Breaking free from the crystal lattice:Structural biology in solution to study protein degraders

Structural biology offers a versatile arsenal of techniques and methods to investigate the structure and conformational dynamics of proteins and their assemblies. The growing field of targeted protein degradation centres on the premise of developing small molecules, termed degraders, to induce proximity between an E3 ligase and a protein of interest to be signalled for degradation. This new drug modality brings with it new opportunities and challenges to structural biologists. Here we discuss how several structural biology techniques, including nuclear magnetic resonance, cryo-electron microscopy, structural mass spectrometry and small angle scattering, have been explored to complement X-ray crystallography in studying degraders and their ternary complexes. Together the studies covered in this review make a case for the invaluable perspectives that integrative structural biology techniques in solution can bring to understanding ternary complexes and designing degraders

Signal loss due to oligomerization in ELISA analysis of amyloid-beta can be recovered by a novel sample pre-treatment method

According to the predominant theories, soluble amyloid-beta (Aβ) aggregates are the principal neurotoxic agents in Alzheimer’s disease pathology, making them a popular target for the development of therapeutics and diagnostic markers. One of the most commonly used methods for determining the concentration of Aβ is ELISA. However, ELISA was developed for monomeric proteins and may be ill-suited for detecting aggregates. Therefore, we investigated the effect of aggregation on the ELISA measurement and developed a novel chemical pre-treatment method, designed to disaggregate Aβ peptides, to improve the ELISA measurement of the total Aβ concentration. Synthetic Aβ40 monomers, Aβ42 oligomers and biological samples from mice and humans were subjected to a chemical pre-treatment protocol with: trifluoroacetic acid (TFA), formic acid (FA) or hexafluoroisopropanol (HFIP) prior to ELISA analysis. In our study we have shown that: • Aβ oligomerization leads to epitope masking and steric hindrance and results in an underestimation of the total Aβ content with ELISA. • Chemically pre-treating samples to disaggregate oligomers can (partially) recover the signal loss. • This novel sample pre-treatment method could provide a more accurate ELISA measurement of the total Aβ concentration in samples with a high oligomer content

Opposite Structural Effects of Epigallocatechin-3-gallate and Dopamine Binding to α-Synuclein

The intrinsically disordered and amyloidogenic protein α-synuclein (AS) has been linked to several neurodegenerative states, including Parkinson's disease. Here, nanoelectrospray-ionization mass spectrometry (nano-ESI-MS), ion mobility (IM), and native top-down electron transfer dissociation (ETD) techniques are employed to study AS interaction with small molecules known to modulate its aggregation, such as epigallocatechin-3-gallate (EGCG) and dopamine (DA). The complexes formed by the two ligands under identical conditions reveal peculiar differences. While EGCG engages AS in compact conformations, DA preferentially binds to the protein in partially extended conformations. The two ligands also have different effects on AS structure as assessed by IM, with EGCG leading to protein compaction and DA to its extension. Native top-down ETD on the protein-ligand complexes shows how the different observed modes of binding of the two ligands could be related to their known opposite effects on AS aggregation. The results also show that the protein can bind either ligand in the absence of any covalent modifications, such as oxidation

Multidimensional Dynamics of the Proteome in the Neurodegenerative and Aging Mammalian Brain

Neurodegenerative diseases are characterized by the abnormal accumulation of aggregated proteins in the brain. Using in vivo pulse isotope labeling, we screened the proteome for changes in protein turnover and abundance in multiple mouse models of neurodegeneration. These data suggest that the disease state of pathologically affected tissue is characterized by a proteome-wide increase in protein turnover and repair. In contrast, in healthy wild-type mice, aging in the mammalian brain is associated with a global slowdown in protein turnover.Peer reviewe

Sample deposition onto cryo-EM grids: from sprays to jets and back

Despite the great strides made in the field of single-particle cryogenic electron microscopy (cryo-EM) in microscope design, direct electron detectors and new processing suites, the area of sample preparation is still far from ideal. Traditionally, sample preparation involves blotting, which has been used to achieve high resolution, particularly for well behaved samples such as apoferritin. However, this approach is flawed since the blotting process can have adverse effects on some proteins and protein complexes, and the long blot time increases exposure to the damaging air-water interface. To overcome these problems, new blotless approaches have been designed for the direct deposition of the sample on the grid. Here, different methods of producing droplets for sample deposition are compared. Using gas dynamic virtual nozzles, small and high-velocity droplets were deposited on cryo-EM grids, which spread sufficiently for high-resolution cryo-EM imaging. For those wishing to pursue a similar approach, an overview is given of the current use of spray technology for cryo-EM grid preparation and areas for enhancement are pointed out. It is further shown how the broad aspects of sprayer design and operation conditions can be utilized to improve grid quality reproducibly

Structure of an Hsp90-Cdc37-Cdk4 complex

Activation of many protein kinases depends on their interaction with the Hsp90 molecular chaperone system. Recruitment of protein kinase clients to the Hsp90 chaperone system is mediated by the cochaperone adaptor protein Cdc37, which acts as a scaffold, simultaneously binding protein kinases and Hsp90. We have now expressed and purified an Hsp90-Cdc37-Cdk4 complex, defined its stoichiometry, and determined its 3D structure by single-particle electron microscopy. Comparison with the crystal structure of Hsp90 allows us to identify the locations of Cdc37 and Cdk4 in the complex and suggests a mechanism by which conformational changes in the kinase are coupled to the Hsp90 ATPase cycle

MIND: A Double-Linear Model To Accurately Determine Monoisotopic Precursor Mass in High-Resolution Top-Down Proteomics

Top-down proteomics approaches are becoming ever more popular, due to the advantages offered by knowledge of the intact protein mass in correctly identifying the various proteoforms that potentially arise due to point mutation, alternative splicing, post-translational modifications, etc. Usually, the average mass is used in this context; however, it is known that this can fluctuate significantly due to both natural and technical causes. Ideally, one would prefer to use the monoisotopic precursor mass, but this falls below the detection limit for all but the smallest proteins. Methods that predict the monoisotopic mass based on the average mass are potentially affected by imprecisions associated with the average mass. To address this issue, we have developed a framework based on simple, linear models that allows prediction of the monoisotopic mass based on the exact mass of the most-abundant (aggregated) isotope peak, which is a robust measure of mass, insensitive to the aforementioned natural and technical causes. This linear model was tested experimentally, as well as in silico, and typically predicts monoisotopic masses with an accuracy of only a few parts per million. A confidence measure is associated with the predicted monoisotopic mass to handle the off-by-one-Da prediction error. Furthermore, we introduce a correction function to extract the “true” (i.e., theoretically) most-abundant isotope peak from a spectrum, even if the observed isotope distribution is distorted by noise or poor ion statistics. The method is available online as an R shiny app: https://valkenborg-lab.shinyapps.io/mind

Type III restriction endonucleases are heterotrimeric:comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA

Fundamental aspects of the biochemistry of Type III restriction endonucleases remain unresolved despite being characterized by numerous research groups in the past decades. One such feature is the subunit stoichiometry of these hetero-oligomeric enzyme complexes, which has important implications for the reaction mechanism. In this study, we present a series of results obtained by native mass spectrometry and size exclusion chromatography with multi-angle light scattering consistent with a 1:2 ratio of Res to Mod subunits in the EcoP15I, EcoPI and PstII complexes as the main holoenzyme species and a 1:1 stoichiometry of specific DNA (sDNA) binding by EcoP15I and EcoPI. Our data are also consistent with a model where ATP hydrolysis activated by recognition site binding leads to release of the enzyme from the site, dissociation from the substrate via a free DNA end and cleavage of the DNA. These results are discussed critically in the light of the published literature, aiming to resolve controversies and discuss consequences in terms of the reaction mechanism

Contactin-associated protein-2 antibodies in non-paraneoplastic cerebellar ataxia

Background Relatively few studies have searched for potentially pathogenic antibodies in non-paraneoplastic patients with cerebellar ataxia. Methods and Results We first screened sera from 52 idiopathic ataxia patients for binding of serum IgG antibodies to cerebellar neurons. One strong-binding serum was selected for immunoprecipitation and mass spectrometry, which resulted in the identification of contactin-associated protein 2 (CASPR2) as a major antigen. CASPR2 antibodies were then found by a cell-based assay in 9/88 (10%) ataxia patients, compared to 3/144 (2%) multiple sclerosis or dementia controls (p=0.011). CASPR2 is strongly expressed in the cerebellum, only partly in association with voltage-gated potassium channels. Conclusions Prospective studies are now needed to see whether identification of CASPR2 antibodies has relevance for the diagnosis and treatment of idiopathic cerebellar ataxia