Emergence of fractal geometries in the evolution of a metabolic enzyme.

Fractals are patterns that are self-similar across multiple length-scales1. Macroscopic fractals are common in nature2-4; however, so far, molecular assembly into fractals is restricted to synthetic systems5-12. Here we report the discovery of a natural protein, citrate synthase from the cyanobacterium Synechococcus elongatus, which self-assembles into Sierpinski triangles. Using cryo-electron microscopy, we reveal how the fractal assembles from a hexameric building block. Although different stimuli modulate the formation of fractal complexes and these complexes can regulate the enzymatic activity of citrate synthase in vitro, the fractal may not serve a physiological function in vivo. We use ancestral sequence reconstruction to retrace how the citrate synthase fractal evolved from non-fractal precursors, and the results suggest it may have emerged as a harmless evolutionary accident. Our findings expand the space of possible protein complexes and demonstrate that intricate and regulatable assemblies can evolve in a single substitution.

Regulation of inositol 5-phosphatase activity by the C2 domain of SHIP1 and SHIP2.

SHIP1, an inositol 5-phosphatase, plays a central role in cellular signaling. As such, it has been implicated in many conditions. Exploiting SHIP1 as a drug target will require structural knowledge and the design of selective small molecules. We have determined apo, and magnesium and phosphate-bound structures of the phosphatase and C2 domains of SHIP1. The C2 domains of SHIP1 and the related SHIP2 modulate the activity of the phosphatase domain. To understand the mechanism, we performed activity assays, hydrogen-deuterium exchange mass spectrometry, and molecular dynamics on SHIP1 and SHIP2. Our findings demonstrate that the influence of the C2 domain is more pronounced for SHIP2 than SHIP1. We determined 91 structures of SHIP1 with fragments bound, with some near the interface between the two domains. We performed a mass spectrometry screen and determined four structures with covalent fragments. These structures could act as starting points for the development of potent, selective probes.

Cryo-EM of soft-landed ß-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 ß-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.

From structural polymorphism to structural metamorphosis of the coat protein of flexuous filamentous potato virus Y.

The structural diversity and tunability of the capsid proteins (CPs) of various icosahedral and rod-shaped viruses have been well studied and exploited in the development of smart hybrid nanoparticles. However, the potential of CPs of the wide-spread flexuous filamentous plant viruses remains to be explored. Here, we show that we can control the shape, size, RNA encapsidation ability, symmetry, stability and surface functionalization of nanoparticles through structure-based design of CP from potato virus Y (PVY). We provide high-resolution insight into CP-based self-assemblies, ranging from large polymorphic or monomorphic filaments to smaller annular, cubic or spherical particles. Furthermore, we show that we can prevent CP self-assembly in bacteria by fusion with a cleavable protein, enabling controlled nanoparticle formation in vitro. Understanding the remarkable structural diversity of PVY CP not only provides possibilities for the production of biodegradable nanoparticles, but may also advance future studies of CP's polymorphism in a biological context.

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.

Development of a PNGase Rc Column for Online Deglycosylation of Complex Glycoproteins during HDX-MS.

Protein glycosylation is one of the most common PTMs and many cell surface receptors, extracellular proteins, and biopharmaceuticals are glycosylated. However, HDX-MS analysis of such important glycoproteins has so far been limited by difficulties in determining the HDX of the protein segments that contain glycans. We have developed a column containing immobilized PNGase Rc (from Rudaea cellulosilytica) that can readily be implemented into a conventional HDX-MS setup to allow improved analysis of glycoproteins. We show that HDX-MS with the PNGase Rc column enables efficient online removal of N-linked glycans and the determination of the HDX of glycosylated regions in several complex glycoproteins. Additionally, we use the PNGase Rc column to perform a comprehensive HDX-MS mapping of the binding epitope of a mAb to c-Met, a complex glycoprotein drug target. Importantly, the column retains high activity in the presence of common quench-buffer additives like TCEP and urea and performed consistent across 114 days of extensive use. Overall, our work shows that HDX-MS with the integrated PNGase Rc column can enable fast and efficient online deglycosylation at harsh quench conditions to provide comprehensive analysis of complex glycoproteins.

The beauty and complexity of the small heat shock proteins: a report on the proceedings of the fourth workshop on small heat shock proteins.

The Fourth Cell Stress Society International workshop on small heat shock proteins (sHSPs), a follow-up to successful workshops held in 2014, 2016 and 2018, took place as a virtual meeting on the 17-18 November 2022. The meeting was designed to provide an opportunity for those working on sHSPs to reconnect and discuss their latest work. The diversity of research in the sHSP field is reflected in the breadth of topics covered in the talks presented at this meeting. Here we summarise the presentations at this meeting and provide some perspectives on exciting future topics to be addressed in the field.

Dimerization of European Robin Cryptochrome 4a.

Homo-dimer formation is important for the function of many proteins. Although dimeric forms of cryptochromes (Cry) have been found by crystallography and were recently observed in vitro for European robin Cry4a, little is known about the dimerization of avian Crys and the role it could play in the mechanism of magnetic sensing in migratory birds. Here, we present a combined experimental and computational investigation of the dimerization of robin Cry4a resulting from covalent and non-covalent interactions. Experimental studies using native mass spectrometry, mass spectrometric analysis of disulfide bonds, chemical cross-linking, and photometric measurements show that disulfide-linked dimers are routinely formed, that their formation is promoted by exposure to blue light, and that the most likely cysteines are C317 and C412. Computational modeling and molecular dynamics simulations were used to generate and assess a number of possible dimer structures. The relevance of these findings to the proposed role of Cry4a in avian magnetoreception is discussed.

Fortuitously compatible protein surfaces primed allosteric control in cyanobacterial photoprotection.

Highly specific interactions between proteins are a fundamental prerequisite for life, but how they evolve remains an unsolved problem. In particular, interactions between initially unrelated proteins require that they evolve matching surfaces. It is unclear whether such surface compatibilities can only be built by selection in small incremental steps, or whether they can also emerge fortuitously. Here, we used molecular phylogenetics, ancestral sequence reconstruction and biophysical characterization of resurrected proteins to retrace the evolution of an allosteric interaction between two proteins that act in the cyanobacterial photoprotection system. We show that this interaction between the orange carotenoid protein (OCP) and its unrelated regulator, the fluorescence recovery protein (FRP), evolved when a precursor of FRP was horizontally acquired by cyanobacteria. FRP's precursors could already interact with and regulate OCP even before these proteins first encountered each other in an ancestral cyanobacterium. The OCP-FRP interaction exploits an ancient dimer interface in OCP, which also predates the recruitment of FRP into the photoprotection system. Together, our work shows how evolution can fashion complex regulatory systems easily out of pre-existing components.

Shape-Morphing of an Artificial Protein Cage with Unusual Geometry Induced by a Single Amino Acid Change.

Artificial protein cages are constructed from multiple protein subunits. The interaction between the subunits, notably the angle formed between them, controls the geometry of the resulting cage. Here, using the artificial protein cage, "TRAP-cage", we show that a simple alteration in the position of a single amino acid responsible for Au(I)-mediated subunit-subunit interactions in the constituent ring-shaped building blocks results in a more acute dihedral angle between them. In turn, this causes a dramatic shift in the structure from a 24-ring cage with an octahedral symmetry to a 20-ring cage with a C2 symmetry. This symmetry change is accompanied by a decrease in the number of Au(I)-mediated bonds between cysteines and a concomitant change in biophysical properties of the cage.

Expansion and Neofunctionalization of Actinoporin-like Genes in Mediterranean Mussel (Mytilus galloprovincialis).

Pore-forming toxins are an important component of the venom of many animals. Actinoporins are potent cytolysins that were first detected in the venom of sea anemones; however, they are occasionally found in animals other than cnidarians and are expanded in a few predatory gastropods. Here, we report the presence of 27 unique actinoporin-like genes with monophyletic origin in Mytilus galloprovincialis, which we have termed mytiporins. These mytiporins exhibited a remarkable level of molecular diversity and gene presence-absence variation, which warranted further studies aimed at elucidating their functional role. We structurally and functionally characterized mytiporin-1 and found significant differences from the archetypal actinoporin fragaceatoxin C. Mytiporin-1 showed weaker permeabilization activity, no specificity towards sphingomyelin, and weak activity in model lipid systems with negatively charged lipids. In contrast to fragaceatoxin C, which forms octameric pores, functional mytiporin-1 pores on negatively charged lipid membranes were hexameric. Similar hexameric pores were observed for coluporin-26 from Cumia reticulata and a conoporin from Conus andremenezi. This indicates that also other molluscan actinoporin-like proteins differ from fragaceatoxin C. Although the functional role of mytiporins in the context of molluscan physiology remains to be elucidated, the lineage-specific gene family expansion event that characterizes mytiporins indicates that strong selective forces acted on their molecular diversification. Given the tissue distribution of mytiporins, this process may have broadened the taxonomic breadth of their biological targets, which would have important implications for digestive processes or mucosal immunity.

Hyperphosphorylated tau self-assembles into amorphous aggregates eliciting TLR4-dependent responses.

Soluble aggregates of the microtubule-associated protein tau have been challenging to assemble and characterize, despite their important role in the development of tauopathies. We found that sequential hyperphosphorylation by protein kinase A in conjugation with either glycogen synthase kinase 3ß or stress activated protein kinase 4 enabled recombinant wild-type tau of isoform 0N4R to spontaneously polymerize into small amorphous aggregates in vitro. We employed tandem mass spectrometry to determine the phosphorylation sites, high-resolution native mass spectrometry to measure the degree of phosphorylation, and super-resolution microscopy and electron microscopy to characterize the morphology of aggregates formed. Functionally, compared with the unmodified aggregates, which require heparin induction to assemble, these self-assembled hyperphosphorylated tau aggregates more efficiently disrupt membrane bilayers and induce Toll-like receptor 4-dependent responses in human macrophages. Together, our results demonstrate that hyperphosphorylated tau aggregates are potentially damaging to cells, suggesting a mechanism for how hyperphosphorylation could drive neuroinflammation in tauopathies.

Complementing machine learning-based structure predictions with native mass spectrometry.

The advent of machine learning-based structure prediction algorithms such as AlphaFold2 (AF2) and RoseTTa Fold have moved the generation of accurate structural models for the entire cellular protein machinery into the reach of the scientific community. However, structure predictions of protein complexes are based on user-provided input and may require experimental validation. Mass spectrometry (MS) is a versatile, time-effective tool that provides information on post-translational modifications, ligand interactions, conformational changes, and higher-order oligomerization. Using three protein systems, we show that native MS experiments can uncover structural features of ligand interactions, homology models, and point mutations that are undetectable by AF2 alone. We conclude that machine learning can be complemented with MS to yield more accurate structural models on a small and large scale.

Mass-selective and ice-free electron cryomicroscopy protein sample preparation via native electrospray ion-beam deposition.

Despite tremendous advances in sample preparation and classification algorithms for electron cryomicroscopy (cryo-EM) and single-particle analysis (SPA), sample heterogeneity remains a major challenge and can prevent access to high-resolution structures. In addition, optimization of preparation conditions for a given sample can be time-consuming. In the current work, it is demonstrated that native electrospray ion-beam deposition (native ES-IBD) is an alternative, reliable approach for the preparation of extremely high-purity samples, based on mass selection in vacuum. Folded protein ions are generated by native electrospray ionization, separated from other proteins, contaminants, aggregates, and fragments, gently deposited on cryo-EM grids, frozen in liquid nitrogen, and subsequently imaged by cryo-EM. We demonstrate homogeneous coverage of ice-free cryo-EM grids with mass-selected protein complexes. SPA reveals that the complexes remain folded and assembled, but variations in secondary and tertiary structures are currently limiting information in 2D classes and 3D EM density maps. We identify and discuss challenges that need to be addressed to obtain a resolution comparable to that of the established cryo-EM workflow. Our results show the potential of native ES-IBD to increase the scope and throughput of cryo-EM for protein structure determination and provide an essential link between gas-phase and solution-phase protein structures.

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 reimplementing 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 AND IMPLEMENTATION: Biobox is freely available on https://github.com/degiacom/biobox, along with its API and interactive Jupyter notebook tutorials.

The binding of the small heat-shock protein αB-crystallin to fibrils of α-synuclein is driven by entropic forces.

Molecular chaperones are key components of the cellular proteostasis network whose role includes the suppression of the formation and proliferation of pathogenic aggregates associated with neurodegenerative diseases. The molecular principles that allow chaperones to recognize misfolded and aggregated proteins remain, however, incompletely understood. To address this challenge, here we probe the thermodynamics and kinetics of the interactions between chaperones and protein aggregates under native solution conditions using a microfluidic platform. We focus on the binding between amyloid fibrils of α-synuclein, associated with Parkinson's disease, to the small heat-shock protein αB-crystallin, a chaperone widely involved in the cellular stress response. We find that αB-crystallin binds to α-synuclein fibrils with high nanomolar affinity and that the binding is driven by entropy rather than enthalpy. Measurements of the change in heat capacity indicate significant entropic gain originates from the disassembly of the oligomeric chaperones that function as an entropic buffer system. These results shed light on the functional roles of chaperone oligomerization and show that chaperones are stored as inactive complexes which are capable of releasing active subunits to target aberrant misfolded species.

Ion mobility-mass spectrometry shows stepwise protein unfolding under alkaline conditions.

Although native mass spectrometry is widely applied to monitor chemical or thermal protein denaturation, it is not clear to what extent it can inform about alkali-induced unfolding. Here, we probe the relationship between solution- and gas-phase structures of proteins under alkaline conditions. Native ion mobility-mass spectrometry reveals that globular proteins are destabilized rather than globally unfolded, which is supported by solution studies, providing detailed insights into alkali-induced unfolding events. Our results pave the way for new applications of MS to monitor structures and interactions of proteins at high pH.

Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry.

In solution, the charge of a protein is intricately linked to its stability, but electrospray ionization distorts this connection, potentially limiting the ability of native mass spectrometry to inform about protein structure and dynamics. How the behavior of intact proteins in the gas phase depends on the presence and distribution of ionizable surface residues has been difficult to answer because multiple chargeable sites are present in virtually all proteins. Turning to protein engineering, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, while coexisting conformers can be distinguished using ion mobility separation. An excess of ionizable side chains, on the other hand, effectively modulates protein ion stability. In fact, moving a single ionizable group can dramatically alter the gas-phase conformation of a protein ion. We conclude that although the sum of the charges is governed solely by Coulombic terms, their locations affect the stability of the protein in the gas phase.

Single-molecule fluorescence-based approach reveals novel mechanistic insights into human small heat shock protein chaperone function.

Small heat shock proteins (sHsps) are a family of ubiquitous intracellular molecular chaperones; some sHsp family members are upregulated under stress conditions and play a vital role in protein homeostasis (proteostasis). It is commonly accepted that these chaperones work by trapping misfolded proteins to prevent their aggregation; however, fundamental questions regarding the molecular mechanism by which sHsps interact with misfolded proteins remain unanswered. The dynamic and polydisperse nature of sHsp oligomers has made studying them challenging using traditional biochemical approaches. Therefore, we have utilized a single-molecule fluorescence-based approach to observe the chaperone action of human alphaB-crystallin (αBc, HSPB5). Using this approach we have, for the first time, determined the stoichiometries of complexes formed between αBc and a model client protein, chloride intracellular channel 1. By examining the dispersity and stoichiometries of these complexes over time, and in response to different concentrations of αBc, we have uncovered unique and important insights into a two-step mechanism by which αBc interacts with misfolded client proteins to prevent their aggregation.

Software Requirements for the Analysis and Interpretation of Native Ion Mobility Mass Spectrometry Data.

The past few years have seen a dramatic increase in applications of native mass and ion mobility spectrometry, especially for the study of proteins and protein complexes. This increase has been catalyzed by the availability of commercial instrumentation capable of carrying out such analyses. As in most fields, however, the software to process the data generated from new instrumentation lags behind. Recently, a number of research groups have started addressing this by developing software, but further improvements are still required in order to realize the full potential of the data sets generated. In this perspective, we describe practical aspects as well as challenges in processing native mass spectrometry (MS) and ion mobility-MS data sets and provide a brief overview of currently available tools. We then set out our vision of future developments that would bring the community together and lead to the development of a common platform to expedite future computational developments, provide standardized processing approaches, and serve as a location for the deposition of data for this emerging field. This perspective has been written by members of the European Cooperation in Science and Technology Action on Native MS and Related Methods for Structural Biology (EU COST Action BM1403) as an introduction to the software tools available in this area. It is intended to serve as an overview for newcomers and to stimulate discussions in the community on further developments in this field, rather than being an in-depth review. Our complementary perspective (http://dx.doi.org/10.1021/acs.analchem.9b05791) focuses on computational approaches used in this field.