Cryo-EM analysis of human mitochondrial Hsp90 in multiple tetrameric states

Hsp90 is a ubiquitous molecular chaperone that mediates the folding and maturation of hundreds of "client" proteins. Although Hsp90s generally function as homodimers, recent discoveries suggested that the mitochondrion specific Hsp90 (TRAP1) also forms functionally relevant tetramers. The structural mechanism of tetramer formation remains elusive. Here we used a combination of solution, biochemical and cryo-electron microscopy (cryo-EM) approaches to confirm that, independent of nucleotide state, a subpopulation of TRAP1 exists as tetramers. Unexpectedly, cryo-EM reveals multiple tetramer conformations having TRAP1 dimers arranged in orthogonal, parallel, or antiparallel configurations. The cryo-EM structure of one of the orthogonal tetrameric states was determined at 3.5 [A] resolution. Each of the two TRAP1 dimers is in a symmetric AMP{middle dot}PNP-bound closed state with the tetramer being stabilized through three distinct dimer-dimer interaction sites. In unique ways, each of the three TRAP1 domains contributes to tetramer formation. In addition to tetramerization via direct dimer-dimer contacts, our structure suggests that additional stabilization could come from domain swapping between the dimers. These results expand our understanding of TRAP1 biology beyond the conventional view of a functional dimer and provide a platform to further explore the function and regulation of tetrameric TRAP1 in mitochondria.