Composition and cryoEM structure of the GC-C–Hsp90–Cdc37 regulatory complex.

(A) Cartoon representation of the components of GC-C signaling and Hsp90–Cdc37 regulation and the zippered and activated GC-C. GC-C is colored in red, guanylin/uroguanylin (Gn/Uro) in yellow, Hsp90 in blue and teal, and Cdc37 in purple. Extracellular domains (ECD), transmembrane domain (TM), pseudokinase domain (PK), dimerization domain (DD), and guanylyl cyclase domain (GC) are labelled. In the rightmost cartoon, the regions unobserved in the cryoEM density are in a lighter shade with a dashed outline. (B) The refined and sharpened cryoEM density map of GC-C–Hsp90–Cdc37, colored as in A, with a transparent overlay of an unsharpened map with additional DD density resolved. Cdc37 coil-coiled and middle domain (MD) are labelled. (C) Reference free 2D averages for the GC-C–Hsp90– Cdc37 complex. (D) The refined and sharpened cryoEM density map of GC-C–Hsp90–Cdc37, colored as in A and B, labelled with all domains as in A and B, with the addition of Hsp90 N-terminal domain (NTD), middle domain (MD), and C-terminal domain (CTD). (E) Ribbon representation of a model of GC-C–Hsp90–Cdc37 complex, colored and labelled as in A, B, and C.

Cdc37 mediated GC-C recruitment and Hsp90 loading interfaces.

(A) Ribbon representation of a model of GC-C–Hsp90–Cdc37 complex. GC-C is colored in red, Hsp90 in blue and teal, and Cdc37 in purple. Pseudokinase (PK), coil-coiled, middle (MD), C-terminal (CTD), and N-terminal (NTD) domains are labelled. (B) The Cdc37–GC-C interface in ribbon representation, with interacting residues drawn in sticks, colored as in A. (C) The unfolded N-lobe of GC-C PK domain as it passes between the Hsp90 dimer, in ribbon representation, with interacting residues drawn in sticks, colored as in A and B. This region’s sequence is: VKLDTMIFGVIEYCERG.

CryoEM data collection, refinement, and validation statistics.

GC-C–Hsp90–Cdc37 complex cryoEM data processing.

(A) Workflow for cryoEM data processing. SDS-PAGE gel, representative micrograph, reference free 2D averages, and cryoEM maps at the various stages of processing. (B) Local resolution estimation of the finalised cryoEM map. (C) FSC curve of the reconstruction using gold-standard refinement calculated from unmasked and masked half maps. Map-model FSC curve. (D) Orientational distribution of the reconstruction. (E) Directional FSC curves from 3DFSC (Aiyer et al., 2021).

Representative density of GC-C–Hsp90–Cdc37.

(A) Representative density of Hsp90. (B) Representative density of Cdc37. (C) Representative density of GC-C.

Conservation of Cdc37 mediated Hsp90 regulation.

(A) Ribbon representation of a models of client–Hsp90β–Cdc37 complexes. GC-C is colored in red, Cdk4 in yellow (5FWK), RAF1 in green (7Z37), B-raf in orange (7ZR0), Hsp90β in light blue and teal, and Cdc37 in light purple. (B) A structural overlay of the structures in A. (C) A sequence alignment of the pseudokinase domain of GC-C and the kinase domains of Cdk4, RAF1, and B-raf. Sequence numbering per GC-C, with a blue line depicting regions resolved in the cryoEM density.

Regulatory mechanisms for mGC activity.

A schematic of mGC ligand induced activity, phosphorylation, and destabilization, leading to formation of the mGC–Hsp90–Cdc37 complex structurally characterized in this work. This core regulatory complex would then lead to refolding of the PK and reactivation of the receptor (i), recruitment of PP5 and dephosphorylation of the receptor (ii), or recruitment of E3 ligases and removal of the receptor (iii). An mGC is depicted in red, ligand in yellow, Hsp90 in blue and teal, Cdc37 in purple, a phosphatase in green, and an E3 ligase in orange.