Meet the authors: Guy Zoltsman and Rina Rosenzweig.

We talk to first and last authors Guy Zoltsman and Rina Rosenzweig about their paper, "A unique chaperoning mechanism in Class A JDPs recognizes and stabilizes mutant p53," how every result may be important in the right context, and the importance to Rina that her lab is an encouraging and collaborative place.

A unique chaperoning mechanism in class A JDPs recognizes and stabilizes mutant p53.

J-domain proteins (JDPs) constitute a large family of molecular chaperones that bind a broad spectrum of substrates, targeting them to Hsp70, thus determining the specificity of and activating the entire chaperone functional cycle. The malfunction of JDPs is therefore inextricably linked to myriad human disorders. Here, we uncover a unique mechanism by which chaperones recognize misfolded clients, present in human class A JDPs. Through a newly identified ß-hairpin site, these chaperones detect changes in protein dynamics at the initial stages of misfolding, prior to exposure of hydrophobic regions or large structural rearrangements. The JDPs then sequester misfolding-prone proteins into large oligomeric assemblies, protecting them from aggregation. Through this mechanism, class A JDPs bind destabilized p53 mutants, preventing clearance of these oncoproteins by Hsp70-mediated degradation, thus promoting cancer progression. Removal of the ß-hairpin abrogates this protective activity while minimally affecting other chaperoning functions. This suggests the class A JDP ß-hairpin as a highly specific target for cancer therapeutics.

The Hsp70 chaperone network.

The 70-kDa heat shock proteins (Hsp70s) are ubiquitous molecular chaperones that act in a large variety of cellular protein folding and remodelling processes. They function virtually at all stages of the life of proteins from synthesis to degradation and are thus crucial for maintaining protein homeostasis, with direct implications for human health. A large set of co-chaperones comprising J-domain proteins and nucleotide exchange factors regulate the ATPase cycle of Hsp70s, which is allosterically coupled to substrate binding and release. Moreover, Hsp70s cooperate with other cellular chaperone systems including Hsp90, Hsp60 chaperonins, small heat shock proteins and Hsp100 AAA+ disaggregases, together constituting a dynamic and functionally versatile network for protein folding, unfolding, regulation, targeting, aggregation and disaggregation, as well as degradation. In this Review we describe recent advances that have increased our understanding of the molecular mechanisms and working principles of the Hsp70 network. This knowledge showcases how the Hsp70 chaperone system controls diverse cellular functions, and offers new opportunities for the development of chemical compounds that modulate disease-related Hsp70 activities.

NudC guides client transfer between the Hsp40/70 and Hsp90 chaperone systems.

In the eukaryotic cytosol, the Hsp70 and the Hsp90 chaperone machines work in tandem with the maturation of a diverse array of client proteins. The transfer of nonnative clients between these systems is essential to the chaperoning process, but how it is regulated is still not clear. We discovered that NudC is an essential transfer factor with an unprecedented mode of action: NudC interacts with Hsp40 in Hsp40-Hsp70-client complexes and displaces Hsp70. Then, the interaction of NudC with Hsp90 allows the direct transfer of Hsp40-bound clients to Hsp90 for further processing. Consistent with this mechanism, NudC increases client activation in vitro as well as in cells and is essential for cellular viability. Together, our results show the complexity of the cooperation between the major chaperone machineries in the eukaryotic cytosol.

Bringing dynamic molecular machines into focus by methyl-TROSY NMR.

Large macromolecular assemblies, so-called molecular machines, are critical to ensuring proper cellular function. Understanding how proper function is achieved at the atomic level is crucial to advancing multiple avenues of biomedical research. Biophysical studies often include X-ray diffraction and cryo-electron microscopy, providing detailed structural descriptions of these machines. However, their inherent flexibility has complicated an understanding of the relation between structure and function. Solution NMR spectroscopy is well suited to the study of such dynamic complexes, and continued developments have increased size boundaries; insights into function have been obtained for complexes with masses as large as 1 MDa. We highlight methyl-TROSY (transverse relaxation optimized spectroscopy) NMR, which enables the study of such large systems, and include examples of applications to several cellular machines. We show how this emerging technique contributes to an understanding of cellular function and the role of molecular plasticity in regulating an array of biochemical activities.

HSP40 proteins use class-specific regulation to drive HSP70 functional diversity.

The ubiquitous heat shock protein 70 (HSP70) family consists of ATP-dependent molecular chaperones, which perform numerous cellular functions that affect almost all aspects of the protein life cycle from synthesis to degradation1-3. Achieving this broad spectrum of functions requires precise regulation of HSP70 activity. Proteins of the HSP40 family, also known as J-domain proteins (JDPs), have a key role in this process by preselecting substrates for transfer to their HSP70 partners and by stimulating the ATP hydrolysis of HSP70, leading to stable substrate binding3,4. In humans, JDPs constitute a large and diverse family with more than 40 different members2, which vary in their substrate selectivity and in the nature and number of their client-binding domains5. Here we show that JDPs can also differ fundamentally in their interactions with HSP70 chaperones. Using nuclear magnetic resonance spectroscopy6,7 we find that the major class B JDPs are regulated by an autoinhibitory mechanism that is not present in other classes. Although in all JDPs the interaction of the characteristic J-domain is responsible for the activation of HSP70, in DNAJB1 the HSP70-binding sites in this domain are intrinsically blocked by an adjacent glycine-phenylalanine rich region-an inhibition that can be released upon the interaction of a second site on DNAJB1 with the HSP70 C-terminal tail. This regulation, which controls substrate targeting to HSP70, is essential for the disaggregation of amyloid fibres by HSP70-DNAJB1, illustrating why no other class of JDPs can substitute for class B in this function. Moreover, this regulatory layer, which governs the functional specificities of JDP co-chaperones and their interactions with HSP70s, could be key to the wide range of cellular functions of HSP70.

Structural study of UFL1-UFC1 interaction uncovers the role of UFL1 N-terminal helix in ufmylation.

Ufmylation plays a crucial role in various cellular processes including DNA damage response, protein translation, and ER homeostasis. To date, little is known about how the enzymes responsible for ufmylation coordinate their action. Here, we study the details of UFL1 (E3) activity, its binding to UFC1 (E2), and its relation to UBA5 (E1), using a combination of structural modeling, X-ray crystallography, NMR, and biochemical assays. Guided by Alphafold2 models, we generate an active UFL1 fusion construct that includes its partner DDRGK1 and solve the crystal structure of this critical interaction. This fusion construct also unveiled the importance of the UFL1 N-terminal helix for binding to UFC1. The binding site suggested by our UFL1-UFC1 model reveals a conserved interface, and competition between UFL1 and UBA5 for binding to UFC1. This competition changes in the favor of UFL1 following UFM1 charging of UFC1. Altogether, our study reveals a novel, terminal helix-mediated regulatory mechanism, which coordinates the cascade of E1-E2-E3-mediated transfer of UFM1 to its substrate and provides new leads to target this modification.

Bacterial adaptation to cold: Conservation of a short J-domain co-chaperone and its protein partners in environmental proteobacteria.

Bacterial genomes are a huge reservoir of genes encoding J-domain protein co-chaperones that recruit the molecular chaperone DnaK to assist protein substrates involved in survival, adaptation, or fitness. The atc operon of the aquatic mesophilic bacterium Shewanella oneidensis encodes the proteins AtcJ, AtcA, AtcB, and AtcC, and all of them, except AtcA, are required for growth at low temperatures. AtcJ is a short J-domain protein that interacts with DnaK, but also with AtcC through its 21 amino acid C-terminal domain. This interaction network is critical for cold growth. Here, we show that AtcJ represents a subfamily of short J-domain proteins that (i) are found in several environmental, mostly aquatic, ß- or É£-proteobacteria and (ii) contain a conserved PX7 W motif in their C-terminal extension. Using a combination of NMR, biochemical and genetic approaches, we show that the hydrophobic nature of the tryptophan of the S. oneidensis AtcJ PX7 W motif determines the strong AtcJ-AtcC interaction essential for cold growth. The AtcJ homologues are encoded by operons containing at least the S. oneidensis atcA, atcB, and atcC homologues. These findings suggest a conserved network of DnaK and Atc proteins necessary for low-temperature growth and, given the variation in the atc operons, possibly for other biological functions.

Cross-Polarization Schemes for Improved Heteronuclear Transfers Involving Labile Protons in Biomolecular Solution NMR.

INEPT-based experiments are widely used for 1 H→15 N transfers, but often fail when involving labile protons due to solvent exchanges. J-based cross polarization (CP) strategies offer a more efficient alternative to perform such transfers, particularly when leveraging the Hwater ↔ ${ \leftrightarrow }$ HN exchange process to boost the 1 H→15 N transfer process. This leveraging, however, demands the simultaneous spin-locking of both Hwater and HN protons by a strong 1 H RF field, while fulfilling the γH B1,H =γN B1,N Hartmann-Hahn matching condition. Given the low value of γN /γH , however, these demands are often incompatible-particularly when experiments are executed by the power-limited cryogenic probes used in contemporary high field NMR. The present manuscript discusses CP alternatives that can alleviate this limitation, and evaluates their performance on urea, amino acids, and intrinsically disordered proteins. These alternatives include new CP variants based on frequency-swept and phase-modulated pulses, designed to simultaneously fulfill the aforementioned conflicting conditions. Their performances vis-à-vis current options are theoretically analyzed with Liouville-space simulations, and experimentally tested with double and triple resonance transfer experiments.

Tetramerization of Phosphoprotein is Essential for Respiratory Syncytial Virus Budding while its N Terminal Region Mediates Direct Interactions with the Matrix Protein.

It was shown previously that the Matrix (M), Phosphoprotein (P), and the Fusion (F) proteins of Respiratory syncytial virus (RSV) are sufficient to produce virus-like particles (VLPs) that resemble the RSV infection-induced virions. However, the exact mechanism and interactions among the three proteins are not known. This work examines the interaction between P and M during RSV assembly and budding. We show that M interacts with P in the absence of other viral proteins in cells using a Split Nano Luciferase assay. By using recombinant proteins, we demonstrate a direct interaction between M and P. By using Nuclear Magnetic Resonance (NMR) we identify three novel M interaction sites on P, namely site I in the αN2 region, site II in the 115-125 region, and the oligomerization domain (OD). We show that the OD, and likely the tetrameric structural organization of P, is required for virus-like filament formation and VLP release. Although sites I and II are not required for VLP formation, they appear to modulate P levels in RSV VLPs.Importance Human RSV is the commonest cause of infantile bronchiolitis in the developed world and of childhood deaths in resource-poor settings. It is a major unmet target for vaccines and anti-viral drugs. The lack of knowledge of RSV budding mechanism presents a continuing challenge for VLP production for vaccine purpose. We show that direct interaction between P and M modulates RSV VLP budding. This further emphasizes P as a central regulator of RSV life cycle, as an essential actor for transcription and replication early during infection and as a mediator for assembly and budding in the later stages for virus production.

Assessing Site-Specific Enhancements Imparted by Hyperpolarized Water in Folded and Unfolded Proteins by 2D HMQC NMR.

Hyperpolarized water can be a valuable aid in protein NMR, leading to amide group 1H polarizations that are orders of magnitude larger than their thermal counterparts. Suitable procedures can exploit this to deliver 2D 1H-15N correlations with good resolution and enhanced sensitivity. These enhancements depend on the exchange rates between the amides and the water, thereby yielding diagnostic information about solvent accessibility. This study applied this "HyperW" method to four proteins exhibiting a gamut of exchange behaviors: PhoA(350-471), an unfolded 122-residue fragment; barstar, a fully folded ribonuclease inhibitor; R17, a 13.3 kDa system possessing folded and unfolded forms under slow interconversion; and drkN SH3, a protein domain whose folded and unfolded forms interchange rapidly and with temperature-dependent population ratios. For PhoA4(350-471) HyperW sensitivity enhancements were ≥300×, as expected for an unfolded protein sequence. Though fully folded, barstar also exhibited substantial enhancements; these, however, were not uniform and, according to CLEANEX experiments, reflected the solvent-exposed residues. R17 showed the expected superposition of ≥100-fold enhancements for its unfolded form, coexisting with more modest enhancements for their folded counterparts. Unexpected, however, was the behavior of drkN SH3, for which HyperW enhanced the unfolded but, surprisingly, enhanced even more certain folded protein sites. These preferential enhancements were repeatedly and reproducibly observed. A number of explanations-including three-site exchange magnetization transfers between water and the unfolded and folded states; cross-correlated relaxation processes from hyperpolarized "structural" waters and labile side-chain protons; and the possibility that faster solvent exchange rates characterize certain folded sites over their unfolded counterparts-are considered to account for them.

Structural and Biochemical Properties of Hsp40/Hsp70 Chaperone System.

Hsp70s are ubiquitous molecular chaperones that act in a myriad of cellular functions, affecting virtually all aspects in the life of proteins from synthesis to degradation. Hsp70 proteins act in the cell in cooperation with a large set of dedicated co-chaperones consisting of J-domain proteins and nucleotide exchange factors that regulate the Hsp70 chaperone cycle. Recent studies have made significant progress towards obtaining a better understanding of the mechanisms through which Hsp70s and their co-chaperones operate, providing insights into structural, kinetic, and functional features of the various members of this network. In this chapter we describe the emerging working principles of the Hsp70 machine and its co-chaperones, and highlight how mechanistic aspects of this network are tied to distinct protein folding functions.

Hsp70 biases the folding pathways of client proteins.

The 70-kDa heat shock protein (Hsp70) family of chaperones bind cognate substrates to perform a variety of different processes that are integral to cellular homeostasis. Although detailed structural information is available on the chaperone, the structural features of folding competent substrates in the bound form have not been well characterized. Here we use paramagnetic relaxation enhancement (PRE) NMR spectroscopy to probe the existence of long-range interactions in one such folding competent substrate, human telomere repeat binding factor (hTRF1), which is bound to DnaK in a globally unfolded conformation. We show that DnaK binding modifies the energy landscape of the substrate by removing long-range interactions that are otherwise present in the unbound, unfolded conformation of hTRF1. Because the unfolded state of hTRF1 is only marginally populated and transiently formed, it is inaccessible to standard NMR approaches. We therefore developed a (1)H-based CEST experiment that allows measurement of PREs in sparse states, reporting on transiently sampled conformations. Our results suggest that DnaK binding can significantly bias the folding pathway of client substrates such that secondary structure forms first, followed by the development of longer-range contacts between more distal parts of the protein.

Mapping the conformation of a client protein through the Hsp70 functional cycle.

The 70 kDa heat shock protein (Hsp70) chaperone system is ubiquitous, highly conserved, and involved in a myriad of diverse cellular processes. Its function relies on nucleotide-dependent interactions with client proteins, yet the structural features of folding-competent substrates in their Hsp70-bound state remain poorly understood. Here we use NMR spectroscopy to study the human telomere repeat binding factor 1 (hTRF1) in complex with Escherichia coli Hsp70 (DnaK). In the complex, hTRF1 is globally unfolded with up to 40% helical secondary structure in regions distal to the binding site. Very similar conformational ensembles are observed for hTRF1 bound to ATP-, ADP- and nucleotide-free DnaK. The patterns in substrate helicity mirror those found in the unfolded state in the absence of denaturants except near the site of chaperone binding, demonstrating that DnaK-bound hTRF1 retains its intrinsic structural preferences. To our knowledge, our study presents the first atomic resolution structural characterization of a client protein bound to each of the three nucleotide states of DnaK and establishes that the large structural changes in DnaK and the associated energy that accompanies ATP binding and hydrolysis do not affect the overall conformation of the bound substrate protein.

ClpB N-terminal domain plays a regulatory role in protein disaggregation.

ClpB/Hsp100 is an ATP-dependent disaggregase that solubilizes and reactivates protein aggregates in cooperation with the DnaK/Hsp70 chaperone system. The ClpB-substrate interaction is mediated by conserved tyrosine residues located in flexible loops in nucleotide-binding domain-1 that extend into the ClpB central pore. In addition to the tyrosines, the ClpB N-terminal domain (NTD) was suggested to provide a second substrate-binding site; however, the manner in which the NTD recognizes and binds substrate proteins has remained elusive. Herein, we present an NMR spectroscopy study to structurally characterize the NTD-substrate interaction. We show that the NTD includes a substrate-binding groove that specifically recognizes exposed hydrophobic stretches in unfolded or aggregated client proteins. Using an optimized segmental labeling technique in combination with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR, the interaction of client proteins with both the NTD and the pore-loop tyrosines in the 580-kDa ClpB hexamer has been characterized. Unlike contacts with the tyrosines, the NTD-substrate interaction is independent of the ClpB nucleotide state and protein conformational changes that result from ATP hydrolysis. The NTD interaction destabilizes client proteins, priming them for subsequent unfolding and translocation. Mutations in the NTD substrate-binding groove are shown to have a dramatic effect on protein translocation through the ClpB central pore, suggesting that, before their interaction with substrates, the NTDs block the translocation channel. Together, our findings provide both a detailed characterization of the NTD-substrate complex and insight into the functional regulatory role of the ClpB NTD in protein disaggregation.

Together, Rpn10 and Dsk2 can serve as a polyubiquitin chain-length sensor.

As a signal for substrate targeting, polyubiquitin meets various layers of receptors upstream to the 26S proteasome. We obtained structural information on two receptors, Rpn10 and Dsk2, alone and in complex with (poly)ubiquitin or with each other. A hierarchy of affinities emerges with Dsk2 binding monoubiquitin tighter than Rpn10 does, whereas Rpn10 prefers the ubiquitin-like domain of Dsk2 to monoubiquitin, with increasing affinities for longer polyubiquitin chains. We demonstrated the formation of ternary complexes of both receptors simultaneously with (poly)ubiquitin and found that, depending on the ubiquitin chain length, the orientation of the resulting complex is entirely different, providing for alternate signals. Dynamic rearrangement provides a chain-length sensor, possibly explaining how accessibility of Dsk2 to the proteasome is limited unless it carries a properly tagged cargo. We propose a mechanism for a malleable ubiquitin signal that depends both on chain length and combination of receptors to produce tetraubiquitin as an efficient signal threshold.

Solution NMR Spectroscopy Provides an Avenue for the Study of Functionally Dynamic Molecular Machines: The Example of Protein Disaggregation.

Solution-based NMR spectroscopy has been an important tool for studying the structure and dynamics of relatively small proteins and protein complexes with aggregate molecular masses under approximately 50 kDa. The development of new experiments and labeling schemes, coupled with continued improvements in hardware, has significantly reduced this size limitation, enabling atomic-resolution studies of molecular machines in the 1 MDa range. In this Perspective, some of the important advances are highlighted in the context of studies of molecular chaperones involved in protein disaggregation. New insights into the structural biology of disaggregation obtained from NMR studies are described, focusing on the unique capabilities of the methodology for obtaining atomic-resolution descriptions of dynamic systems.

Misfolded PrP impairs the UPS by interaction with the 20S proteasome and inhibition of substrate entry.

Prion diseases are associated with the conversion of cellular prion protein (PrP(C)) to toxic ß-sheet isoforms (PrP(Sc)), which are reported to inhibit the ubiquitin-proteasome system (UPS). Accordingly, UPS substrates accumulate in prion-infected mouse brains, suggesting impairment of the 26S proteasome. A direct interaction between its 20S core particle and PrP isoforms was demonstrated by immunoprecipitation. ß-PrP aggregates associated with the 20S particle, but did not impede binding of the PA26 complex, suggesting that the aggregates do not bind to its ends. Aggregated ß-PrP reduced the 20S proteasome's basal peptidase activity, and the enhanced activity induced by C-terminal peptides from the 19S ATPases or by the 19S regulator itself, including when stimulated by polyubiquitin conjugates. However, the 20S proteasome was not inhibited when the gate in the α-ring was open due to a truncation mutation or by association with PA26/PA28. These PrP aggregates inhibit by stabilising the closed conformation of the substrate entry channel. A similar inhibition of substrate entry into the proteasome may occur in other neurodegenerative diseases where misfolded ß-sheet-rich proteins accumulate.

Rpn1 and Rpn2 coordinate ubiquitin processing factors at proteasome.

Substrates tagged with (poly)ubiquitin for degradation can be targeted directly to the 26 S proteasome where they are proteolyzed. Independently, ubiquitin conjugates may also be delivered by bivalent shuttles. The majority of shuttles attach to the proteasome through a ubiquitin-like domain (UBL) while anchoring cargo at a C-terminal polyubiquitin-binding domain(s). We found that two shuttles of this class, Rad23 and Dsk2, dock at two different receptor sites embedded within a single subunit of the 19 S proteasome regulatory particle, Rpn1. Their association/dissociation constants and affinities for Rpn1 are similar. In contrast, another UBL-containing protein, the deubiquitinase Ubp6, is also anchored by Rpn1, yet it dissociates slower, thus behaving as an occasional proteasome subunit that is distinct from the transiently associated shuttles. Two neighboring subunits, Rpn10 and Rpn13, show a marked preference for polyubiquitin over UBLs. Rpn10 attaches to the central solenoid portion of Rpn1, although this association is stabilized by the presence of a third subunit, Rpn2. Rpn13 binds directly to Rpn2. These intrinsic polyubiquitin receptors may compete with substrate shuttles for their polyubiquitin-conjugate cargos, thereby aiding release of the emptied shuttles. By binding multiple ubiquitin-processing factors simultaneously, Rpn1 is uniquely suited to coordinate substrate recruitment, deubiquitination, and movement toward the catalytic core. The broad range of affinities for ubiquitin, ubiquitin-like, and non-ubiquitin signals by adjacent yet nonoverlapping sites all within the base represents a hub of activity that coordinates the intricate relay of substrates within the proteasome, and consequently it influences substrate residency time and commitment to degradation.

Protein disaggregation machineries in the human cytosol.

Proteins carry out the vast majority of functions in cells, but can only do so when properly folded. Following stress or mutation, proteins can lose their proper fold, resulting in misfolding, inactivity, and aggregation-posing a threat to cellular health. In order to counteract protein aggregation, cells have evolved a remarkable subset of molecular chaperones, called protein disaggregases, which collaboratively possess the ability to forcibly untangle protein aggregates. Here, we review the different chaperone disaggregation machineries present in the human cytosol and their mechanisms of action. Understanding, how these disaggregases function, is both universally and clinically important, as protein aggregation has been linked to multiple, debilitating neurodegenerative diseases.