Arrangement and Symmetry of the Fungal E3BP-Containing Core of the Pyruvate Dehydrogenase Complex

Abstract

The pyruvate dehydrogenase complex (PDC) is a multienzyme protein complex central to aerobic respiration, connecting glycolysis to mitochondrial oxidation of pyruvate. Its three main components (E1, E2, E3) exhibit a fascinating organization with multiple levels of symmetry. Despite its central metabolic role, important questions regarding its composition and regulation remain. Similar to the E3-binding protein (E3BP) of mammalian PDC, there is a “Protein X” (PX) that selectively recruits E3 to the fungal PDC, but its divergent sequence suggests a distinct structural mechanism. To explain the variation in stoichiometry reported for PX in fungal PDC, we have used cryo-electron microscopy to reconstruct PDC densities from the filamentous fungus Neurospora crassa, and show how PX is present interior to the PDC core instead of substituting E2 subunits as in mammals. We show how steric occlusion limits PX binding, resulting in predominantly tetrahedral symmetry, which explains the observations of a variety of stoichiometries in Saccharomyces cerevisiae. The PX-binding site is conserved in - and specific to - fungi, and complements possible C-terminal binding motifs in PX that are absent in mammalian E3BP. Consideration of multiple symmetries thus reveals a differential structural basis for E3BP-like function in fungal PDC, and suggests the PX oligomer stability and size are potential mechanisms that might adjust PDC composition in response to external cues.