Abstract
The enzyme soluble guanylyl cyclase (sGC) is a heterodimer composed of an α subunit and a heme-containing β subunit. It participates in signaling by generating cGMP in response to nitric oxide (NO). Heme insertion into the β1 subunit of sGC (sGCβ) is critical for function, and heat shock protein 90 (HSP90) associates with heme-free sGCβ (apo-sGCβ) to drive its heme insertion. Here, we tested the accuracy and relevance of a modeled apo-sGCβ-HSP90 complex by constructing sGCβ variants predicted to have an impaired interaction with HSP90. Using site-directed mutagenesis, purified recombinant proteins, mammalian cell expression, and fluorescence approaches, we found that (i) three regions in apo-sGCβ predicted by the model mediate direct complex formation with HSP90 both in vitro and in mammalian cells; (ii) such HSP90 complex formation directly correlates with the extent of heme insertion into apo-sGCβ and with cyclase activity; and (iii) apo-sGCβ mutants possessing an HSP90-binding defect instead bind to sGCα in cells and form inactive, heme-free sGC heterodimers. Our findings uncover the molecular features of the cellular apo-sGCβ-HSP90 complex and reveal its dual importance in enabling heme insertion while preventing inactive heterodimer formation during sGC maturation.