions as an atypical Ser/Thr phosphatase and is a regulator of oxidative stress response, necroptosis, and autophagy. Here we present several crystal structures of PGAM5 including the activating N-terminal regulatory sequences, providing a model for structural plasticity, dimerization of the catalytic domain, and the assembly into an enzymatically active dodecameric form. Oligomeric states observed in structures were supported by hydrogen exchange mass spectrometry, size-exclusion chromatography, and analytical ultracentrifugation experiments in solution. We report that the catalytically important N-terminal WDPNWD motif acts as a structural integrator assembling PGAM5 into a dodecamer, allosterically activating the phosphatase by promoting an ordering of the catalytic loop. Additionally the observed active site plasticity enabled visualization of essential conformational rearrangements of catalytic elements. The comprehensive biophysical characterization offers detailed structural models of this key mitochondrial phosphatase that has been associated with the development of diverse diseases.

Highlights

Phosphoglycerate mutase family member 5 (PGAM5) is a mitochondrial membrane protein that belongs to the phosphoglycerate mutase (PGAM) branch of the histidine acid phosphatase superfamily, known as two-histidine phosphatase (2H-phosphatase)
PGAM5 is an essential regulatory component that promotes oxidative stress-induced necrosis/necroptosis in cancer cells through an association with the RIP1-RIP3-MLKL necrosome in mitochondria. This mitochondria attack complex initiates RIPK3 phosphorylation on PGAM5, which leads to the dephosphorylation and activation of DRP1, a guanosine triphosphatase that regulates an early and obligatory mitochondrial fission step for execution of programmed necroptosis (Lin et al, 2013; Lu et al, 2016; Moriwaki and Chan, 2013; Wang et al, 2012; Zhuang et al, 2013)
The catalytic domain of PGAM5 adopted a classical phosphoglycerate mutase-like fold with the canonical three-layer sandwich topology, which featured a six-stranded b sheet flanked on both sides by a helices with the first four parallel strands arranged in an alternated b/a manner (Figures 1A and 1B)

Summary

Introduction

Phosphoglycerate mutase family member 5 (PGAM5) is a mitochondrial membrane protein that belongs to the phosphoglycerate mutase (PGAM) branch of the histidine acid phosphatase superfamily, known as two-histidine phosphatase (2H-phosphatase). PGAM5 is an essential regulatory component that promotes oxidative stress-induced necrosis/necroptosis in cancer cells through an association with the RIP1-RIP3-MLKL necrosome in mitochondria This mitochondria attack complex initiates RIPK3 phosphorylation on PGAM5, which leads to the dephosphorylation and activation of DRP1, a guanosine triphosphatase that regulates an early and obligatory mitochondrial fission step for execution of programmed necroptosis (Lin et al, 2013; Lu et al, 2016; Moriwaki and Chan, 2013; Wang et al, 2012; Zhuang et al, 2013).

Methods

Results

Conclusion