A central role for extended polypeptide linkers in establishing the spatial architecture of a pyruvate dehydrogenase complex

Abstract

The pyruvate dehydrogenase complex is among the largest multifunctional enzymes found in cells (>11MDa). It catalyzes the production of acetyl-CoA from pyruvate and is comprised of three component enzymes: E1, E2, and E3. Previous work from our laboratory resulted in determination of the molecular architectures of sub-complexes comprising the 60-mer E2 from Bacillus stearothermophilus decorated with either 60 copies of E1 or E3. An annular gap of ~75–95 Å separates the catalytic domains of the E2 from an outer shell formed of E1’s or E3’s. To test the mechanisms involved in the maintenance of this annular gap and its significance for active site coupling, cryo-electron tomography was employed to determine the spatial relationship of E1 and E2 components within individual, non-averaged, E1E2 sub-complexes. In addition to verifying the presence of the annular space in three dimensions, stochastic variation in the exact position of each individual E1 density relative to the E2 core was measured. Biophysical studies of a peptide, corresponding to the inner linker region, suggests this region is flexible, yet highly extended, and maintains a long persistence length (~70–89 Å). Together these results demonstrate that the inner linker region separating the bound E1 and E3 molecules from the E2 core is an extended peptide chain that maintains a long persistence length, sixty of which generate a scaffold for the organization of E1 and E3 molecules in a modular radial shell. This is likely to be critically important to the coupling of E1, E2, and E3 active sites, mediated by movements across the gap by an additional domain at the N-terminal end of the E2 chain.