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

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.

The structure and evolutionary diversity of the fungal E3-binding protein

“…As shown previously(Forsberg et al , 2020; Tüting et al , 2021), the CTD-trimers form a homomeric dimer interface and hydrophobic pocket to which the CBD of fungal E3BP binds (Fig. 2B).…”

“…E3BP and E2 are homologous based on similarities in domain topology. Computational modeling of C. Thermophilum E3BP also predicted that its CBD was a partial E2 fold (Tüting et al, 2021). The reconstruction of the N.crassa CBD validates this model and confirms that fungal E3BP arose by neo-functionalization of a duplicated 2-oxoacid acetyltransferase gene in fungi as well as in mammals.…”

“…3B). A conserved Phe (N.crassa F494) appears crucial by protruding directly into the binding site, as predicted in C. thermophilum (Tüting et al, 2021). Additionally, K266 from both participating CTD monomers interact with M2 residues N325 and Q326 from the CBD (Fig.…”

“…Like mammals, fungi also utilize E3BP to recruit E3 (Maeng et al, 1994(Maeng et al, , 1996Stoops et al, 1997). Unlike mammals however, fungal E3BP binds to the interior of the E2 core assembly instead of substituting core components (Forsberg et al, 2020;Skalidis et al, 2021;Tüting et al, 2021)(Fig. 1B).…”

Comparative Network Biology Discovers Protein Complexes That Underline Cellular Differentiation in Anabaena sp.

“…Over the years, the combination of cryo-EM with CoFrac–MS has facilitated the development of systems' structural proteomics by reducing the requirement for a pure and homogeneous sample ( 64 , 65 ). A fully assembled structural information of a certain protein complex can be obtained by utilizing the cryo-EM, crosslinking, and MS in native cell extracts, such as pyruvate dehydrogenase complex ( 66 , 67 ). Combined with these assays, the active nanostructures of protein complexes involved in heterocyst differentiation can be further investigated in the future.…”