Cryo-EM snapshots of a native lysate provide structural insights into a metabolon-embedded transacetylase reaction

Christian Tüting,Ioannis Skalidis,Johannes Müller,Farzad Hamdi,Marija Sorokina,Panagiotis L Kastritis,Fotis L Kyrilis,Yashar Sadian

doi:10.1038/s41467-021-27287-4

Abstract

Found across all kingdoms of life, 2-keto acid dehydrogenase complexes possess prominent metabolic roles and form major regulatory sites. Although their component structures are known, their higher-order organization is highly heterogeneous, not only across species or tissues but also even within a single cell. Here, we report a cryo-EM structure of the fully active Chaetomium thermophilum pyruvate dehydrogenase complex (PDHc) core scaffold at 3.85 Å resolution (FSC = 0.143) from native cell extracts. By combining cryo-EM with macromolecular docking and molecular dynamics simulations, we resolve all PDHc core scaffold interfaces and dissect the residing transacetylase reaction. Electrostatics attract the lipoyl domain to the transacetylase active site and stabilize the coenzyme A, while apolar interactions position the lipoate in its binding cleft. Our results have direct implications on the structural determinants of the transacetylase reaction and the role of flexible regions in the context of the overall 10 MDa PDHc metabolon architecture.

Highlights

Found across all kingdoms of life, 2-keto acid dehydrogenase complexes possess prominent metabolic roles and form major regulatory sites
As a pyruvate dehydrogenase complex (PDHc) core scaffold, we describe the native assembly of E2 and E3BP which is essential to understand the localization of E1 and E3, respectively, as well as the relative proximity of all proteins and the lipoyl domain (LD)
We have optimized our previously established single-step fractionation protocol[21] to enrich for the endogenous, active 10 MDa PDHc metabolon, a critical complex involved in primary metabolism that performs “the link reaction”

Summary

Introduction

Found across all kingdoms of life, 2-keto acid dehydrogenase complexes possess prominent metabolic roles and form major regulatory sites Their component structures are known, their higher-order organization is highly heterogeneous, across species or tissues and even within a single cell. The flexible N-ter of E2 and E3BP tether E1 and E3, respectively, in relative proximity to the core[21], forming a local reaction chamber, the pyruvate dehydrogenase factory organization[21]. In this reaction chamber, the lipoyl domain (LD). The acetyl-moiety is transferred to a Coenzyme A (CoA), which enters the Krebs cycle

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