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The FK506‐binding protein 51 (FKBP51) is a promising target in a variety of disorders including depression, chronic pain, and obesity. Previous FKBP51‐targeting strategies were restricted to occupation of the FK506‐binding site, which does not affect core functions of FKBP51. Here, we report the discovery of the first FKBP51 proteolysis targeting chimera (PROTAC) that enables degradation of FKBP51 abolishing its scaffolding function. Initial synthesis of 220 FKBP‐focused PROTACs yielded a plethora of active PROTACs for FKBP12, six for FKBP51, and none for FKBP52. Structural analysis of a binary FKBP12:PROTAC complex revealed the molecular basis for negative cooperativity. Linker‐based optimization of first generation FKBP51 PROTACs led to the PROTAC SelDeg51 with improved cellular activity, selectivity, and high cooperativity. The structure of the ternary FKBP51:SelDeg51:VCB complex revealed how SelDeg51 establishes cooperativity by dimerizing FKBP51 and the von Hippel‐Lindau protein (VHL) in a glue‐like fashion. SelDeg51 efficiently depletes FKBP51 and reactivates glucocorticoid receptor (GR)‐signalling, highlighting the enhanced efficacy of full protein degradation compared to classical FKBP51 binding.

Human histone deacetylase 4 (HDAC4) is a key epigenetic regulator involved in a number of important cellular processes. This makes HDAC4 a promising target for the treatment of several cancers and neurodegenerative diseases, in particular Huntington's disease. HDAC4 is highly regulated by phosphorylation and oxidation, which determine its nuclear or cytosolic localization, and exerts its function through multiple interactions with other proteins, forming multiprotein complexes of varying composition. The catalytic domain of HDAC4 is known to interact with the SMRT/NCOR corepressor complex when the structural zinc‐binding domain (sZBD) is intact and forms a closed conformation. Crystal structures of the HDAC4 catalytic domain have been reported showing an open conformation of HDAC4 when bound to certain ligands. Here, we investigated the relevance of this HDAC4 conformation under physiological conditions in solution. We show that proper zinc chelation in the sZBD is essential for enzyme function. Loss of the structural zinc ion not only leads to a massive decrease in enzyme activity, but it also has serious consequences for the overall structural integrity and stability of the protein. However, the Zn²⁺ free HDAC4 structure in solution is incompatible with the open conformation. In solution, the open conformation of HDAC4 was also not observed in the presence of a variety of structurally divergent ligands. This suggests that the open conformation of HDAC4 cannot be induced in solution, and therefore cannot be exploited for the development of HDAC4‐specific inhibitors.

Pericentric heterochromatin (PCH) forms spatio-temporarily distinct compartments and affects chromosome organization and stability. Albeit some of its components are known, an elucidation of its proteome and how it differs between tissues in vivo is lacking. Here, we find that PCH compartments are dynamically organized in a tissue-specific manner, possibly reflecting compositional differences. As the mouse brain and liver exhibit very different PCH architecture, we isolated native PCH fractions from these tissues, analyzed their protein compositions using quantitative mass spectrometry, and compared them to identify common and tissue-specific PCH proteins. In addition to heterochromatin-enriched proteins, the PCH proteome includes RNA/transcription and membrane-related proteins, which showed lower abundance than PCH-enriched proteins. Thus, we applied a cut-off of PCH-unspecific candidates based on their abundance and validated PCH-enriched proteins. Amongst the hits, MeCP2 was classified into brain PCH-enriched proteins, while linker histone H1 was not. We found that H1 and MeCP2 compete to bind to PCH and regulate PCH organization in opposite ways. Altogether, our workflow of unbiased PCH isolation, quantitative mass spectrometry, and validation-based analysis allowed the identification of proteins that are common and tissue-specifically enriched at PCH. Further investigation of selected hits revealed their opposing role in heterochromatin higher-order architecture in vivo.

In recent years, there has been a tremendous evolution in the high‐throughput, tandem mass spectrometry‐based analysis of intact proteins, also known as top‐down proteomics (TDP). Both hardware and software have developed to the point that the technique has largely entered the mainstream, and large‐scale, ambitious, multi‐laboratory initiatives have started to make their appearance in the literature. For this, however, more convenient and robust data sharing and reuse will be required. Walzer et al. have created TopDownApp, a customisable, open platform for visualisation and analysis of TDP data, which they hope will be a step in this direction. As they point out, other benefits of such data sharing and interoperability would include reanalysis of published datasets, as well as the prospect of using large amounts of data to train machine learning algorithms. In time, this work could prove to be a valuable resource in the move towards a future of greater TDP data findability, accessibility, interoperability and reusability.

In order to understand protein structure to a sufficient extent for, e.g., drug discovery, no single technique can provide satisfactory information on both the lowest-energy conformation and on dynamic changes over time (the ‘four-dimensional’ protein structure). Instead, a combination of complementary techniques is required. Mass spectrometry methods have shown promise in addressing protein dynamics, but often rely on the use of high-end commercial or custom instruments. Here, we apply well-established chemistry to conformation-sensitive oxidative protein labelling on a timescale of a few seconds, followed by analysis through a routine protein analysis workflow. For a set of model proteins, we show that site selectivity of labelling can indeed be rationalised in terms of known structural information, and that conformational changes induced by ligand binding are reflected in the modification pattern. In addition to conventional bottom-up analysis, further insights are obtained from intact mass measurement and native mass spectrometry. We believe that this method will provide a valuable and robust addition to the ‘toolbox’ of mass spectrometry researchers studying higher-order protein structure.

In recent years, there has been a growing understanding that a significant fraction of the eukaryotic proteome is intrinsically disordered, and that these conformationally dynamic proteins play a myriad of vital biological roles in both normal and pathological states. In this review, selected examples of intrinsically disordered proteins are highlighted, with particular attention for a few which are relevant in neurological disorders and in viral infection. Next, the underlying causes for intrinsic disorder are discussed, along with computational methods used to predict whether a given amino acid sequence is likely to adopt a folded or unfolded state in solution. Finally, biophysical methods for the analysis of intrinsically disordered proteins will be discussed, as well as the unique challenges they pose in this context due to their highly dynamic nature.