How do Hsp90 and Cdc37 assist protein folding in Cdk4?

Part of the phosphotransferase family, protein kinases are involved in the modification of approximately 1/3 of all proteins via phosphorylation. Misregulation of these enzymes can prove detrimental to cell growth and survival and so activity requires tight regulation; for about 60% of kinases, their active state and stabilisation are only achieved with the protein-folding molecular chaperone Hsp90 and its co-chaperone Cdc37.

Hsp90 is a dimer, with each monomer having a CTD accountable for dimerization, an MD associated with client binding, and an NTD to which ATP binds: ATP binding promotes the transition from an open state to a closed state. Cdc37 is a monomer, with a CTD of unknown function, and NTD and globular MB that form interactions with kinases and Hsp90, respectively. Knowledge of the mechanism of assisting kinase function and the specificity that allows chaperones to discriminate between closely related kinases is lacking; this is a potential area for future research.

A key kinase client Hsp90-Cdc37 complex is Cdk4, a crucial protein in the G1-phase progression of the cell cycle. The atomic structure of the Hsp90-Cdc37-Cdk4 complex (Figure 1) revealed in a recent paper gives an updated model of the mechanism of kinase-chaperone interactions.

Figure 1. Cartoon representation of the Hsp90-Cdc37-Cdk4 kinase complex. 

Hsp90 monomers (green and cyan) wrap around the Cdk4 (yellow) MD and Cdc37 (magenta) forms stabilising interactions with both Hsp90 and Cdc37.

This updated structure was obtained by co-expressing human homologs of the three proteins in insect cells; cyro-electron microscopy was performed, giving 3.9 Å density map of the complex. A refined atomic model of Hsp90β was built into the map, adopting a closed conformation; the resulting regions included a globular density and a long coiled-coil like bulge.

Searching against protein folds led to a high-quality fit of the Cdk4 C-lobe accounting for the globular region. No suitable density was present for the folded kinase N-lobe; however, when kinase density was traced from the C-lobe to the N terminus, a distinct tubular region going through the lumen of Hsp90 was determined, indicating that the N-lobe threads through Hsp90 in an intensely altered conformation, stabilised by the chaperone. Further investigation by threading the Cdk4 sequence into this region uncovered that the two lobes are completely separated and the β4-β5 sheet is unfolded, with β5 forming hydrophobic interactions and two salt bridges with a client binding site of Hsp90. This unfolded structure is unusual for kinases to adopt and is only possible because of the stabilising interactions with Hsp90 and Cdc37.

Figure 2. Cdk4 is completely unfolded in the complex (A-C left to right). 

(A) Hsp90 (green/cyan) lumen and Cdk4 (yellow) C-lobe shown as a surface image. (B) Unfolded β4-β5 sheet, with Cdk4 threaded through the Hsp90 lumen. (C) Hsp90-Cdk4 β5 interactions. Dotted lines between residues represent salt bridges. 

The coiled-coil density was taken as the Cdc37 NTD following sequence analysis, and transitioned from helical to strand-like throughout the chain, wrapping around the Hsp90 MD. Essentially, Cdc37 mimics a section of the kinase N-lobe by addition of a β-strand to the β-sheet to stabilise an open conformation of the kinase.

Phosphorylation of Ser¹³ within the Cdc37 chain is crucial for kinase binding and eventual function. However the specific function of the phosphorylation was only brought to light after the research by K. Verba et al. - previously it was only know that this residue is highly conserved and that its location with the NTD suggests it has a role in kinase binding however specific interactions within the complex are now known (Figure 3). These interactions have been found to stabilise the coiled-coil NTD and regulate Cdc37 function. It has been suggested that phosphorylation of this residue is needed for the formation of productive Hsp90-Cdc37-Cdk4 complexes and its subsequent dephosphorylation is required for the activation and release of the processed kinase (C.K. Vaughan et al, 2008).

Figure 3. Electrostatic interactions between phosphorylated Cdc37-Ser¹³ and nearby residues. 

Salt bridges (shown as dashed yellow lines) form between Ser¹³ and Cdc37-Arg³⁶ and Cdc-His³³, and also between Cdc37-Ser¹³ and Hsp90-Lys⁴⁰⁶, thus contributing the overall electrostatic nature of Hsp90-Cdc37 interactions.

The identification of the unfolded structure adopted by Cdk4 provides an insight into why no sequence motif for Hsp90 interactions has been discovered – it is proposed that the tendency of a kinase to unfold into this open state determines whether a kinase is an Hsp90 client, as opposed to any specific binding sequence. The property of a kinase being able to remain in a stable open intermediate has a functional and regulatory benefit. Cdk4 transitions between the inactive and active state through an open, unfolded state; this has been shown to be more energetically favourable than the standard rigid-body transition adopted by most proteins. Hsp90 client kinases generally have a lower stability allowing them to enter the unfolded state, encouraging chaperone binding which then stabilises this state.

Also crucial for kinase binding are the ATP binding sites within Hsp90. Without ATP, Hsp90 adopts an open formation (Figure 4), but upon ATP binding the HSP90 dimer changes conformation and closes (Figure 5). ATP binds after Cdc37 and Cdk4 have bound to Hsp90; the conformation change traps the unfolded Cdk4 region, allowing the kinase to fold. 

Figure 4. Open conformation of the Hsp90 dimer

Figure 5. Formation of the closed state of Hsp90 upon ATP binding. ATP molecules are represented as ball and stick figure; each Hsp90 monomer has a binding site for an ATP molecule.

In some cancers raised levels of Cdc37 have been detected, with overexpression being associated with increased kinase pathway activation and cellular proliferation rates through more Hsp90-Cdk4 and Cdc37-Cdk4 complexes. Drug treatment studies on kinase inhibitors have suggested that efficacy might be partially attributable to the compounds’ activity in preventing Kinase-Cdc37 binding and thus formation of the complex. Cdc37 inhibitors are therefore a potential lead for improved therapeutics. (S. R. McAlpine et al. 2016, pg 35-6)

Unless otherwise stated, information is taken from a paper by K. Verba et al, 2016