The role of cryptic ancestral symmetry in histone folding mechanisms across Eukarya and Archaea.

Histones compact and store DNA in both Eukarya and Archaea, forming heterodimers in Eukarya and homodimers in Archaea. Despite this, the folding mechanism of histones across species remains unclear. Our study addresses this gap by investigating 11 types of histone and histone-like proteins across humans, Drosophila, and Archaea through multiscale molecular dynamics (MD) simulations, complemented by NMR and circular dichroism experiments. We confirm and elaborate on the widely applied "folding upon binding" mechanism of histone dimeric proteins and report a new alternative conformation, namely, the inverted non-native dimer, which may be a thermodynamically metastable configuration. Protein sequence analysis indicated that the inverted conformation arises from the hidden ancestral head-tail sequence symmetry underlying all histone proteins, which is congruent with the previously proposed histone evolution hypotheses. Finally, to explore the potential formations of homodimers in Eukarya, we utilized MD-based AWSEM and AI-based AlphaFold-Multimer models to predict their structures and conducted extensive all-atom MD simulations to examine their respective structural stabilities. Our results suggest that eukaryotic histones may also form stable homodimers, whereas their disordered tails bring significant structural asymmetry and tip the balance towards the formation of commonly observed heterotypic dimers.

Branched ubiquitin chain binding and deubiquitination by UCH37 facilitate proteasome clearance of stress-induced inclusions.

UCH37, also known as UCHL5, is a highly conserved deubiquitinating enzyme (DUB) that associates with the 26S proteasome. Recently, it was reported that UCH37 activity is stimulated by branched ubiquitin (Ub) chain architectures. To understand how UCH37 achieves its unique debranching specificity, we performed biochemical and Nuclear Magnetic Resonance (NMR) structural analyses and found that UCH37 is activated by contacts with the hydrophobic patches of both distal Ubs that emanate from a branched Ub. In addition, RPN13, which recruits UCH37 to the proteasome, further enhances branched-chain specificity by restricting linear Ub chains from having access to the UCH37 active site. In cultured human cells under conditions of proteolytic stress, we show that substrate clearance by the proteasome is promoted by both binding and deubiquitination of branched polyubiquitin by UCH37. Proteasomes containing UCH37(C88A), which is catalytically inactive, aberrantly retain polyubiquitinated species as well as the RAD23B substrate shuttle factor, suggesting a defect in recycling of the proteasome for the next round of substrate processing. These findings provide a foundation to understand how proteasome degradation of substrates modified by a unique Ub chain architecture is aided by a DUB.

Top-down analysis of novel synthetic branched proteins.

A strategy for top-down analysis of branched proteins has been reported earlier, which relies on electron transfer dissociation assisted by collisional activation, and software designed for graphic interpretation of tandem mass spectra and adapted for branched proteins. In the present study, the strategy is applied to identify unknown and novel products of reactions in which rationally mutated proteoforms of Rub1 are used to probe the selectivity of E1 and E2 enzymes normally active in ubiquitination. To test and demonstrate this application, components and attachment sites of three branched dimers are deduced and the mutations are confirmed.

Top-Down Analysis of Branched Proteins Using Mass Spectrometry.

Post-translational modifications by the covalent attachment of Rub1 (NEDD8), ubiquitin, SUMO, and other small signaling proteins have profound impacts on the functions and fates of cellular proteins. Investigations of the relationship of these bioactive structures and their functions are limited by analytical methods that are scarce and tedious. A novel strategy is reported here for the analysis of branched proteins by top-down mass spectrometry and illustrated by application to four recombinant proteins and one synthetic peptide modified by covalent bonds with ubiquitin or Rub1. The approach allows an analyte to be recognized as a branched protein; the participating proteins to be identified; the site of conjugation to be defined; and other chemical, native, and recombinant modifications to be characterized. In addition to the high resolution and high accuracy provided by the mass spectrometer, success is based on sample fragmentation by electron-transfer dissociation assisted by collisional activation and on software designed for graphic interpretation and adapted for branched proteins. The strategy allows for structures of unknown, two-component branched proteins to be elucidated directly the first time and can potentially be extended to more complex systems.

Alanine scan of core positions in ubiquitin reveals links between dynamics, stability, and function.

Mutations at solvent-inaccessible core positions in proteins can impact function through many biophysical mechanisms including alterations to thermodynamic stability and protein dynamics. As these properties of proteins are difficult to investigate, the impacts of core mutations on protein function are poorly understood for most systems. Here, we determined the effects of alanine mutations at all 15 core positions in ubiquitin on function in yeast. The majority (13 of 15) of alanine substitutions supported yeast growth as the sole ubiquitin. Both the two null mutants (I30A and L43A) were less stable to temperature-induced unfolding in vitro than wild type (WT) but were well folded at physiological temperatures. Heteronuclear NMR studies indicated that the L43A mutation reduces temperature stability while retaining a ground-state structure similar to WT. This structure enables L43A to bind to common ubiquitin receptors in vitro. Many of the core alanine ubiquitin mutants, including one of the null variants (I30A), exhibited an increased accumulation of high-molecular-weight species, suggesting that these mutants caused a defect in the processing of ubiquitin-substrate conjugates. In contrast, L43A exhibited a unique accumulation pattern with reduced levels of high-molecular-weight species and undetectable levels of free ubiquitin. When conjugation to other proteins was blocked, L43A ubiquitin accumulated as free ubiquitin in yeast. Based on these findings, we speculate that ubiquitin's stability to unfolding may be required for efficient recycling during proteasome-mediated substrate degradation.