HSP90 Interaction with eNOS and nNOS

HSP90s are multi‐function chaperonins expressed at high levels in mammalian cells; homologues are important in most eukaryotes. HSP90s have a broad range of client proteins, and have been implicated in protein folding, and many proteins are stabilized by interaction with HSP90. HSP90 recognizes early stages denaturation and prevents the formation of large aggregates, allowing clients to regain native conformation. The HSP90‐client complex also includes the P23 co‐chaperone, a small heat shock protein. Binding is ATP independent, but HSP90 has significant ATPase activity connected with client release. Binding of eNOS and nNOS to HSP90 can be observed using interferometric methods. Both eNOS and nNOS bind to immobilized HSP90 with moderate affinity (~200–500 nmolar). Both isoforms have a complex report of binding. Above Kd, both bind with near diffusion limited rates, and both show a second slow phase during which the shift of the interference pattern is significantly reduced, implying either release or a major conformational change while bound to the sensor. HSP90 is a potent activator of both cytochrome c reduction and NO synthesis in both isoforms. This can be explained at the molecular level by changes in population of enzyme conformations. eNOS and nNOS have three major FMN fluorescence lifetime states corresponding to FMN‐FAD, FMN‐heme, and essentially non interacting configurations. Like calmdodulin, HSP90 releases the FMN binding domain from a tight complex with the dehydrogenase domains of NOS, promoting the longer lived states at the expense of the short lived (90ps) FMN‐FAD pair of the input state. HSP90‐NOS interactions revealed via fluorescence exhibit temporal fluctuations reminiscent of the complex report of binding, and are responsive to ATP.