Phosphorylated and Unphosphorylated Serine 13 of CDC37 Stabilize Distinct Interactions between Its Client and HSP90 Binding Domains

Abstract

Folding and maturation of most protein kinases require chaperone assistance. In higher eukaryotes, CDC37 is the predominant cochaperone that facilitates the transfer of kinase clients to HSP90. Kinase recognition is thought to occur through the N-terminal domain, which has, thus far, eluded structure determination. Client processing also requires the phosphorylation of the N-terminal tail at Ser13 by protein kinase CK2 (casein kinase 2). How phosphorylation alters the molecular properties of CDC37 is not understood. We show that the phosphorylation at Ser13 induces a large shift toward a more compact structure, based on ANS fluorescence, while modestly increasing secondary structure. Moreover, this transition requires interactions of the N-terminal domain and the remainder of CDC37. The stabilizing property of the phosphorylation event can be recreated in trans by a (phospho-Ser13) peptide derived from the N-terminal tail. However, the phosphorylation-induced transition is not dependent on the transferred phosphate group but rather the loss of serine-like properties at position 13. The complete absence of the N-terminal tail results in reduced secondary structure and unresponsiveness to subsequent addition of peptides. The N-terminal tail may therefore serve as an intramolecular chaperone that ensures that CDC37 assumes one of two readily interconvertible states in a manner that impacts the interaction of the client binding N-domain and the MC-domains, involved in dimerization and HSP90 binding.

Atomic structure of Hsp90-Cdc37-Cdk4 reveals that Hsp90 traps and stabilizes an unfolded kinase

“…In our structure there is density for the phosphorylated-serine13, which forms a salt bridge with Cdc37:R36 and Cdc37:H33, stabilizing the very N-terminus of the coiled coil (Fig 5B middle insert). This provides a molecular rationale for previous observations of a Cdc37 conformational change upon phosphorylation(28). Of note, this phosphate also contributes to the overall electrostatic nature of Hsp90-Cdc37 interactions, forming a salt bridge with Hsp90:K406.…”

Atomic structure of Hsp90:Cdc37:Cdk4 reveals Hsp90 regulates kinase via dramatic unfolding

“…In our structure there is density for the phosphorylated-serine13, which forms a salt bridge with Cdc37:R36 and Cdc37:H33, stabilizing the very N-terminus of the coiled coil ( Fig 5B middle insert). This provides a molecular rationale for previous observations of a Cdc37 conformational change upon phosphorylation (27). Of note, this phosphate also contributes to the overall electrostatic nature of Hsp90-Cdc37 interactions, forming a salt bridge with Hsp90:K406.…”

Molecular Mechanism of Protein Kinase Recognition and Sorting by the Hsp90 Kinome-Specific Cochaperone Cdc37

“…The S13A mutation in Cdc37 (Cdc37 S13A ) is known to impact client interaction and maturation, likely at later stages of stable ternary complexes involving Hsp90, while its impact on the formation of the unstable binary complex is low. This allows complex recovery when the overexpression of both client and Cdc37 increases the population of binary complex intermediates (Liu and Landgraf, 2015). However, while the impairment in Cdc37 complex recovery is far more pronounced for the AEE mutant, the mutant still discriminates between wild-type and bRaf V600E .…”