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Conformational Disease Landscape of DNAJB1-PRKACA

Phillip Aoto
Appointment Period: 2016-2018, Grant Year: [31, 32]

Philip Aoto

DNAJB1-PRKACA (LiverC) is the recently discovered driver of the untreatable liver cancer fibrolamellar hepatocellular carcinoma (FLHCC). It is a fusion protein between the catalytic subunit of protein kinase A (PKA-C) and the J-domain of heat shock protein 40 (Hsp40). The molecular mechanism leading to oncogenesis is not precisely known, but it is likely that aberrant activity or scaffolding function of liverC contributes to disease. The chimera is the driver of the cancer and thus is the obvious therapeutic target for treatment of FLHCC. An understanding of the dynamic conformational landscape of liverC will clarify the mechanism of disease and can be utilized in the identification of novel and highly specific allosteric druggable pockets. The aims and recent progress for the project are as follows:

Aim 1: Characterize the conformational landscape of liverC by molecular dynamics (MD) simulations and Markov state models (MSM) in order to elucidate the molecular mechanism of disease and to identify hidden druggable allosteric pockets. An exploration of the liverC conformational landscape by MD simulations/MSM will identify, in conjunction with biochemical characterization (aim 2), disease relevant states to help understand the molecular basis of FLHCC. Conformations of liverC that do not promote disease will be investigated for hidden allosteric pockets. Small molecules will be screened in silico to target these pockets and characterized biochemically and by crystallography. Progress: MD simulations show significant increase in dynamics of the N-terminal helix in liverC. Mutual information network analysis of the simulations identifies a loss of a key allosteric coupling between the N-terminal helix and the active site. This disruption leads to an observed systemwide loss in HDX (concerted breathing motions) in liverC. To return normal allosteric regulation to the N-terminal helix (an important regulatory element) we will attempt to restrict the flexibility of the helix by binding small molecules. We have also found that changes in protonation state of key titratable groups in wildtype PKAc and liverC lead to differential changes in conformational state. This is an undiscovered mechanism for driving conformational change in PKAc. In addition, these protonation stabilized metastable states offer unprobed conformation space as therapeutic targets. Intriguingly, we have found that these states have unique solvent exposed pockets amenable to binding by small molecule.

Aim 2: Investigate the interaction of binding partners with liverC to elucidate their effect on disease and to expand on our understanding of the conformational landscape. Interactions with binding partners of liverC will be characterized. Results from Aim 2 will be used within Aim 1 to design small molecules that (de)stabilize the liverC conformation conducive for interaction, depending on if the complex represents an active or inactive disease state. Progress: The J-domain in LiverC contains a surface exposed Hsp70 recognition motif and preliminary docking simulations have found that the ATPase domain of Hsp70 docks in an orientation amenable to interaction with the motif. A nuclear translocation protein(AKIP1a) has also been shown by peptide array to interact with Hsp70 and LiverC separately and may contribute to liverC:Hsp70 docking. We plan to further investigate the exact binding surface of the complexes by HDX and/or NMR. Integrative modeling will be performed with experimental data to create a refined docking model. Concurrently, crystallization of the complexes will be attempted. Preliminary size-exclusion data also indicates that LiverC preferentially forms a heterotetramer with two R-subunits and one wildtype PKAc. The role of this heterotetramer in disease will be investigated. Progress is in parallel to work by collaborators on the cryoEM structure of the complex. APEX2 labeling in HEK293 cells will be used to identify differences in binding partners and to verify the existence of a liverC heterotetramer.

PUBLICATIONS (resulting from this training):

Trainee recently appointed to grant, publications are still in progress.