Client Recognition Research Articles

Conserved conformational selection mechanism of Hsp70 chaperone-substrate interactions.

Molecular recognition is integral to biological function and frequently involves preferred binding of a molecule to one of several exchanging ligand conformations in solution. In such a process the bound structure can be selected from the ensemble of interconverting ligands a priori (conformational selection, CS) or may form once the ligand is bound (induced fit, IF). Here we focus on the ubiquitous and conserved Hsp70 chaperone which oversees the integrity of the cellular proteome through its ATP-dependent interaction with client proteins. We directly quantify the flux along CS and IF pathways using solution NMR spectroscopy that exploits a methyl TROSY effect and selective isotope-labeling methodologies. Our measurements establish that both bacterial and human Hsp70 chaperones interact with clients by selecting the unfolded state from a pre-existing array of interconverting structures, suggesting a conserved mode of client recognition among Hsp70s and highlighting the importance of molecular dynamics in this recognition event.

Functional Role and Hierarchy of the Intermolecular Interactions in Binding of Protein Kinase Clients to the Hsp90-Cdc37 Chaperone: Structure-Based Network Modeling of Allosteric Regulation.

A fundamental role of the Hsp90-Cdc37 chaperone machinery in mediating conformational development and activation of diverse protein kinase clients is essential for signal transduction. Structural and biochemical studies have demonstrated that characterization of global conformational changes and allosteric interactions in the Hsp90-Cdc37-kinase complexes are central to our understanding of the mechanisms underlying kinase recruitment and processing by the Hsp90-Cdc37 chaperone. The recent cryo-electron microscopy structure of the Hsp90-Cdc37-Cdk4 kinase complex has provided a framework for dissecting regulatory principles underlying differentiation and recruitment of protein kinase clients to the chaperone machinery. In this work, we have characterized functional role and hierarchy of the intermolecular interactions in binding of protein kinase clients to the Hsp90-Cdc37 system. The network analysis revealed important relationships between structural stability, global centrality, and functional significance of regulatory hotspots in chaperone regulation and client recognition. A unique asymmetric topography of the intermolecular communities in the chaperone-kinase complex has quantified a central mediating role of Cdc37 in client recognition and allosteric regulation of the chaperone-kinase complex. Modeling of allosteric pathways in the chaperone complex has further clarified structural and energetic signatures of allosteric hotspots, particularly linking sites of post-translational modifications in Hsp90 with their role in allosteric interactions and client regulation. The results of this integrative computational study are compared with a wide range of structural, biochemical, and cell-based experiments, offering a robust network-centric model of allosteric regulation and client kinase recognition by the Hsp90-Cdc37 chaperone machine.

Mechanistic Basis for Client Recognition and Amyloid Inhibition of NMNAT

Imbalance of proteostasis leads to abnormal protein aggregations as a common pathological hallmark in a variety of devastating human neurodegenerative diseases such as AD and PD. Chaperones play essential roles in maintaining proteostasis and preventing protein from abnormal aggregation. NMNAT, a NAD synthase firstly identified as a key protector in Wallerian axon degeneration, was recently found to possess chaperone activity and exhibits protective effects in cells and animal models of neurodegeneration. However, it remains enigmatic how an enzyme evolves chaperone activity and prevents pathological amyloid aggregation. Here, we found that NMNAT recognizes a full spectrum of client amyloid proteins (e.g. α-syn, Tau, Aβ and IAPP), and prevents their aggregation and cytotoxicity. By using multiple biophysical approaches, we identified the client binding pocket of NMNAT which is partially overlapped with its catalytic pocket, providing the structural basis of how NMNAT exhibits both chaperone and enzyme activity. We found that both electrostatic and hydrophobic interactions are crucial for the client-chaperone recognition. In addition to binding various amyloid client proteins, NMNAT shows high binding affinity and selectively to phosphorylated Tau, which is closely related to diseases. Moreover, NMNAT can inhibit both liquid-liquid phase separation and amyloid aggregation of phosphorylated Tau. Our work helps to explain how a chaperone halt its client proteins from aggregation and highlight the importance of NMNAT as a maintenance factor in neurodegenerative diseases. Reference:

Atomistic simulations and network-based modeling of the Hsp90-Cdc37 chaperone binding with Cdk4 client protein: A mechanism of chaperoning kinase clients by exploiting weak spots of intrinsically dynamic kinase domains.

A fundamental role of the Hsp90 and Cdc37 chaperones in mediating conformational development and activation of diverse protein kinase clients is essential in signal transduction. There has been increasing evidence that the Hsp90-Cdc37 system executes its chaperoning duties by recognizing conformational instability of kinase clients and modulating their folding landscapes. The recent cryo-electron microscopy structure of the Hsp90-Cdc37-Cdk4 kinase complex has provided a framework for dissecting regulatory principles underlying differentiation and recruitment of protein kinase clients to the chaperone machinery. In this work, we have combined atomistic simulations with protein stability and network-based rigidity decomposition analyses to characterize dynamic factors underlying allosteric mechanism of the chaperone-kinase cycle and identify regulatory hotspots that control client recognition. Through comprehensive characterization of conformational dynamics and systematic identification of stabilization centers in the unbound and client- bound Hsp90 forms, we have simulated key stages of the allosteric mechanism, in which Hsp90 binding can induce instability and partial unfolding of Cdk4 client. Conformational landscapes of the Hsp90 and Cdk4 structures suggested that client binding can trigger coordinated dynamic changes and induce global rigidification of the Hsp90 inter-domain regions that is coupled with a concomitant increase in conformational flexibility of the kinase client. This process is allosteric in nature and can involve reciprocal dynamic exchanges that exert global effect on stability of the Hsp90 dimer, while promoting client instability. The network-based rigidity analysis and emulation of thermal unfolding of the Cdk4-cyclin D complex and Hsp90-Cdc37-Cdk4 complex revealed weak spots of kinase instability that are present in the native Cdk4 structure and are targeted by the chaperone during client recruitment. Our findings suggested that this mechanism may be exploited by the Hsp90-Cdc37 chaperone to recruit and protect intrinsically dynamic kinase clients from degradation. The results of this investigation are discussed and interpreted in the context of diverse experimental data, offering new insights into mechanisms of chaperone regulation and binding.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo.

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results. This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB's capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB's chaperone function in vivo.

Crystal Structure of the ERp44-Peroxiredoxin 4 Complex Reveals the Molecular Mechanisms of Thiol-Mediated Protein Retention

ERp44 controls the localization and transport of diverse proteins in the early secretory pathway. The mechanisms that allow client recognition and the source of the oxidative power for forming intermolecular disulfides are as yet unknown. Here we present the structure of ERp44 bound to a client, peroxiredoxin 4. Our data reveal that ERp44 binds theoxidized form of peroxiredoxin 4 via thiol-disulfide interchange reactions. The structure explains the redox-dependent recognition and characterizes theessential non-covalent interactions at the interface. The ERp44-Prx4 covalent complexes can be reduced by glutathione and protein disulfide isomerase family members in the ER, allowing the two components to recycle. This work provides insights into the mechanisms of thiol-mediated protein retention and indicates the key roles of ERp44 in this biochemical cycle to optimize oxidative folding and redox homeostasis.

Coupled folding and binding with 2D Window-Exchange Umbrella Sampling.

Intrinsically disordered regions of proteins can gain structure by binding to a partner. This process, of coupled folding and binding (CFaB), is a fundamental part of many important biological processes. Structure-based models have proven themselves capable of revealing fundamental aspects of how CFaB occurs, however, typical methods to enhance the sampling of these transitions, such as replica exchange, do not adequately sample the transition state region of this extremely rare process. Here, we use a variant of Umbrella Sampling to enforce sampling of the transition states of CFaB of HdeA monomers at neutral pH, an extremely rare process that occurs over timescales ranging from seconds to hours. Using high resolution sampling in the transition state region, we cluster states along the principal transition path to obtain a detailed description of coupled binding and folding for the HdeA dimer, revealing new insight into the ensemble of states that are accessible to client recognition. We then demonstrate that exchanges between umbrella sampling windows, as done in previous work, significantly improve relaxation in variables orthogonal to the restraints used. Altogether, these results show that Window-Exchange Umbrella Sampling is a promising approach for systems that exhibit flexible binding, and can reveal transition state ensembles of these systems in high detail. © 2015 Wiley Periodicals, Inc.

Structure and Assembly Pathway of the Ribosome Quality Control Complex

SummaryDuring ribosome-associated quality control, stalled ribosomes are split into subunits and the 60S-housed nascent polypeptides are poly-ubiquitinated by Listerin. How this low-abundance ubiquitin ligase targets rare stall-generated 60S among numerous empty 60S is unknown. Here, we show that Listerin specificity for nascent chain-60S complexes depends on nuclear export mediator factor (NEMF). The 3.6 Å cryo-EM structure of a nascent chain-containing 60S-Listerin-NEMF complex revealed that NEMF makes multiple simultaneous contacts with 60S and peptidyl-tRNA to sense nascent chain occupancy. Structural and mutational analyses showed that ribosome-bound NEMF recruits and stabilizes Listerin’s N-terminal domain, while Listerin’s C-terminal RWD domain directly contacts the ribosome to position the adjacent ligase domain near the nascent polypeptide exit tunnel. Thus, highly specific nascent chain targeting by Listerin is imparted by the avidity gained from a multivalent network of context-specific individually weak interactions, highlighting a new principle of client recognition during protein quality control.

‘Riding the Rollercoaster’: Veterinary Experiences of Managing Laminitis in Practice

Reasons for performing studyLittle is reported about the successes and challenges experienced by veterinary surgeons while managing cases of laminitis. Such information could enhance the effectiveness of communication of management and preventive health strategies for laminitis.ObjectivesTo explore veterinary surgeons’ experiences of dealing with laminitis and examine how veterinary–client communication influences the treatment and management of cases.Study designFocus group discussions conducted among first‐opinion equine veterinary surgeons.MethodsFirst‐opinion ambulatory veterinary practices from Cheshire (n = 15), Lancashire (n = 14) and the Rutland region (n = 14) were identified. Letters of invitation were sent and all practices were telephoned to invite veterinary surgeons to participate in the study. Key questions relating to laminitis were explored within semi‐structured focus group discussions: veterinary perceptions of laminitis information, reasons for horse‐owners’ seeking veterinary advice, veterinary perceptions of horse‐owners’ knowledge, attitudes and practices, situations where the advice of other professionals may be sought. All discussions were recorded and transcribed verbatim. A thematic analysis was conducted using NVivo software.ResultsThree focus group discussions were held with veterinary surgeons with a range of experience; 2 in the North West (9 vets) and one in Rutland (4 vets). Emerging themes relating to veterinary surgeons’ experience of dealing with laminitis included; veterinary–client communication, challenges faced by vets, managing clients perceptions and expectations, client recognition and management of laminitis, feeding and obesity management, issues of compliance, causes of laminitis, strategies to prevent laminitis, diagnosing, treating and prognosticating in laminitis cases, sources of veterinary information and collaboration with other professionals.ConclusionsThe findings illustrate key features of the veterinary–client relationship in supporting owners through the variable course of this disease. This study demonstrates the complexity of issues that veterinary surgeons may encounter when managing cases of laminitis and communicating healthcare messages to the client.Ethical animal research: This project was approved by the University of Liverpool Ethics for Research Committee. Sources of funding: The authors gratefully acknowledge support and funding from the BVA Animal Welfare Foundation charitable trust. Competing interests: None.

Structure-Function Relationships of the Collagen-Specific Chaperone Hsp47

Hsp47 is an essential collagen specific chaperone that is crucial for proper formation of the collagen triple helix. KO-mice die at an early emybronic state and missense mutations are linked to severe forms of osteogenesis imperfecta (Ishida & Nagata, 2011). On the other hand, Hsp47 is strongly upregulated in fibrotic pathogenies and has in proof-of-principle studies been successfully targeted . It is a very unusual chaperone as it specifically recognises the folded conformation and releases ist client upon changes in pH and not in an ATP-cycle dependent manner. We have deterined the crystal structure of Hsp47 alone and in complex with homotrimeric collagen model peptides (Widmer et al., 2012), revealing a 2:1 stoichiometry Hsp47:collagen-trimer. The specific recognition of an arginine at the Y-position of the triple helix is explained by a saltbridge to an aspartic acid of Hsp47. Based on these structures we have undergone further investigation in order to shed light on the pH-triggered client release and to gain further insight into client recognition. We have undergone a systematic site-directed mutagenesis study of histidine residues in the binding interface. Interestingly, only few side-chains are predicted to change significantly their protonation state by a pH change from 7 to 6. We have furthermore investigated the influence of the reactive center loop, which is disordered in most crystal structures. An open question is how many binding sites for Hsp47 exist in procollagen. EM studies reveal a large number of specific binding sites, explaining how Hsp47 clamps and stabilises the triple helix and prevents lateral aggregation.

Quantitative Analysis of Hsp90-Client Interactions Reveals Principles of Substrate Recognition

HSP90 is a molecular chaperone that associates with numerous substrate proteins called clients. It plays many important roles in human biology and medicine, but determinants of client recognition by HSP90 have remained frustratingly elusive. We systematically and quantitatively surveyed most human kinases, transcription factors, and E3 ligases for interaction with HSP90 and its cochaperone CDC37. Unexpectedly, many more kinases than transcription factors bound HSP90. CDC37 interacted with kinases, but not with transcription factors or E3 ligases. HSP90::kinase interactions varied continuously over a 100-fold range and provided a platform to study client protein recognition. In wild-type clients, HSP90 did not bind particular sequence motifs, but rather associated with intrinsically unstable kinases. Stabilization of the kinase in either its active or inactive conformation with diverse small molecules decreased HSP90 association. Our results establish HSP90 client recognition as a combinatorial process: CDC37 provides recognition of the kinase family, whereas thermodynamic parameters determine client binding within the family.

Molecular basis for the action of the collagen-specific chaperone Hsp47/SERPINH1 and its structure-specific client recognition

Collagen is the most abundant protein in animals and is a major component of the extracellular matrix in tissues such as skin and bone. A distinctive structural feature of all collagen types is a unique triple-helical structure formed by tandem repeats of the consensus sequence Xaa-Yaa-Gly, in which Xaa and Yaa frequently are proline and hydroxyproline, respectively. Hsp47/SERPINH1 is a procollagen-specific molecular chaperone that, unlike other chaperones, specifically recognizes the folded conformation of its client. Reduced functional levels of Hsp47 were reported in severe recessive forms of osteogenesis imperfecta, and homozygous knockout is lethal in mice. Here we present crystal structures of Hsp47 in its free form and in complex with homotrimeric synthetic collagen model peptides, each comprising one Hsp47-binding site represented by an arginine at the Yaa-position of a Xaa-Yaa-Gly triplet. Two of these three binding sites in the triple helix are occupied by Hsp47 molecules, which bind in a head-to-head fashion, thus making extensive contacts with the leading and trailing strands of the collagen triple helix. The important arginine residue within the Xaa-Arg-Gly triplet is recognized by a conserved aspartic acid. The structures explain the stabilization of the triple helix as well as the inhibition of collagen-bundle formation by Hsp47. In addition, we propose a pH-dependent substrate release mechanism based on a cluster of histidine residues.

It's frustrating! Managing emotional dissonance in aged care work

Performing paid care work requires workers to simultaneously negotiate care and waged relationships. Mediating contradictions between the two often causes workers to experience frustration, a form of emotional dissonance. Drawing on semi‐structured interviews with 100 workers in Australian aged care organisations, this research identifies aspects of the work environment that enable care workers to successfully juggle or synthesise the emotional demands of their work. The research demonstrates that employers have an important role in alleviating frustration, and can do this by creating work environments which: give care workers relative autonomy; allow care workers to construct themselves as professional carers providing quality care; provide workers with support to manage their emotions and develop emotional resilience; and promote client recognition of workers' roles. Providing such work environments has the potential to not only alleviate workers' frustration, but also to increase job satisfaction and retain workers in aged care.

Job satisfaction and social rewards in the social services

This article investigates the sources of job satisfaction among practitioners and managers employed in the Norwegian public social services and the professionals´ perception of social rewards in particular. Being valued, receiving praise and positive feedback are considered to be important aspects of job satisfaction. Nevertheless the expertise and competence of social workers is not always acknowledged. A central question raised is whether the workers´ job satisfaction is influenced by their opportunities for support and recognition, compared to other (intrinsic and organisational) rewarding aspects available to social service workers. The empirical data come from a 2004 quantitative survey among social workers in local welfare agencies. Despite conflicting demands and lack of resources in the front line services, findings indicate that managers and practitioners perceive their work as overall equally satisfying. Still, the managers find their job more interesting and challenging due to their position, reporting higher feelings of accomplishment and control over work. Receiving public approval and co-worker support are positively associated with job satisfaction within both work positions, while superior support and client recognition were found to be significantly rewarding aspects to the practitioners only. The final discussion addresses the challenges for an organizational climate that sustain the worth and contribution of social professionals

The Endoplasmic Reticulum Chaperone Cosmc Directly Promotes in Vitro Folding of T-synthase

The T-synthase is the key beta 3-galactosyltransferase essential for biosynthesis of core 1 O-glycans (Gal beta 1-3GalNAc alpha 1-Ser/Thr) in animal cell glycoproteins. Here we describe the novel ability of an endoplasmic reticulum-localized molecular chaperone termed Cosmc to specifically interact with partly denatured T-synthase in vitro to cause partial restoration of activity. By contrast, a mutated form of Cosmc observed in patients with Tn syndrome has reduced chaperone function. The chaperone activity of Cosmc is specific, does not require ATP in vitro, and is effective toward T-synthase but not another beta-galactosyltransferase. Cosmc represents the first ER chaperone identified to be required for folding of a glycosyltransferase.

A speech and character combined recognition engine for mobile devices

PurposeThe purpose of this paper is to describe a speech and character combined recognition engine (SCCRE) developed for working on personal digital assistants (PDAs) or on mobile devices. Also, the architecture of a distributed recognition system for providing a more convenient user interface is discussed.Design/methodology/approachIn SCCRE, feature extraction for speech and for character is carried out separately, but the recognition is performed in an engine. The client recognition engine essentially employs a continuous hidden Markov model (CHMM) structure and this CHMM structure consists of variable parameter topology in order to minimize the number of model parameters and to reduce recognition time. This model also adopts the proposed successive state and mixture splitting (SSMS) method for generating context independent model. SSMS optimizes the number of mixtures through splitting in mixture domain and the number of states through splitting in time domain.FindingsThe recognition results show that the developed engine can reduce the total number of Gaussian up to 40 per cent compared with the fixed parameter models at the same recognition performance when applied to speech recognition for mobile devices. It shows that SSMS can reduce the size of memory for models to 65 per cent and that for processing to 82 per cent. Moreover, the recognition time decreases 17 per cent with the SMS model while maintaining the recognition rate.Originality/valueThe proposed system will be very useful for many on‐line multimodal interfaces such as PDAs and mobile applications.

Partners in surveillance and quality control

Partners in surveillance and quality control

Sgt1p Is a Unique Co-chaperone That Acts as a Client Adaptor to Link Hsp90 to Skp1p

Sgt1p is a conserved, essential protein required for kinetochore assembly in both yeast and animal cells. Sgt1p has homology to both TPR and p23 domains, sequences often found in proteins that interact with and regulate the molecular chaperone, Hsp90. The presence of these domains and the recent findings that Sgt1p interacts with Hsp90 has led to the speculation that Sgt1p and Hsp90 form a co-chaperone complex. To test this possibility, we have used purified recombinant proteins to characterize the in vitro interactions between yeast Sgt1p and Hsp82p (an Hsp90 homologue in yeast). We show that Sgt1p interacts directly with Hsp82p via its p23 homology region in a nucleotide-dependent manner. However, Sgt1p binding does not alter the enzymatic activity of Hsp82p, suggesting that it is distinct from other co-chaperones. We find that Sgt1p can form a ternary chaperone complex with Hsp82p and Sti1p, a well characterized Hsp90 co-chaperone. Sgt1p interacts with its binding partner Skp1p through its TPR domains and links Skp1p to the core Hsp82p-Sti1p co-chaperone complex. The multidomain nature of Sgt1p and its ability to bridge the interaction between Skp1p and Hsp82p argue that Sgt1p acts as a "client adaptor" recruiting specific clients to Hsp82p co-chaperone complexes.

Specific Recognition of the Collagen Triple Helix by Chaperone HSP47: MINIMAL STRUCTURAL REQUIREMENT AND SPATIAL MOLECULAR ORIENTATION

The unique folding of procollagens in the endoplasmic reticulum is achieved with the assistance of procollagen-specific molecular chaperones. Heat-shock protein 47 (HSP47) is an endoplasmic reticulum-resident chaperone that plays an essential role in normal procollagen folding, although its molecular function has not yet been clarified. Recent advances in studies on the binding specificity of HSP47 have revealed that Arg residues at Yaa positions in collagenous Gly-Xaa-Yaa repeats are critical for its interactions (Koide, T., Takahara, Y., Asada, S., and Nagata, K. (2002) J. Biol. Chem. 277, 6178-6182; Tasab, M., Jenkinson, L., and Bulleid, N. J. (2002) J. Biol. Chem. 277, 35007-35012). In the present study, we further examined the client recognition mechanism of HSP47 by taking advantage of systems employing engineered collagen model peptides. First, in vitro binding studies using conformationally constrained collagen-like peptides revealed that HSP47 only recognized correctly folded triple helices and that the interaction with the corresponding single-chain polypeptides was negligible. Second, a binding study using heterotrimeric model clients for HSP47 demonstrated a minimal requirement for the number of Arg residues in the triple helix. Finally, a cross-linking study using photoreactive collagenous peptides provided information about the spatial orientation of an HSP47 molecule in the chaperone-collagen complex. The obtained results led to the development of a new model of HSP47-collagen complexes that differs completely from the previously proposed "flying capstan model" (Dafforn, T. R., Della, M., and Miller, A. D. (2001) J. Biol. Chem. 276, 49310-49319).