Terminal regions confer plasticity to the tetrameric assembly of human HspB2 and HspB3

Heterogeneity in small heat shock proteins (sHsps) spans multiple spatiotemporal regimes – from fast fluctuations of part of the protein, to conformational variability of tertiary structure, plasticity of the interfaces, and polydispersity of the inter-converting, and co-assembling oligomers. This heterogeneity and dynamic nature of sHsps has significantly hindered their structural characterisation. Atomic-coordinates are particularly lacking for vertebrate sHsps, where most available structures are of extensively truncated homomers. sHsps play important roles in maintaining protein levels in the cell and therefore in organismal health and disease. HspB2 and HspB3 are vertebrate sHsps that are found co-assembled in neuromuscular cells, and variants thereof are associated with disease. Here, we present the structure of human HspB2/B3, which crystallised as a hetero-tetramer in a 3:1 ratio. In the HspB2/B3 tetramer, the four a-crystallin domains (ACDs) assemble into a flattened tetrahedron which is pierced by two non-intersecting approximate dyads. Assembly is mediated by flexible “nuts and bolts” involving IXI/V motifs from terminal regions filling ACD pockets. Parts of the N-terminal region bind in an unfolded conformation into the anti-parallel shared ACD dimer grooves. Tracts of the terminal regions are not resolved, most likely due to their disorder in the crystal lattice. This first structure of a full-length human sHsp heteromer reveals the heterogeneous interactions of the terminal regions and suggests a plasticity that is important for the cytoprotective functions of sHsps

Recommendations for reporting ion mobility Mass Spectrometry measurements

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

EM∩IM: Software for relating ion mobility mass spectrometry and electron microscopy data

We present EM∩IM, software that allows the calculation of collision cross-sections from electron density maps obtained for example by means of transmission electron microscopy. This allows the assessment of structures other than those described by atomic coordinates with ion mobility mass spectrometry data, and provides a new means for contouring and validating electron density maps. EM∩IM thereby facilitates the use of data obtained in the gas phase within structural biology studies employing diverse experimental methodologies

Subunit exchange of multimeric protein complexes Real-time monitoring of subunit exchange between small heat shock proteins by using electrospray-mass spectrometry.

The subunit exchange of the small heat shock proteins (sHSPs) PsHSP18.1 from pea and TaHSP16.9 from wheat has been monitored in real-time using nanoelectrospray mass spectrometry. By preserving the noncovalent interactions between subunits in the mass spectrometer, we show that these proteins are dodecameric. After mixing PsHSP18.1 and TaHSP16.9, a distribution of heterododecamers is formed. A comparison with spectra obtained from statistical modeling demonstrates that after equilibration the distribution of these heterocomplexes is governed by the starting ratio of the two components rather than an inherent preference for certain stoichiometries. This finding suggests that the two different sHSP subunits interact in a very similar manner. Following the kinetics of this reaction by mass spectrometry reveals that exchange proceeds via sequential incorporation of subunits with dimeric species being the principal units of exchange. Therefore, we conclude that sHSP complexes are in rapid dissociation/reassociation equilibria with suboligomeric forms. More generally, these experiments illustrate a powerful approach for the real-time analysis of the evolution of transient species and their relative populations during the subunit exchange of multimeric protein complexes

Collision cross sections for structural proteomics

Ion mobility mass spectrometry (IM-MS) allows the structural interrogation of biomolecules by reporting their collision cross sections (CCSs). The major bottleneck for exploiting IM-MS in structural proteomics lies in the lack of speed at which structures and models can be related to experimental data. Here we present IMPACT (Ion Mobility Projection Approximation Calculation Tool), which overcomes these twin challenges, providing accurate CCSs up to 106 times faster than alternative methods. This allows us to assess the CCS space presented by the entire structural proteome, interrogate ensembles of protein conformers, and monitor molecular dynamics trajectories. Our data demonstrate that the CCS is a highly informative parameter and that IM-MS is of considerable practical value to structural biologists

Quaternary dynamics of αB-crystallin as a direct consequence of localised tertiary fluctuations in the C-terminus

The majority of proteins exist in vivo within macromolecular assemblies whose functions are dependent on dynamical processes spanning a wide range of timescales. One such assembly is formed by the molecular chaperone αB-crystallin that exists in a variety of exchanging oligomeric states, centred on a mass of approximately 560 kDa. For many macromolecular assemblies, including αB-crystallin, the inherent dynamics, heterogeneity and high mass contribute to difficulties in quantitative studies. Here we demonstrate a strategy based on correlating solution-state nuclear magnetic resonance spectroscopy and mass spectrometry data to characterize simultaneously the organization and dynamics of the polydisperse αB-crystallin ensemble. We show that protomeric dimers assemble into oligomers via the binding of extended C-termini, with each monomer donating and receiving one terminus. Moreover, we establish that the C-termini undergo millisecond fluctuations which regulate the inter-conversion of oligomeric forms. The combined biophysical approach allows construction of an energy profile for a single monomer that completely describes the equilibrium dynamics of the ensemble. It also facilitates an analysis of dynamics spanning the millisecond to hour timescales and secondary to quaternary structural levels and provides an approach for, simultaneously, obtaining detailed structural, thermodynamic and kinetic information on a heterogeneous protein assembly

Separating and visualising protein assemblies by means of preparative mass spectrometry and microscopy

Many multi-protein assemblies exhibit characteristics which hamper their structural and dynamical characterization. These impediments include low copy number, heterogeneity, polydispersity, hydrophobicity, and intrinsic disorder. It is becoming increasingly apparent that both novel and hybrid structural biology approaches need to be developed to tackle the most challenging targets. Nanoelectrospray mass spectrometry has matured over the last decade to enable the elucidation of connectivity and composition of large protein assemblies. Moreover, comparing mass spectrometry data with transmission electron microscopy images has enabled the mapping of subunits within topological models. Here we describe a preparative form of mass spectrometry designed to isolate specific protein complexes from within a heterogeneous ensemble, and to ‘soft-land’ these target complexes for ex situ imaging. By building a retractable probe incorporating a versatile target holder, and modifying the ion optics of a commercial mass spectrometer, we show that we can steer the macromolecular ion beam onto a target for imaging by means of transmission electron microscopy and atomic force microscopy. Our data for the tetradecameric chaperonin GroEL show that not only are the molecular volumes of the landed particles consistent with the overall dimensions of the complex, but also that their gross topological features can be maintained

Integrating mass spectrometry with MD simulations reveals the role of lipids in Na+/H+ antiporters

Na+/H+ antiporters are found in all kingdoms of life and exhibit catalysis rates that are among the fastest of all known secondary-active transporters. Here we combine ion mobility mass spectrometry and molecular dynamics simulations to study the conformational stability and lipid-binding properties of the Na+/H+ exchanger NapA from Thermus thermophilus and compare this to the prototypical antiporter NhaA from Escherichia coli and the human homologue NHA2. We find that NapA and NHA2, but not NhaA, form stable dimers and do not selectively retain membrane lipids. By comparing wild-type NapA with engineered variants, we show that the unfolding of the protein in the gas phase involves the disruption of inter-domain contacts. Lipids around the domain interface protect the native fold in the gas phase by mediating contacts between the mobile protein segments. We speculate that elevator-type antiporters such as NapA, and likely NHA2, use a subset of annular lipids as structural support to facilitate large-scale conformational changes within the membrane

The Tetrameric Plant Lectin BanLec Neutralizes HIV through Bidentate Binding to Specific Viral Glycans

Select lectins have powerful anti-viral properties that effectively neutralize HIV-1 by targeting the dense glycan shield on the virus. Here, we reveal the mechanism by which one of the most potent lectins, BanLec, achieves its inhibition. We identify that BanLec recognizes a subset of high-mannose glycans via bidentate interactions spanning the two binding sites present on each BanLec monomer that were previously considered separate carbohydrate recognition domains. We show that both sites are required for high-affinity glycan binding and virus neutralization. Unexpectedly we find that BanLec adopts a tetrameric stoichiometry in solution whereby the glycan-binding sites are positioned to optimally target glycosylated viral spikes. The tetrameric architecture, together with bidentate binding to individual glycans, leads to layers of multivalency that drive viral neutralization through enhanced avidity effects. These structural insights will prove useful in engineering successful lectin therapeutics targeting the dense glycan shield of HIV