Structural basis for the folding of PINK1 by the HSP90–CDC37 chaperone complex

Reviewer #1 (Public review):

Summary:

The ubiquitin kinase PINK1 accumulates on damaged mitochondria to signal the initiation of mitophagy. While we know what PINK1 looks like when it is stabilised on damaged mitochondria, not much is known about how it gets there. In this study, Okatsu et al. solve a cryoEM structure of partially folded PINK1 in complex with its chaperones HSP90 and CDC37 to a resolution of 3.08 Å. This structure captures PINK1 in a state whereby the C-lobe of its kinase domain is folded, while the N-lobe remains unfolded and stabilised by an HSP90 dimer. According to the authors' model, their structure represents cytosolic PINK1 on its way to the mitochondria. This structure also demonstrates how PINK1 is folded in a step-wise mechanism and proposes a role for residues that are mutated in Parkinson's disease.

Strengths:

PINK1 is known to be a client of the HSP90 chaperone system. Here, Okatsu et al. present a solid structural dataset showing how PINK1 interacts with HSP90 and CDC37, and they describe key residues and motifs predicted to facilitate the interactions between PINK1 and the chaperones. Notably, two key residues within interacting regions on PINK1 are also mutated in Parkinson's disease. The structure by Okatsu et al. is in line with another recently published structure of the same complex (Tian et al. Nat Comms, 2025), which appears very similar, further supporting the findings. Together, these two studies represent the first observations of cytosolic PINK1 in a semi-folded state, which provides a novel insight into PINK1 at an earlier stage within the signalling cascade.

Weaknesses:

This paper is not the first to describe the structure of the PINK1-HPS90-CDC37 complex. A study by Tian et al. was published in early December 2025 in Nature Communications, reporting a 2.84 Å structure of PINK1-HSP90-CDC37, as well as a structure of PINK1 with HSP90 and another HSP90 co-chaperone, FKBP51. It would be important to acknowledge this comparable study and to discuss how the structure in this study compares with the Tian et al. structures and whether it reveals any additional information.

Although they make claims about the functional relevance of PINK1-interacting residues, the study by Okatsu et al. does not include any biochemical or functional validation of the structure. To support their claims, the authors should test the PINK1-HSP90-CDC37 interaction using their recombinant proteins for mutants of the conserved hydrophobic PINK1 residues in the PINK1 c-lobe, H352, L353, H360, I382, D384, as well as the PINK1 HPNI motif, especially the PD mutation H271Q. The PINK1 PD mutation L347P, which interacts with the CDC37 HPNI moti,f is also worth testing.

A major question that arises from this work is whether the PINK1-HSP90-CDC37 complex is newly translated PINK1 on its way to mitochondria (as suggested by the authors) or PINK1 that has already entered mitochondria, been cleaved and then retrotranslocated. This latter scenario is the favoured model proposed by Tian et al. (Nat Comms) based on their biochemical experiments. The discrepancies between the two models should at least be discussed, and the authors should also attempt to demonstrate experimentally whether their model is correct. This question is important to address because it would allow this structural information to be placed in the greater context of PINK1 signalling.

It is also unclear what the consequences are of disrupted PINK1-HSP90-CDC37 interactions on the PINK1 signalling process more broadly - does PINK1 accumulate in the cytosol? Is there less of it? Can it still be degraded via the N-end rule? What happens during mitophagy? Perhaps some of these questions can be answered with cell-based studies using a selection of the PINK1 mutants mentioned above that disrupt the PINK1-HSP90-CDC37 complex formation.

Reviewer #2 (Public review):

Summary:

Okatsu et al report the cryoEM structure of the PINK1-HSP90-CDC37 complex at 3.08A. To do so, they mutated the PARL cleavage site (F104M) and removed the N-terminal 103 a.a. The construct was co-expressed with HSP90beta and CDC37 in insect cells, as performed previously for other kinase-HSP90-CDC37 complexes (e.g. Raf1). Molybdate was added to prevent cycling between open and closed HSP90 conformations. The initial characterization by single particle cryoEM reveals two HSP90 conformations: closed with CDC37 dissociated, and open with the CTD of HSP90 separated. Thus, the authors crosslinked the complex, which yielded a more homogenous closed structure with clearly visible density for HSP90, CDC37, and PINK1. The structure shows an immature or partially folded kinase domain conformation for PINK1, with the C-lobe bound to HSP90 and the N-lobe unfolded. The C-lobe binds to HSP90 via the HPNI motif in CDC37, which mimics the HPNI motif found in the N-lobe of kinases, and which is conserved across kinases. The main novelty here is the interaction between the C-terminal extension (CTE) of PINK1, which must adopt another conformation than in the folded state, which would otherwise clash with HSP90. The interaction with the CTE is notably mediated by the flexible charged linker (FCL) of HSP90, which is partially disordered. In this conformation, HSP90 would clash with TOM20 binding.

Strengths:

Overall, this is well-executed structural biology work, which brings insight into the elements required to fold PINK1. The protein engineering used in this study is of great value and will help others in the field explore the function of PINK1 folding. Understanding the mode of activation of PINK1 is important, and this work brings forward hypotheses that are worthy of testing.

Weaknesses:

In the absence of functional assays, the study does not bring much novelty or biological insights. Furthermore, there are already several structures of HSP90-CDC37 bound to partially folded kinases, and a simple superposition of these structures on the model of HsPINK1 allows similar conclusions to be drawn, i.e. that it would bind a folded C-lobe and unfolded N-lobe. Furthermore, a very similar structure of PINK1 bound to HSP90-CDC37 (and FKBP5) was published in Nature Communications in December 2025 by another group. The main novelty from this work (and the paper published in December) is that the CTE adopts a different conformation compared to the mature form, but the implications of this are not explored. Furthermore, the authors propose that HSP90 would compete with TOM20, but what dictates the outcome of this competition? More importantly, how do these results help understand how PINK1 become active? Again, this is not explored.