No abstract

Methyl groups are powerful probes for the analysis of structure, dynamics and function of supramolecular assemblies, using both solution- and solid-state NMR. Widespread application of the methodology has been limited due to the challenges associated with assigning spectral resonances to specific locations within a biomolecule. Here, we present Methyl Assignment by Graph Matching (MAGMA), for the automatic assignment of methyl resonances. A graph matching protocol examines all possibilities for each resonance in order to determine an exact assignment that includes a complete description of any ambiguity. MAGMA gives 100% accuracy in confident assignments when tested against both synthetic data, and 9 cross-validated examples using both solution- and solid-state NMR data. We show that this remarkable accuracy enables a user to distinguish between alternative protein structures. In a drug discovery application on HSP90, we show the method can rapidly and efficiently distinguish between possible ligand binding modes. By providing an exact and robust solution to methyl resonance assignment, MAGMA can facilitate significantly accelerated studies of supramolecular machines using methyl-based NMR spectroscopy.

Deep learning-based approaches to protein structure prediction, such as AlphaFold2 and RoseTTAFold, can now define many protein structures with atomic-level accuracy. The AlphaFold Protein Structure Database (AFDB) contains a predicted structure for nearly every protein in the human proteome, including proteins that have intrinsically disordered regions (IDRs), which do not adopt a stable structure and rapidly interconvert between conformations. Although it is generally assumed that IDRs have very low AlphaFold2 confidence scores that reflect low-confidence structural predictions, we show here that AlphaFold2 assigns confident structures to nearly 15% of human IDRs. The amino-acid sequences of IDRs with high-confidence structures do not show significant similarity to the Protein Data Bank; instead, these IDR sequences exhibit a higher degree of positional amino-acid sequence conservation and are more enriched in charged and hydrophobic residues than IDRs with low-confidence structures. We compared the AlphaFold2 predictions to experimental NMR data for a subset of IDRs known to fold under specific conditions, finding that AlphaFold2 tends to capture the folded state structure. We note, however, that these AlphaFold2 predictions cannot detect functionally relevant structural plasticity within IDRs and cannot offer an ensemble representation of IDRs. Nevertheless, AlphaFold2 assigns high-confidence scores to about 60% of a set of 350 IDRs that have been reported to conditionally fold, suggesting that AlphaFold2 has learned to identify conditionally folded IDRs, which is unexpected, since IDRs were minimally represented in the training data. Leveraging this ability to discover IDRs that conditionally fold, we find that up to 80% of IDRs in archaea and bacteria are predicted to conditionally fold, but less than 20% of eukaryotic IDRs. Our results suggest that a large majority of IDRs in the proteomes of human and other eukaryotes would be expected to function in the absence of conditional folding.

The small heat-shock protein HSP27 is a redox-sensitive molecular chaperone that is expressed throughout the human body. Here we describe redox-induced changes to the structure, dynamics, and function of HSP27 and its conserved -crystallin domain, and provide the first structural characterization of a small heat-shock protein monomer. While HSP27 assembles into oligomers, we show that the transiently populated monomers released upon reduction are highly active chaperones in vitro, but are kinetically unstable and susceptible to uncontrolled aggregation. By using relaxation dispersion and high-pressure nuclear magnetic resonance spectroscopy, we reveal that the pair of strands that mediate dimerization become partially disordered in the monomer. Strikingly, we note that numerous HSP27 mutations associated with inherited neuropathies cluster to this unstructured region. The high degree of sequence conservation in the -crystallin domain amongst mammalian sHSPs suggests that partially unfolded monomers may be a general, functional feature of these molecular chaperones.

The small heat-shock protein HSP27 is a redox-sensitive molecular chaperone that is expressed throughout the human body. Here, we describe redox-induced changes to the structure, dynamics, and function of HSP27 and its conserved α-crystallin domain (ACD). While HSP27 assembles into oligomers, we show that the monomers formed upon reduction are highly active chaperones in vitro, but are susceptible to self-aggregation. By using relaxation dispersion and high-pressure nuclear magnetic resonance (NMR) spectroscopy, we observe that the pair of β-strands that mediate dimerisation partially unfold in the monomer. We note that numerous HSP27 mutations associated with inherited neuropathies cluster to this dynamic region. High levels of sequence conservation in ACDs from mammalian sHSPs suggest that the exposed, disordered interface present in free monomers or oligomeric subunits may be a general, functional feature of sHSPs.

In previous work, we showed that intrinsically disordered regions (IDRs) of proteins contain sequence-distributed molecular features that are conserved over evolution, despite little sequence similarity that can be detected in alignments (Zarin et al. 2019). Here, we aim to use these molecular features to predict specific biological functions for individual IDRs and identify the molecular features within them that are associated with these functions. We find that the predictable functions are diverse. Examining the associated molecular features, we note some that are consistent with previous reports, and identify others that were previously unknown. We experimentally confirm that elevated isoelectric point and hydrophobicity, features that are positively associated with mitochondrial localization, are necessary for mitochondrial targeting function. Remarkably, increasing isoelectric point in a synthetic IDR restores weak mitochondrial targeting. We believe feature analysis represents a new systematic approach to understand how biological functions of IDRs are specified by their protein sequences.

Bioinformatics and biophysical studies of intrinsically disordered proteins and regions (IDRs) note the high entropy at individual sequence positions and in conformations sampled in solution. This prevents application of the canonical sequence-structure-function paradigm to IDRs and motivates the development of new methods to extract information from IDR sequences. We argue that the information in IDR sequences cannot be fully revealed through positional conservation, which largely measures stable structural contacts and interaction motifs. Instead, considerations of evolutionary conservation of molecular features can reveal the full extent of information in IDRs. Experimental quantification of the large conformational entropy of IDRs is challenging but can be approximated through the extent of conformational sampling measured by a combination of NMR spectroscopy and lower-resolution structural biology techniques, which can be further interpreted with simulations. Conformational entropy and other biophysical features can be modulated by post-translational modifications that provide functional advantages to IDRs by tuning their energy landscapes and enabling a variety of functional interactions and modes of regulation. The diverse mosaic of functional states of IDRs and their conformational features within complexes demands novel metrics of information, which will reflect the complicated sequence-conformational ensemble-function relationship of IDRs.

Many membraneless organelles are thought to be biomolecular condensates formed by phase separation of proteins and other biopolymers. Post-translational modifications (PTMs) can impact protein phase separation behavior, although for many PTMs this aspect of their function is unknown. Olinked β-D-N-acetylglucosaminylation (O-GlcNAcylation) is an abundant form of intracellular glycosylation whose roles in regulating biomolecular condensate assembly and dynamics have not been delineated. Using an in vitro approach, we found that O-GlcNAcylation reduces the phase separation propensity of the EWS N-terminal low complexity region (LCR N ) under different conditions, including in the presence of the arginine-and glycine-rich RNA-binding domains (RBD). O-GlcNAcylation enhances fluorescence recovery after photobleaching (FRAP) within EWS LCR N condensates and causes the droplets to exhibit more liquid-like relaxation following fusion. Following extended incubation times, EWS LCR N +RBD condensates exhibit diminished FRAP, indicating a loss of fluidity, while condensates containing the O-GlcNAcylated LCR N do not. In HeLa cells, EWS is less O-GlcNAcylated following OGT knockdown, which correlates with its increased accumulation in a filter retardation assay. Relative to the human proteome, O-GlcNAcylated proteins are enriched with regions that are predicted to phase separate, suggesting a general role of O-GlcNAcylation in regulation of biomolecular condensates.