Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states1,2. Healthy metazoan cells effectively eliminate intracellular protein aggregates3,4, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems5,6, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro4,7. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control.

In recent years, chemical crosslinking of protein complexes and the identification of crosslinked residues by mass spectrometry (XL-MS; sometimes abbreviated as CX-MS) has become an important technique bridging mass spectrometry (MS) and structural biology. By now, XL-MS is well established and supported by publicly available resources as a convenient and versatile part of the structural biologist's toolbox. The combination of XL-MS with cryoelectron microscopy (cryo-EM) and/or integrative modeling is particularly promising to study the topology and structure of large protein assemblies. Among the targets studied so far are proteasomes, ribosomes, polymerases, chromatin remodelers, and photosystem complexes. Here we provide an overview of recent advances in XL-MS, the current state of the field, and a cursory outlook on future challenges. Chemical Crosslinking as a Tool for Structural BiologyStructural biology makes use of many different techniques to elucidate the 3D structures of proteins and protein complexes. While high-resolution structures have traditionally been obtained by X-ray crystallography, cryo-EM is increasingly able to also generate (near) atomic-resolution models. In recent years, techniques and applications of MS have also rapidly progressed. Earlier studies were largely focused on the large-scale identification and quantification of proteins, whereas recent methods also support queries into the composition, stoichiometry, and spatial arrangement of subunits in a complex. These developments have now further progressed toward generating information that contributes, as part of hybrid structural strategies, to the structure elucidation of large molecular assemblies including protein complexes that perform essential processes in the cell. XL-MS is a particularly powerful mass spectrometric technique in this respect, because it provides several layers of information. Identifying protein-protein contacts through XL-MS confirms physical proximity between subunits because the proteins must be close enough in space to be crosslinked. Localizing the side chains that are connected restricts this proximity to certain regions (e.g., domains or even single helices or loops). Finally, the structure of the connected side chains and the crosslinker moiety impart a distance restraint that can be used for molecular modeling purposes because an upper Trends Chemical crosslinking followed by the mass spectrometric analysis of cross linked peptides (XL MS) identifies con tact sites between residues within a single or between multiple proteins.The application of XL MS to many bio logically relevant molecular machines has been shown, with a rapidly growing number of successful studies reported in the past 2 3 years.Crosslinking data are useful in integra tive modeling workflows by providing distance restraints on the surface of folded proteins and complexes. XL MS has been shown to be particularly powerful in combination with 3D cryo electron microscopy. Sciences ; 41 (2016), 1. -S. 20-32 https://dx.doi.org/10.1016...

SummaryEukaryotic translation initiation requires the recruitment of the large, multiprotein eIF3 complex to the 40S ribosomal subunit. We present X-ray structures of all major components of the minimal, six-subunit Saccharomyces cerevisiae eIF3 core. These structures, together with electron microscopy reconstructions, cross-linking coupled to mass spectrometry, and integrative structure modeling, allowed us to position and orient all eIF3 components on the 40S⋅eIF1 complex, revealing an extended, modular arrangement of eIF3 subunits. Yeast eIF3 engages 40S in a clamp-like manner, fully encircling 40S to position key initiation factors on opposite ends of the mRNA channel, providing a platform for the recruitment, assembly, and regulation of the translation initiation machinery. The structures of eIF3 components reported here also have implications for understanding the architecture of the mammalian 43S preinitiation complex and the complex of eIF3, 40S, and the hepatitis C internal ribosomal entry site RNA.

Small Heat Shock Proteins (sHSPs) are a diverse family of molecular chaperones that prevent protein aggregation by binding clients destabilized during cellular stress. Here we probe the architecture and dynamics of complexes formed between an oligomeric sHSP and client by employing unique mass spectrometry strategies. We observe over 300 different stoichiometries of interaction, demonstrating that an ensemble of structures underlies the protection these chaperones confer to unfolding clients. This astonishing heterogeneity not only makes the system quite distinct in behavior to ATP-dependent chaperones, but also renders it intractable by conventional structural biology approaches. We find that thermally regulated quaternary dynamics of the sHSP establish and maintain the plasticity of the system. This extends the paradigm that intrinsic dynamics are crucial to protein function to include equilibrium fluctuations in quaternary structure, and suggests they are integral to the sHSPs' role in the cellular protein homeostasis network.heterogeneity | mass spectrometry | polydispersity | protein dynamics | proteostasis S mall Heat Shock Proteins (sHSPs) are one of the least well understood classes of molecular chaperones, proteins which act to prevent or reverse improper protein associations (1). The importance of the sHSPs is evidenced by their almost ubiquitous expression (2), the presence of multiple sHSP genes in most organisms (3), and their dramatic up-regulation under stress conditions making them among the most abundant of cellular proteins (4). They are implicated in a range of disease states including cataract, cancer, myopathies, motor neuropathies, and neurodegeneration (5-8). The current view of their chaperone action is that they bind unfolding "client" proteins, thereby preventing their irreversible aggregation (9-12). These sHSP: client complexes then interact with ATP-dependent chaperones to allow refolding of the clients (9-12). Structural interrogation of the complexes they form with clients has however been hampered by their apparent heterogeneity, and their organization remains consequently very poorly defined (13-15).MS is an emergent technology for the structural biology of protein assemblies (16), allowing the interrogation of a wide range of biomolecular systems, including those complicated by polydispersity and dynamics (17, 18). Here we capitalize on these unique advantages to study the complexes formed between pea HSP18.1 and a model client protein, firefly luciferase (Luc). HSP18.1 represents the family of class I cytosolic plant sHSPs which accumulate at heat-shock temperatures (≥38°C) to ≈1% of the total cellular protein (19). Extensive in vitro studies have established that HSP18.1 is able to bind destabilized clients, enabling subsequent refolding by the HSP70 machinery (20,21). With the in vivo clients of HSP18.1 yet to be identified, Luc was chosen as it is extremely thermo-sensitive and has been used extensively in chaperone studies (12). Luc does not interact with HSP18.1 at room te...