How kelch domain-containing protein 3 distinguishes between the C-end degron of herpesviral protein UL49.5 and its mutants - Insights from molecular dynamics.

The C-terminal residues of proteins can function as degrons recognized by ubiquitin ligases for proteasomal degradation. Kelch domain-containing protein 3 (KLHDC3) is a substrate receptor for E3 ubiquitin ligase (Cullin2-RING ligase) that targets the C-terminal degrons. UL49.5 is 96 amino-acid type 1 transmembrane protein from bovine herpesvirus 1. Herpesviruses have evolved highly effective strategies to evade the antiviral immune response. One of these strategies is inhibition of the antigen processing and presentation pathway by MHC I, thereby reducing the presentation of the antigenic peptides on the surface of the infected cell. Recently, it has been demonstrated that UL49.5 triggers TAP degradation via recruiting the E3 ubiquitin ligase to TAP. Moreover, the mutagenesis revealed that the mutations within the UL49.5 C-degron sequence (93RGRG96) affect binding of UL49.5 to KLHDC3. In this work the molecular dynamics of KLHDC3 in complexes with the C-terminal decapeptide of the herpesviral protein UL4.95 and its three mutants has been employed to provide a framework for understanding molecular recognition of UL49.5 by KLHDC3. The findings of this study give insights into the interactions of the various degrons with KLHDC3. During the molecular dynamics, an active RGKG mutant adopts a conformation similar to that of the wild type decapeptide, whereas the conformations of two inactive mutants, KGRG and RGRD are significantly different. Both R93K and G96D mutations impair the interactions of the C-terminal glycine with KLHDC3. The findings of this study expand the existing knowledge about the mechanism of protein recognition by Cullin2-RING ligases thus contributing to the design of antiviral and anticancer drugs that can selectively promote or inhibit degradation of the proteins of interest.

The herpesvirus UL49.5 protein hijacks a cellular C-degron pathway to drive TAP transporter degradation.

The transporter associated with antigen processing (TAP) is a key player in the major histocompatibility class I-restricted antigen presentation and an attractive target for immune evasion by viruses. Bovine herpesvirus 1 impairs TAP-dependent antigenic peptide transport through a two-pronged mechanism in which binding of the UL49.5 gene product to TAP both inhibits peptide transport and triggers its proteasomal degradation. How UL49.5 promotes TAP degradation has, so far, remained unknown. Here, we use high-content siRNA and genome-wide CRISPR-Cas9 screening to identify CLR2KLHDC3 as the E3 ligase responsible for UL49.5-triggered TAP disposal. We propose that the C terminus of UL49.5 mimics a C-end rule degron that recruits the E3 to TAP and engages the cullin-RING E3 ligase in endoplasmic reticulum-associated degradation.

An intrinsic network of polar interactions is responsible for binding of UL49.5 C-degron by the CRL2KLHDC3 ubiquitin ligase.

Bovine herpesvirus type 1 (BoHV-1) is a pathogen of cattle responsible for infectious bovine rhinotracheitis. The BoHV-1 UL49.5 is a transmembrane protein that binds to the transporter associated with antigen processing (TAP) and downregulates cell surface expression of the antigenic peptide complexes with the major histocompatibility complex class I (MHC-I). KLHDC3 is a kelch domain-containing protein 3 and a substrate receptor of a cullin2-RING (CRL2) E3 ubiquitin ligase. Recently, it has been identified that CRL2KLHDC3 is responsible for UL49.5-triggered TAP degradation via a C-degron pathway and the presence of the degron sequence does not lead to the degradation of UL49.5 itself. The molecular modeling of KLHDC3 in complexes with four UL49.5 C-terminal decapeptides (one native protein and three mutants) revealed their activity to be closely correlated with the conformation which they adopt in KLHDC3 binding cleft. To analyze the interaction between UL49.5 and KLHDC3 in detail, in this work a total of 3.6 µs long molecular dynamics simulations have been performed. The complete UL49.5-KLHDC3 complexes were embedded into the fully hydrated all-atom lipid membrane model with explicit water molecules. The network of polar interactions has been proposed to be responsible for the recognition and binding of the degron in KLHDC3. The interaction network within the binding pocket appeared to be very similar between two CRL2 substrate receptors: KLHDC3 and KLHDC2.

The herpesvirus UL49.5 protein hijacks a cellular C-degron pathway to drive TAP transporter degradation.

The transporter associated with antigen processing (TAP) is a key player in the MHC class I-restricted antigen presentation and an attractive target for immune evasion by viruses. Bovine herpesvirus 1 (BoHV-1) impairs TAP-dependent antigenic peptide transport through a two-pronged mechanism in which binding of the UL49.5 gene product to TAP both inhibits peptide transport and promotes its proteasomal degradation. How UL49.5 promotes TAP degradation is unknown. Here, we use high-content siRNA and genome-wide CRISPR-Cas9 screening to identify CLR2KLHDC3 as the E3 ligase responsible for UL49.5-triggered TAP disposal in human cells. We propose that the C-terminus of UL49.5 mimics a C-end rule degron that recruits the E3 to TAP and engages the CRL2 E3 in ER-associated degradation.

The N-terminal Proline Hinge Motif Controls the Structure of Bovine Herpesvirus 1-encoded Inhibitor of the Transporter Associated with Antigen Processing Required for its Immunomodulatory Function.

Due to unique features, proline residues may control protein structure and function. Here, we investigated the role of 52PPQ54 residues, indicated by the recently established experimental 3D structure of bovine herpesvirus 1-encoded UL49.5 protein as forming a characteristic proline hinge motif in its N-terminal domain. UL49.5 acts as a potent inhibitor of the transporter associated with antigen processing (TAP), which alters the antiviral immune response. Mechanisms employed by UL49.5 to affect TAP remain undetermined on a molecular level. We found that mutations in the 52PPQ54 region had a vast impact on its immunomodulatory function, increasing cell surface MHC class I expression, TAP levels, and peptide transport efficiency. This inhibitory effect was specific for UL49.5 activity towards TAP but not towards the viral glycoprotein M. To get an insight into the impact of proline hinge modifications on structure and dynamics, we performed all-atom and coarse-grained molecular dynamics studies on the native protein and PPQ mutants. The results demonstrated that the proline hinge sequence with its highly rigid conformation served as an anchor into the membrane. This anchor was responsible for the structural and dynamical behavior of the whole protein, constraining the mobility of the C-terminus, increasing the mobility of the transmembrane region, and controlling the accessibility of the C-terminal residues to the cytoplasmic environment. Those features appear crucial for TAP binding and inhibition. Our findings significantly advance the structural understanding of the UL49.5 protein and its functional regions and support the importance of proline motifs for the protein structure.

Molecular insights into the mechanism of sugar-modified enkephalin binding to opioid receptors.

Opioid receptors (delta, kappa, and mu) belong to the G protein-coupled receptor (GPCR) superfamily. They are responsible for pain perception - being activated by opioid peptides such as enkephalins, endorphins and dynorphins and by opiates, such as morphine. Enkephalins are naturally occurring endogenous pentapeptides with the amino acid sequence Tyr-Gly-Gly-Phe-Leu/Met. Both enkephalins are potent agonists of the delta receptor, and to a lesser extent the mu receptor, with little to no effect on the kappa receptor. Like most small peptides, enkephalins are easily catabolised via enzymatic degradation and show poor blood-brain barrier penetration. The attachment of sugars to peptides increases their penetration of the blood-brain barrier but also may affect interactions with receptors. In this study, the [Leu5]enkephalin and [Leu5]enkephalin containing the ß-D-glucuronic acid were investigated to explain how the presence of sugar moiety in the peptide molecule influences its interaction with the opioid receptors. In conclusion, the conjugation of an enkephalin molecule with the glucuronic acid has a direct and strong impact on the receptor-ligand interactions. The enhancement of ligand binding is much stronger in the delta receptor than in the mu receptor; thus, enkephalin conjugated with glucuronic acid shows greater selectivity toward the delta opioid receptor than the original peptide.

Modeling protein structures with the coarse-grained UNRES force field in the CASP14 experiment.

The UNited RESidue (UNRES) force field was tested in the 14th Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP14), in which larger oligomeric and multimeric targets were present compared to previous editions. Three prediction modes were tested (i) ab initio (the UNRES group), (ii) contact-assisted (the UNRES-contact group), and (iii) template-assisted (the UNRES-template group). For most of the targets, the contact restraints were derived from the server models top-ranked by the DeepQA method, while the DNCON2 method was used for 11 targets. Our consensus-fragment procedure was used to run template-assisted predictions. Each group also processed the Nuclear Magnetic Resonance (NMR)- and Small Angle X-Ray Scattering (SAXS)-data assisted targets. The average Global Distance Test Total Score (GDT_TS) of the 'Model 1' predictions were 29.17, 39.32, and 56.37 for the UNRES, UNRES-contact, and UNRES-template predictions, respectively, increasing by 0.53, 2.24, and 3.76, respectively, compared to CASP13. It was also found that the GDT_TS of the UNRES models obtained in ab initio mode and in the contact-assisted mode decreases with the square root of chain length, while the exponent in this relationship is 0.20 for the UNRES-template group models and 0.11 for the best performing AlphaFold2 models, which suggests that incorporation of database information, which stems from protein evolution, brings in long-range correlations, thus enabling the correction of force-field inaccuracies.

PTD4 Peptide Increases Neural Viability in an In Vitro Model of Acute Ischemic Stroke.

Ischemic stroke is a disturbance in cerebral blood flow caused by brain tissue ischemia and hypoxia. We optimized a multifactorial in vitro model of acute ischemic stroke using rat primary neural cultures. This model was exploited to investigate the pro-viable activity of cell-penetrating peptides: arginine-rich Tat(49-57)-NH2 (R49KKRRQRRR57-amide) and its less basic analogue, PTD4 (Y47ARAAARQARA57-amide). Our model included glucose deprivation, oxidative stress, lactic acidosis, and excitotoxicity. Neurotoxicity of these peptides was excluded below a concentration of 50 µm, and PTD4-induced pro-survival was more pronounced. Circular dichroism spectroscopy and molecular dynamics (MD) calculations proved potential contribution of the peptide conformational properties to neuroprotection: in MD, Tat(49-57)-NH2 adopted a random coil and polyproline type II helical structure, whereas PTD4 adopted a helical structure. In an aqueous environment, the peptides mostly adopted a random coil conformation (PTD4) or a polyproline type II helical (Tat(49-57)-NH2) structure. In 30% TFE, PTD4 showed a tendency to adopt a helical structure. Overall, the pro-viable activity of PTD4 was not correlated with the arginine content but rather with the peptide's ability to adopt a helical structure in the membrane-mimicking environment, which enhances its cell membrane permeability. PTD4 may act as a leader sequence in novel drugs for the treatment of acute ischemic stroke.

Investigation of the Effects of Primary Structure Modifications within the RRE Motif on the Conformation of Synthetic Bovine Herpesvirus 1-Encoded UL49.5 Protein Fragments.

Herpesviruses are the most prevalent viruses that infect the human and animal body. They can escape a host immune response in numerous ways. One way is to block the TAP complex so that viral peptides, originating from proteasomal degradation, cannot be transported to the endoplasmic reticulum. As a result, a reduced number of MHC class I molecules appear on the surface of infected cells and, thus, the immune system is not efficiently activated. BoHV-1-encoded UL49.5 protein is one such TAP transporter inhibitor. This protein binds to TAP in such a way that its N-terminal fragment interacts with the loops of the TAP complex, and the C-terminus stimulates proteasomal degradation of TAP. Previous studies have indicated certain amino acid residues, especially the RRE(9-11) motif, within the helical structure of the UL49.5 N-terminal fragment, as being crucial to the protein's activity. In this work, we investigated the effects of modifications within the RRE region on the spatial structure of the UL49.5 N-terminal fragment. The introduced RRE(9-11) variations were designed to abolish or stabilize the structure of the α-helix and, consequently, to increase or decrease protein activity compared to the wild type. The terminal structure of the peptides was established using circular dichroism (CD), 2D nuclear magnetic resonance (NMR), and molecular dynamics (MD) in membrane-mimetic or membrane-model environments. Our structural results show that in the RRE(9-11)AAA and E11G peptides the helical structure has been stabilized, whereas for the RRE(9-11)GGG peptide, as expected, the helix structure has partially unfolded compared to the native structure. These RRE modifications, in the context of the entire UL49.5 proteins, slightly altered their biological activity in human cells.

Improved Consensus-Fragment Selection in Template-Assisted Prediction of Protein Structures with the UNRES Force Field in CASP13.

The method for protein-structure prediction, which combines the physics-based coarse-grained UNRES force field with knowledge-based modeling, has been developed further and tested in the 13th Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP13). The method implements restraints from the consensus fragments common to server models. In this work, the server models to derive fragments have been chosen on the basis of quality assessment; a fully automatic fragment-selection procedure has been introduced, and Dynamic Fragment Assembly pseudopotentials have been fully implemented. The Global Distance Test Score (GDT_TS), averaged over our "Model 1" predictions, increased by over 10 units with respect to CASP12 for the free-modeling category to reach 40.82. Our "Model 1" predictions ranked 20 and 14 for all and free-modeling targets, respectively (upper 20.2% and 14.3% of all models submitted to CASP13 in these categories, respectively), compared to 27 (upper 21.1%) and 24 (upper 18.9%) in CASP12, respectively. For oligomeric targets, the Interface Patch Similarity (IPS) and Interface Contact Similarity (ICS) averaged over our best oligomer models increased from 0.28 to 0.36 and from 12.4 to 17.8, respectively, from CASP12 to CASP13, and top-ranking models of 2 targets (H0968 and T0997o) were obtained (none in CASP12). The improvement of our method in CASP13 over CASP12 was ascribed to the combined effect of the overall enhancement of server-model quality, our success in selecting server models and fragments to derive restraints, and improvements of the restraint and potential-energy functions.

Extension of the UNRES Coarse-Grained Force Field to Membrane Proteins in the Lipid Bilayer.

The physics-based UNRES coarse-grained force field for the simulations of protein structure and dynamics has been extended to treat membrane proteins. The lipid bilayer has been modeled by introducing a continuous nonpolar phase with the water-interface region of appropriate thickness. The potentials for average electrostatic and correlation interactions of the peptide groups have been rescaled to account for the reduction of the dielectric permittivity compared to the water phase and new potentials for protein side-chain-side-chain interactions inside and across the lipid phase have been introduced. The model was implemented in the UNRES package for coarse-grained simulations of proteins, and the package with the new functionality was tested for total energy conservation and thermostat behavior in microcanonical and canonical molecular dynamics simulations runs, respectively. The method was validated by running unrestricted ab initio blind-prediction tests of 10 short α-helical membrane proteins, all runs started from the extended structures. The modified UNRES force field was able to predict correctly the overall folds of the membrane proteins studied.

Evaluation of the scale-consistent UNRES force field in template-free prediction of protein structures in the CASP13 experiment.

The recent NEWCT-9P version of the coarse-grained UNRES force field for proteins, with scale-consistent formulas for the local and correlation terms, has been tested in the CASP13 experiment of the blind-prediction of protein structure, in the ab initio, contact-assisted, and data-assisted modes. Significant improvement of the performance has been observed with respect to the CASP11 and CASP12 experiments (by over 10 GDT_TS units for the ab initio mode predictions and by over 15 GDT_TS units for the contact-assisted prediction, respectively), which is a result of introducing scale-consistent terms and improved handling of contact-distance restraints. As in previous CASP exercises, UNRES ranked higher in the free modeling category than in the general category that included template based modeling targets. Use of distance restraints from the predicted contacts, albeit many of them were wrong, resulted in the increase of GDT_TS by over 8 units on average and introducing sparse restraints from small-angle X-ray/neutron scattering and chemical cross-link-mass-spectrometry experiments, and ambiguous restraints from nuclear magnetic resonance experiments has also improved the predictions by 8.6, 9.7, and 10.7 GDT_TS units on average, respectively.

Structure determination of UL49.5 transmembrane protein from bovine herpesvirus 1 by NMR spectroscopy and molecular dynamics.

The transporter associated with antigen processing (TAP) directly participates in the immune response as a key component of the cytosolic peptide to major histocompatibility complex (MHC) class I protein loading machinery. This makes TAP an important target for viruses avoiding recognition by CD8+ T lymphocytes. Its activity can be suppressed by the UL49.5 protein produced by bovine herpesvirus 1, although the mechanism of this inhibition has not been understood so far. Therefore, the main goal of our study was to investigate the 3D structure of bovine herpesvirus 1 - encoded UL49.5 protein. The final structure of the inhibitor was established using circular dichroism (CD), 2D nuclear magnetic resonance (NMR), and molecular dynamics (MD) in membrane mimetic environments. In NMR studies, UL49.5 was represented by two fragments: the extracellular region (residues 1-35) and the transmembrane-intracellular fragment (residues 36-75), displaying various functions during viral invasion. After the empirical structure determination, a molecular docking procedure was used to predict the complex of UL49.5 with the TAP heterodimer. Our results revealed that UL49.5 adopted a highly flexible membrane-proximal helical structure in the extracellular part. In the transmembrane region, we observed two short α-helices. Furthermore, the cytoplasmic part had an unordered structure. Finally, we propose three different orientations of UL49.5 in the complex with TAP. Our studies provide, for the first time, the experimental structural information on UL49.5 and structure-based insight in its mechanism of action which might be helpful in designing new drugs against viral infections.

An analysis and evaluation of the WeFold collaborative for protein structure prediction and its pipelines in CASP11 and CASP12.

Every two years groups worldwide participate in the Critical Assessment of Protein Structure Prediction (CASP) experiment to blindly test the strengths and weaknesses of their computational methods. CASP has significantly advanced the field but many hurdles still remain, which may require new ideas and collaborations. In 2012 a web-based effort called WeFold, was initiated to promote collaboration within the CASP community and attract researchers from other fields to contribute new ideas to CASP. Members of the WeFold coopetition (cooperation and competition) participated in CASP as individual teams, but also shared components of their methods to create hybrid pipelines and actively contributed to this effort. We assert that the scale and diversity of integrative prediction pipelines could not have been achieved by any individual lab or even by any collaboration among a few partners. The models contributed by the participating groups and generated by the pipelines are publicly available at the WeFold website providing a wealth of data that remains to be tapped. Here, we analyze the results of the 2014 and 2016 pipelines showing improvements according to the CASP assessment as well as areas that require further adjustments and research.

Use of the UNRES force field in template-assisted prediction of protein structures and the refinement of server models: Test with CASP12 targets.

Knowledge-based methods are, at present, the most effective ones for the prediction of protein structures; however, their results heavily depend on the similarity of a target sequence to those of proteins with known structures. On the other hand, the physics-based methods, although still less accurate and more expensive to execute, are independent of databases and give reasonable results where the knowledge-based methods fail because of weak sequence similarity. Therefore, a plausible approach seems to be the use of knowledge-based methods to determine the sections of the structures that correspond to sufficient sequence similarity and physics-based methods to determine the remaining structure. By participating in the 12th Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP12) as the KIAS-Gdansk group, we tested our recently developed hybrid approach, in which protein-structure prediction is carried out by using the physics-based UNRES coarse-grained energy function, with restraints derived from the server models. Best predictions among all groups were obtained for 2 targets and 80% of our models were in the upper 50% of the models submitted to CASP. Our method was also able to exclude, with about 70% confidence, the information from the servers that performed poorly on a given target. Moreover, the method resulted in the best models of 2 refinement targets and performed remarkably well on oligomeric targets.

Performance of protein-structure predictions with the physics-based UNRES force field in CASP11.

Participating as the Cornell-Gdansk group, we have used our physics-based coarse-grained UNited RESidue (UNRES) force field to predict protein structure in the 11th Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP11). Our methodology involved extensive multiplexed replica exchange simulations of the target proteins with a recently improved UNRES force field to provide better reproductions of the local structures of polypeptide chains. All simulations were started from fully extended polypeptide chains, and no external information was included in the simulation process except for weak restraints on secondary structure to enable us to finish each prediction within the allowed 3-week time window. Because of simplified UNRES representation of polypeptide chains, use of enhanced sampling methods, code optimization and parallelization and sufficient computational resources, we were able to treat, for the first time, all 55 human prediction targets with sizes from 44 to 595 amino acid residues, the average size being 251 residues. Complete structures of six single-domain proteins were predicted accurately, with the highest accuracy being attained for the T0769, for which the CαRMSD was 3.8 Å for 97 residues of the experimental structure. Correct structures were also predicted for 13 domains of multi-domain proteins with accuracy comparable to that of the best template-based modeling methods. With further improvements of the UNRES force field that are now underway, our physics-based coarse-grained approach to protein-structure prediction will eventually reach global prediction capacity and, consequently, reliability in simulating protein structure and dynamics that are important in biochemical processes. AVAILABILITY AND IMPLEMENTATION: Freely available on the web at http://www.unres.pl/ CONTACT: has5@cornell.edu.

Vasopressin V1a and V1b receptor modulators: a patent review (2012 - 2014).

INTRODUCTION: The vasopressin V1a and V1b receptors are involved in many crucial physiological, reproductive, behavioral and social functions. Consequently, they are also involved in several pathological conditions, thus the ligands capable of selective stimulation/inhibition of these receptors may present therapeutic benefit in a variety of diseases. AREAS COVERED: In this review, the author focuses on the vasopressin V1a and V1b receptors, their biological functions and agonists and antagonists patented in the years 2012 - 2014. This paper is divided according to both the target receptor and the applicant and describes the compounds from the patents along with their biological activity. EXPERT OPINION: In the recent years, pharmaceutical companies have discovered and patented new compounds which act through vasopressin V1a and/or V1b receptors, both peptide and non-peptide. Among the V1bR antagonists published in the last years, the oxindole derivatives appear to be the most promising drug candidates.

Exploring the ligand recognition properties of the human vasopressin V1a receptor using QSAR and molecular modeling studies.

Vaptans are compounds that act as non-peptide vasopressin receptor antagonists. These compounds have diverse chemical structures. In this study, we used a combined approach of protein folding, molecular dynamics simulations, docking, and quantitative structure-activity relationship (QSAR) to elucidate the detailed interaction of the vasopressin receptor V1a (V1aR) with some of its blockers (134). QSAR studies were performed using MLR analysis and were gathered into one group to perform an artificial neural network (ANN) analysis. For each molecule, 1481 molecular descriptors were calculated. Additionally, 15 quantum chemical descriptors were calculated. The final equation was developed by choosing the optimal combination of descriptors after removing the outliers. Molecular modeling enabled us to obtain a reliable tridimensional model of V1aR. The docking results indicated that the great majority of ligands reach the binding site under π-π, π-cation, and hydrophobic interactions. The QSAR studies demonstrated that the heteroatoms N and O are important for ligand recognition, which could explain the structural diversity of ligands that reach V1aR.

Interactions of vasopressin and oxytocin receptors with vasopressin analogues substituted in position 2 with 3,3'-diphenylalanine--a molecular docking study.

Vasopressin and oxytocin receptors belong to the superfamily of G protein-coupled receptors and play an important role in many physiological functions. They are also involved in a number of pathological conditions being important drug targets. In this work, four vasopressin analogues substituted at position 2 with 3,3'-diphenylalanine have been docked into partially flexible vasopressin and oxytocin receptors. The bulky residue at position 2 acts as a structural restraint much stronger in the oxytocin receptor (OTR) than in the vasopressin V2 receptor (V2R), resulting in a different location of the analogues in these receptors. This explains the different, either agonistic or antagonistic, activities of the analogues in V2R and OTR, respectively. In all complexes, the conserved polar residues serve as anchor points for the ligand both in OTR and V2R. Strong interactions of the C-terminus of analogue II ([Mpa(1) ,d-Dpa(2) ,Val(4) ,d-Arg(8) ]VP) with extracellular loop 3 may be responsible for its highest activity at V2R. It also appears that V2R adapts more readily to the docking analogues by conformational changes in the aromatic side chains triggering receptor activation. A weak activity at V1a vasopressin receptor appears to be caused by weak receptor-ligand interactions. Results of this study may facilitate a rational design of new analogues with the highest activity/selectivity at vasopressin and OTRs.

Disruption of ionic interactions between the nucleotide binding domain 1 (NBD1) and middle (M) domain in Hsp100 disaggregase unleashes toxic hyperactivity and partial independence from Hsp70.

Hsp100 chaperones cooperate with the Hsp70 chaperone system to disaggregate and reactivate heat-denatured aggregated proteins to promote cell survival after heat stress. The homology models of Hsp100 disaggregases suggest the presence of a conserved network of ionic interactions between the first nucleotide binding domain (NBD1) and the coiled-coil middle subdomain, the signature domain of disaggregating chaperones. Mutations intended to disrupt the putative ionic interactions in yeast Hsp104 and bacterial ClpB disaggregases resulted in remarkable changes of their biochemical properties. These included an increase in ATPase activity, a significant increase in the rate of in vitro substrate renaturation, and partial independence from the Hsp70 chaperone in disaggregation. Paradoxically, the increased activities resulted in serious growth impediments in yeast and bacterial cells instead of improvement of their thermotolerance. Our results suggest that this toxic activity is due to the ability of the mutated disaggregases to unfold independently from Hsp70, native folded proteins. Complementary changes that restore particular salt bridges within the suggested network suppressed the toxic effects. We propose a novel structural aspect of Hsp100 chaperones crucial for specificity and efficiency of the disaggregation reaction.