Andrea N. Kravats

Anti-adaptors provide multiple modes for regulation of the RssB adaptor protein

RpoS, an RNA polymerase σ factor, controls the response of Escherichia coli and related bacteria to multiple stress responses. During nonstress conditions, RpoS is rapidly degraded by ClpXP, mediated by the adaptor protein RssB, a member of the response regulator family. In response to stress, RpoS degradation ceases. Small anti-adaptor proteins--IraP, IraM, and IraD, each made under a different stress condition--block RpoS degradation. RssB mutants resistant to either IraP or IraM were isolated and analyzed in vivo and in vitro. Each of the anti-adaptors is unique in its interaction with RssB and sensitivity to RssB mutants. One class of mutants defined an RssB N-terminal region close to the phosphorylation site and critical for interaction with IraP but unnecessary for IraM and IraD function. A second class, in the RssB C-terminal PP2C-like domain, led to activation of RssB function. These mutants allowed the response regulator to act in the absence of phosphorylation but did not abolish interaction with anti-adaptors. This class of mutants is broadly resistant to the anti-adaptors and bears similarity to constitutively activated mutants found in a very different PP2C protein. The mutants provide insight into how the anti-adaptors perturb RssB response regulator function and activation.

Interplay between E. coli DnaK, ClpB and GrpE during Protein Disaggregation

The DnaK/Hsp70 chaperone system and ClpB/Hsp104 collaboratively disaggregate protein aggregates and reactivate inactive proteins. The teamwork is specific: Escherichia coli DnaK interacts with E. coli ClpB and yeast Hsp70, Ssa1, interacts with yeast Hsp104. This interaction is between the middle domains of hexameric ClpB/Hsp104 and the DnaK/Hsp70 nucleotide-binding domain (NBD). To identify the site on E. coli DnaK that interacts with ClpB, we substituted amino acid residues throughout the DnaK NBD. We found that several variants with substitutions in subdomains IB and IIB of the DnaK NBD were defective in ClpB interaction in vivo in a bacterial two-hybrid assay and in vitro in a fluorescence anisotropy assay. The DnaK subdomain IIB mutants were also defective in the ability to disaggregate protein aggregates with ClpB, DnaJ and GrpE, although they retained some ability to reactivate proteins with DnaJ and GrpE in the absence of ClpB. We observed that GrpE, which also interacts with subdomains IB and IIB, inhibited the interaction between ClpB and DnaK in vitro, suggesting competition between ClpB and GrpE for binding DnaK. Computational modeling of the DnaK-ClpB hexamer complex indicated that one DnaK monomer contacts two adjacent ClpB protomers simultaneously. The model and the experiments support a common and mutually exclusive GrpE and ClpB interaction region on DnaK. Additionally, homologous substitutions in subdomains IB and IIB of Ssa1 caused defects in collaboration between Ssa1 and Hsp104. Altogether, these results provide insight into the molecular mechanism of collaboration between the DnaK/Hsp70 system and ClpB/Hsp104 for protein disaggregation.

Unfolding and translocation pathway of substrate protein controlled by structure in repetitive allosteric cycles of the ClpY ATPase

Clp ATPases are ring-shaped AAA+ motors in the degradation pathway that perform critical actions of unfolding and translocating substrate proteins (SPs) through narrow pores to deliver them to peptidase components. These actions are effected by conserved diaphragm-forming loops found in the central channel of the Clp ATPase hexamer. Conformational changes, that take place in the course of repetitive ATP-driven cycles, result in mechanical forces applied by the central channel loops onto the SP. We use coarse-grained simulations to elucidate allostery-driven mechanisms of unfolding and translocation of a tagged four-helix bundle protein by the ClpY ATPase. Unfolding is initiated at the tagged C-terminal region via an obligatory intermediate. The resulting nonnative conformation is competent for translocation, which proceeds on a different time scale than unfolding and involves sharp stepped transitions. Completion of the translocation process requires assistance from the ClpQ peptidase. These mechanisms contrast nonallosteric mechanical unfolding of the SP. In atomic force microscopy experiments, multiple unfolding pathways are available and large mechanical forces are required to unravel the SP relative to those exerted by the central channel loops of ClpY. SP threading through a nonallosteric ClpY nanopore involves simultaneous unfolding and translocation effected by strong pulling forces.

Hsp70 and Hsp90 of E. coli Directly Interact for Collaboration in Protein Remodeling

Hsp90 is a highly conserved molecular chaperone that remodels hundreds of client proteins, many involved in the progression of cancer and other diseases. It functions with the Hsp70 chaperone and numerous cochaperones. The bacterial Hsp90 functions with an Hsp70 chaperone, DnaK, but is independent of Hsp90 cochaperones. We explored the collaboration between Escherichia coli Hsp90 and DnaK and found that the two chaperones form a complex that is stabilized by client protein binding. A J-domain protein, CbpA, facilitates assembly of the Hsp90Ec-DnaK-client complex. We identified E. coli Hsp90 mutants defective in DnaK interaction in vivo and show that the purified mutant proteins are defective in physical and functional interaction with DnaK. Understanding how Hsp90 and Hsp70 collaborate in protein remodeling will provide the groundwork for the development of new therapeutic strategies targeting multiple chaperones and cochaperones.

Asymmetric processing of a substrate protein in sequential allosteric cycles of AAA+ nanomachines.

Essential protein quality control includes mechanisms of substrate protein (SP) unfolding and translocation performed by powerful ring-shaped AAA+ (ATPases associated with various cellular activities) nanomachines. These SP remodeling actions are effected by mechanical forces imparted by AAA+ loops that protrude into the central channel. Sequential intra-ring allosteric motions, which underlie repetitive SP-loop interactions, have been proposed to comprise clockwise (CW), counterclockwise (CCW), or random (R) conformational transitions of individual AAA+ subunits. To probe the effect of these allosteric mechanisms on unfoldase and translocase functions, we perform Langevin dynamics simulations of a coarse-grained model of an all-alpha SP processed by the single-ring ClpY ATPase or by the double-ring p97 ATPase. We find that, in all three allosteric mechanisms, the SP undergoes conformational transitions along a common set of pathways, which reveals that the active work provided by the ClpY machine involves single loop-SP interactions. Nevertheless, the rates and yields of SP unfolding and translocation are controlled by mechanism-dependent loop-SP binding events, as illustrated by faster timescales of SP processing in CW allostery compared with CCW and R allostery. The distinct efficacy of allosteric mechanisms is due to the asymmetric collaboration of adjacent subunits, which involves CW-biased structural motions of AAA+ loops and results in CW-compatible torque applied onto the SP. Additional simulations of mutant ClpY rings, which render a subset of subunits catalytically-defective or reduce their SP binding affinity, reveal that subunit-based conformational transitions play the major role in SP remodeling. Based on these results we predict that the minimally functional AAA+ ring includes three active subunits, only two of which are adjacent.

Interaction of E. coli Hsp90 with DnaK Involves the DnaJ Binding Region of DnaK

The 90-kDa heat shock protein (Hsp90) is a widely conserved and ubiquitous molecular chaperone that participates in ATP-dependent protein remodeling in both eukaryotes and prokaryotes. It functions in conjunction with Hsp70 and the Hsp70 cochaperones, an Hsp40 (J-protein) and a nucleotide exchange factor. In Escherichia coli, the functional collaboration between Hsp90Ec and Hsp70, DnaK, requires that the two chaperones directly interact. We used molecular docking to model the interaction of Hsp90Ec and DnaK. The top-ranked docked model predicted that a region in the nucleotide-binding domain (NBD) of DnaK interacted with a region in the middle domain of Hsp90Ec. We then made substitution mutants in DnaK residues suggested by the model to interact with Hsp90Ec. Of the 12 mutants tested, 11 were defective or partially defective in their ability to interact with Hsp90Ecin vivo in a bacterial two-hybrid assay and in vitro in a bio-layer interferometry assay. These DnaK mutants were also defective in their ability to function collaboratively in protein remodeling with Hsp90Ec but retained the ability to act with DnaK cochaperones. Taken together, these results suggest that a specific region in the NBD of DnaK is involved in the interaction with Hsp90Ec, and this interaction is functionally important. Moreover, the region of DnaK that we found to be necessary for Hsp90Ec binding includes residues that are also involved in J-protein binding, suggesting a functional interplay among DnaK, DnaK cochaperones, and Hsp90Ec.

Coarse-Grained Simulations of Topology-Dependent Mechanisms of Protein Unfolding and Translocation Mediated by ClpY ATPase Nanomachines.

Clp ATPases are powerful ring shaped nanomachines which participate in the degradation pathway of the protein quality control system, coupling the energy from ATP hydrolysis to threading substrate proteins (SP) through their narrow central pore. Repetitive cycles of sequential intra-ring ATP hydrolysis events induce axial excursions of diaphragm-forming central pore loops that effect the application of mechanical forces onto SPs to promote unfolding and translocation. We perform Langevin dynamics simulations of a coarse-grained model of the ClpY ATPase-SP system to elucidate the molecular details of unfolding and translocation of an α/β model protein. We contrast this mechanism with our previous studies which used an all-α SP. We find conserved aspects of unfolding and translocation mechanisms by allosteric ClpY, including unfolding initiated at the tagged C-terminus and translocation via a power stroke mechanism. Topology-specific aspects include the time scales, the rate limiting steps in the degradation pathway, the effect of force directionality, and the translocase efficacy. Mechanisms of ClpY-assisted unfolding and translocation are distinct from those resulting from non-allosteric mechanical pulling. Bulk unfolding simulations, which mimic Atomic Force Microscopy-type pulling, reveal multiple unfolding pathways initiated at the C-terminus, N-terminus, or simultaneously from both termini. In a non-allosteric ClpY ATPase pore, mechanical pulling with constant velocity yields larger effective forces for SP unfolding, while pulling with constant force results in simultaneous unfolding and translocation.

50 Interaction and collaboration between DnaK and ClpB during protein disaggregation.

References Berg, J. M., Tymoczko, J. L., & Stryer, L. (2002). Biochemistry (5th ed.). New York, NY: W.H. Freeman. Kanai, R., Ogawa, H., Vilsen, B., Cornelius, F., & Toyoshima, C. (2013). Crystal structure of a Na-bound Na, KATPase preceding the E1P state. Nature, 502, 201–206. Nyblom, M., Poulsen, H., Gourdon, P., Reinhard, L., Andersson, M., Lindahl, E., ... Nissen, P. (2013). Crystal structure of Na+, K(+)-ATPase in the Na(+)-bound state. Science, 342, 123–127. Pan, A. C., Sezer, D., & Roux, B. (2008). Finding transition pathways using the string method with swarms of trajectories. The Journal of Physical Chemistry, 112, 3432–3440. Toyoshima, C., & Cornelius, F. (2013). New crystal structures of PII-type ATPases: Excitement continues. Current Opinion in Structural Biology, 23, 507–514. Yu, H., Ratheal, I. M., Artigas, P., & Roux, B. (2011). Protonation of key acidic residues is critical for the K-selectivity of the Na/K pump. Nature Structural & Molecular Biology, 18, 1159–1163.

Abstract 1180: Targeting the HSP40/HSP70 chaperone axis as a novel strategy to treat castration-resistant prostate cancer

Castration-resistant prostate cancer (CRPC) is frequently characterized by elevated expression of nuclear receptors able to at least partially maintain the androgen receptor (AR) transcriptional program. Elevated expression of a number of constitutively active AR splice variants lacking the ligand binding domain (LBD) (e.g., ARv7, which is ligand-independent and correlates with poor prognosis, reduced survival, and resistance to existing LBD-targeted standard of care therapy) is a frequent occurrence in CRPC. Thus, alternative approaches to disrupt AR signaling in CRPC are of great clinical importance, and a single strategy able to target AR and ARv7 remains a critical unmet need. As a steroid hormone nuclear receptor, the AR exists in an interactive and dynamic cycle with the molecular chaperones (heat shock proteins, HSPs) HSP40/HSP70/HSP90 for proper folding and remodeling of the AR LBD to bind ligand. Notably, HSP90 inhibitors promote AR degradation and display efficacy in prostate cancer xenograft models. Although it has been shown that ARv7 functions independently of HSP90, additional chaperone requirements of LBD-deficient ARv7 are not known. Thus, we tested the hypothesis that both AR and ARv7 are dependent on HSP40/HSP70 and that targeting these chaperones with specific inhibitors (C86 and JG98, respectively) will lead to AR/ARv7 destabilization and loss of transcriptional activity in models of CRPC. To determine if AR proteins associate with HSP40/HSP70, 22Rv1 CRPC cells (expressing endogenous AR and ARv7) were first transfected with FLAG-HSP40 or FLAG-HSP70. Immunoprecipitation with FLAG beads revealed AR and ARv7 associated with both chaperones, indicating potential functional dependence of these nuclear receptors on HSP40/HSP70. To further characterize these interactions, 22Rv1 lysate was probed with biotinylated-C86 and subjected to IP with streptavidin beads. C86 bound a significant fraction of HSP40 complexed with HSP70, AR, and ARv7. Excess unlabeled C86 or JG98 effectively competed away binding of HSP40/HSP70 to biotinylated-C86 with concomitant loss of associated AR and ARv7. Treatment of 22Rv1 cells with C86 or JG98 led to a time and dose-dependent decrease in AR and ARv7 protein, concomitant with a significant loss of viability. We also observed that HSP40/HSP70 inhibition markedly reduced AR and ARv7 transcriptional activity, as indicated by decreased AR (KLK3, TMPRSS2) and ARv7 (UBE2C) target gene expression. Finally, treatment of mice bearing 22Rv1 xenografts with JG231 (an analog of JG98 with enhanced PK properties) led to significantly smaller tumors relative to vehicle treated mice. Together, these data confirm the continued dependence of AR and ARv7 on HSP40/HSP70 molecular chaperones and they demonstrate the feasibility of targeting the HSP40/HSP70 axis to abrogate sustained AR-mediated signaling in CRPC. Citation Format: Michael A. Moses, Yeong Sang Kim, Genesis Rivera-Marquez, Matthew J. Watson, Sunmin Lee, Andrea Kravats, Sue Wickner, Jason Gestwicki, Jane Trepel, Len Neckers. Targeting the HSP40/HSP70 chaperone axis as a novel strategy to treat castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1180. doi:10.1158/1538-7445.AM2017-1180

51 A region in the middle domain of E. coli Hsp90 is important for collaboration with DnaK.

Molecular chaperones are critical members of the cellular protein quality control system that use the energy from ATP hydrolysis to assist in protein remodeling activities. Heat shock protein 90 (Hsp90) is a widely conserved and highly abundant molecular chaperone that assists in the folding and reactivation of a diverse set of client proteins. Since many of these client proteins have been linked to cancer, inhibition of Hsp90 is of interest for cancer therapy. Hsp90 assembles as a highly flexible homodimer and undergoes large-scale structural rearrangements due to ATP binding and hydrolysis in order to remodel client proteins at various stages of folding. Several cochaperones have also been shown to interact with Hsp90 to modulate ATPase activity. In E. coli, the DnaK chaperone system (homologous to the eukaryotic Hsp70 system) has been shown to collaborate and directly interact with Hsp90 (Hsp90Ec) (Genest, Hoskins, Camberg, Doyle, & Wickner, 2011). We identified several residues of Hsp90Ec that are important for interaction with DnaK by making random substitutions in Hsp90Ec and screening for loss of interaction with DnaK by using a bacterial two hybrid assay. Additional mutants in nearby surface exposed residues were also constructed. The Hsp90Ec variants were purified and tested in vitro for ATPase activity and client protein remodeling activity in collaboration with the DnaK system. Our results indicate that a surface exposed region on the middle domain of Hsp90Ec is important for collaboration with DnaK. In order to determine whether this region is functionally conserved, we made a homologous substitution in yeast Hsp90 (Hsp82). The wild type and mutant proteins were purified and compared in protein reactivation assays in vitro. They were also tested in ATPase assays in the absence and presence of several yeast co-chaperones and client proteins. The results indicate a lower rate of client reactivation by the Hsp82 mutant. The mutant also exhibited defective ATPase activity in the presence of some cochaperones, suggesting this region is involved in an important protein-protein interaction or a conformational change. We are currently using molecular modeling to further explore the potential direct interaction between DnaK and the middle domain region of Hsp90Ec.