Cooperative nucleotide binding in Hsp90 and the underlying mechanisms

The function of the molecular chaperone Hsp90 depends on large conformational changes, rearrangement of local motifs, as well as the binding and hydrolysis of ATP. The complexity of the Hsp90 system impedes the detailed investigation of their interplay using standard methods. By the application of three-color single molecule FRET to Hsp90 and a reporter nucleotide, we directly observe cooperativity between the two nucleotide binding pockets in the protein dimer. Through allocating the microscopic states and extracting their kinetics, we identify the mechanisms underlying the cooperativity. Surprisingly, nucleotide binding affects several state transitions, which demonstrates the complexity of cooperativity in protein systems. The co-chaperone Aha1, known to accelerate Hsp90's ATPase activity, adds another layer of complexity by affecting transitions in a nucleotide-dependent and -independent manner.