Hsp70 Oligomerization Is Mediated by an Interaction between the Interdomain Linker and the Substrate-Binding Domain

Oligomerization in the heat shock protein (Hsp) 70 family has been extensively documented both in vitro and in vivo, although the mechanism, the identity of the specific protein regions involved and the physiological relevance of this process are still unclear. We have studied the oligomeric properties of a series of human Hsp70 variants by means of nanoelectrospray ionization mass spectrometry, optical spectroscopy and quantitative size exclusion chromatography. Our results show that Hsp70 oligomerization takes place through a specific interaction between the interdomain linker of one molecule and the substrate-binding domain of a different molecule, generating dimers and higher-order oligomers. We have found that substrate binding shifts the oligomerization equilibrium towards the accumulation of functional monomeric protein, probably by sequestering the helical lid sub-domain needed to stabilize the chaperone: substrate complex. Taken together, these findings suggest a possible role of chaperone oligomerization as a mechanism for regulating the availability of the active monomeric form of the chaperone and for the control of substrate binding and release. © 2013 Aprile et al.FAA was recipient of a graduate fellowship from the Italian Ministry of Education, University and Research. AD is grateful for support from Murray Edwards College, Cambridge, through a Junior Research Fellowship. FS is a Sir Henry Wellcome Fellow. CR acknowledges financial support by the Spanish Ministry of Health according to the 'Plan Nacional de I+D+I 2008-2011', through ISCIII with cofunding by FEDER (CP10/00527). JLPB is a Royal Society University Research Fellow. FAA and PT are grateful for support from Regione Lombardia (NEDD and >Network Tecnologico integrato per lo studio proteomico e trascrittomico di malattie neurodegenerative correlate a deposizioni di amiloidi>). CMD acknowledges support from BBSRC (BB/E019927/1), the Wellcome Trust (094425/Z/10/Z), the European Commission (project LSHM-CT-2006-037525). NC acknowledges support from Human Frontiers Science Program (HFSP) through a Long-term Fellowship (LT000795/2009).Peer Reviewe

Cryo-EM of soft-landed β-galactosidase: Gas-phase and native structures are remarkably similar

Native mass spectrometry (MS) has become widely accepted in structural biology, providing information on stoichiometry, interactions, homogeneity, and shape of protein complexes. Yet, the fundamental assumption that proteins inside the mass spectrometer retain a structure faithful to native proteins in solution remains a matter of intense debate. Here, we reveal the gas-phase structure of β-galactosidase using single-particle cryo–electron microscopy (cryo-EM) down to 2.6-Å resolution, enabled by soft landing of mass-selected protein complexes onto cold transmission electron microscopy (TEM) grids followed by in situ ice coating. We find that large parts of the secondary and tertiary structure are retained from the solution. Dehydration-driven subunit reorientation leads to consistent compaction in the gas phase. By providing a direct link between high-resolution imaging and the capability to handle and select protein complexes that behave problematically in conventional sample preparation, the approach has the potential to expand the scope of both native mass spectrometry and cryo-EM

The adaptability of the ion binding site by the Ag(I)/Cu(I) periplasmic chaperone SilF

The periplasmic chaperone SilF has been identified as part of an Ag(I) detoxification system in Gram negative bacteria. Sil proteins also bind Cu(I), but with reported weaker affinity, therefore leading to the designation of a specific detoxification system for Ag(I). Using isothermal titration calorimetry we show that binding of both ions is not only tighter than previously thought, but of very similar affinities. We investigated the structural origins of ion binding using molecular dynamics and QM/MM simulations underpinned by structural and biophysical experiments. The results of this analysis showed that the binding site adapts to accommodate either ion, with key interactions with the solvent in the case of Cu(I). The implications of this are that Gram negative bacteria do not appear to have evolved a specific Ag(I) efflux system but take advantage of the existing Cu(I) detoxification system. Therefore, there are consequences for how we define a particular metal resistance mechanism and understand its evolution in the environment

Recommendations for reporting ion mobility mass spectrometry measurements

© 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc. Here we present a guide to ion mobility mass spectrometry experiments, which covers both linear and nonlinear methods: what is measured, how the measurements are done, and how to report the results, including the uncertainties of mobility and collision cross section values. The guide aims to clarify some possibly confusing concepts, and the reporting recommendations should help researchers, authors and reviewers to contribute comprehensive reports, so that the ion mobility data can be reused more confidently. Starting from the concept of the definition of the measurand, we emphasize that (i) mobility values (K0) depend intrinsically on ion structure, the nature of the bath gas, temperature, and E/N; (ii) ion mobility does not measure molecular surfaces directly, but collision cross section (CCS) values are derived from mobility values using a physical model; (iii) methods relying on calibration are empirical (and thus may provide method-dependent results) only if the gas nature, temperature or E/N cannot match those of the primary method. Our analysis highlights the urgency of a community effort toward establishing primary standards and reference materials for ion mobility, and provides recommendations to do so. © 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc

Negative Ions Enhance Survival of Membrane Protein Complexes

Membrane protein complexes are commonly introduced to the mass spectrometer solubilized in detergent micelles. The collisional activation used to remove the detergent, however, often causes protein unfolding and dissociation. As in the case for soluble proteins, electrospray in the positive ion mode is most commonly used for the study of membrane proteins. Here we show several distinct advantages of employing the negative ion mode. Negative polarity can yield lower average charge states for membrane proteins solubilized in saccharide detergents, with enhanced peak resolution and reduced adduct formation. Most importantly, we demonstrate that negative ion mode electrospray ionization (ESI) minimizes subunit dissociation in the gas phase, allowing access to biologically relevant oligomeric states. Together, these properties mean that intact membrane protein ions can be generated in a greater range of solubilizing detergents. The formation of negative ions, therefore, greatly expands the possibilities of using mass spectrometry on this intractable class of protein

Negative Ions Enhance Survival of Membrane Protein Complexes

Membrane protein complexes are commonly introduced to the mass spectrometer solubilized in detergent micelles. The collisional activation used to remove the detergent, however, often causes protein unfolding and dissociation. As in the case for soluble proteins, electrospray in the positive ion mode is most commonly used for the study of membrane proteins. Here we show several distinct advantages of employing the negative ion mode. Negative polarity can yield lower average charge states for membrane proteins solubilized in saccharide detergents, with enhanced peak resolution and reduced adduct formation. Most importantly, we demonstrate that negative ion mode electrospray ionization (ESI) minimizes subunit dissociation in the gas phase, allowing access to biologically relevant oligomeric states. Together, these properties mean that intact membrane protein ions can be generated in a greater range of solubilizing detergents. The formation of negative ions, therefore, greatly expands the possibilities of using mass spectrometry on this intractable class of protein

Accommodating protein dynamics in the modeling of chemical crosslinks

Chemical crosslinking can identify the neighborhood relationships between specific amino-acid residues in proteins. The interpretation of crosslinking data is typically performed using single, static atomic structures. However, proteins are dynamic, undergoing motions spanning from local fluctuations of individual residues to global motions of protein assemblies. Here we demonstrate that failure to explicitly accommodate dynamics when interpreting crosslinks structurally can lead to considerable errors. We present a method and associated software, DynamXL, which is able to account directly for flexibility in the context of crosslinking modeling. Our benchmarking on a large dataset of model structures demonstrates significantly improved rationalization of experimental crosslinking data, and enhanced performance in a protein-protein docking protocol. These advances will provide a considerable increase in the structural insights attainable using chemical crosslinking coupled to mass spectrometry

Biobox: a toolbox for biomolecular modelling

Motivation The implementation of biomolecular modelling methods and analyses can be cumbersome, often carried out with in-house software re-implementing common tasks, and requiring the integration of diverse software libraries. Results We present Biobox, a Python-based toolbox facilitating the implementation of biomolecular modelling methods. Availability Biobox is freely available on https://github.com/degiacom/biobox, along with its API and interactive Jupyter notebook tutorials

It takes a dimer to tango: Oligomeric small heat shock proteins dissociate to capture substrate

Small heat-shock proteins (sHsps) are ubiquitous molecular chaperones, and sHsp mutations or altered expression are linked to multiple human disease states. sHsp monomers assemble into large oligomers with dimeric substructure, and the dynamics of sHsp oligomers has led to major questions about the form that captures substrate, a critical aspect of their mechanism of action. We show here that sub-structural dimers of two plant dodecameric sHsps, Ta16.9 and homologous Ps18.1, are functional units in the initial encounter with unfolding substrate. We introduced inter-polypeptide disulfide bonds at the two dodecameric interfaces, dimeric and nondimeric, to restrict how their assemblies can dissociate. When disulfide bonded at the non-dimeric interface, mutants of Ta16.9 and Ps18.1 (TaCT-ACD and PsCT-ACD) were inactive, but when reduced, had wildtype-like chaperone activity, demonstrating that dissociation at non-dimeric interfaces is essential for sHsp activity. Moreover, the size of the TaCT-ACD and PsCT-ACD covalent unit defined a new tetrahedral geometry for these sHsps, different from that observed in the Ta16.9 X-ray structure. Importantly, oxidized Tadimer (disulfide bonded at the dimeric interface) exhibited greatly enhanced ability to protect substrate, indicating that strengthening the dimeric interface increases chaperone efficiency. Temperature-induced size and secondary structure changes revealed that folded sHsp dimers interact with substrate and that dimer stability affects chaperone efficiency. These results yield a model in which sHsp dimers capture substrate before assembly into larger, heterogeneous sHsp–substrate complexes for substrate refolding or degradation and suggest that tuning the strength of the dimer interface can be used to engineer sHsp chaperone efficiency