Marc R. Wilkins

Disruptor of telomeric silencing 1 (Dot1p) is an exquisitely conserved histone methyltransferase and is the sole enzyme responsible for H3K79 methylation in the budding yeast Saccharomyces cerevisiae . It has been shown to be highly phosphorylated in vivo ; however, the upstream kinases that act on Dot1p are almost entirely unknown in yeast and all other eukaryotes. Here, we used in vitro and in vivo kinase discovery approaches to show that mitogen‐activated protein kinase HOG1 (Hog1p) is a bona fide kinase of the Dot1p methyltransferase. In vitro kinase assays showed that Hog1p phosphorylates Dot1p at multiple sites, including at several proline‐adjacent sites that are consistent with known Hog1p substrate preferences. The activity of Hog1p was specifically enhanced at these proline‐adjacent sites on Dot1p upon Hog1p activation by the osmostress‐responsive MAP kinase kinase PBS2 (Pbs2p). Genomic deletion of HOG1 reduced phosphorylation at specific sites on Dot1p in vivo , providing further evidence for Hog1p kinase activity on Dot1p in budding yeast cells. Phenotypic analysis of knockout and phosphosite mutant yeast strains revealed the importance of Hog1p‐catalysed phosphorylation of Dot1p for cellular responses to ultraviolet‐induced DNA damage. In mammalian systems, this kinase–substrate relationship was found to be conserved: human DOT1L (the ortholog of yeast Dot1p) can be phosphorylated by the proline‐directed kinase p38β (also known as MAPK11; the ortholog of yeast Hog1p) at multiple sites in vitro . Taken together, our findings establish Hog1p and p38β as newly identified upstream kinases of the Dot1p/DOT1L H3K79 methyltransferase enzymes in eukaryotes.

Significant recent advances in structural biology, particularly in the field of cryoelectron microscopy, have dramatically expanded our ability to create structural models of proteins and protein complexes. However, many proteins remain refractory to these approaches because of their low abundance, low stability, or—in the case of complexes—simply not having yet been analyzed. Here, we demonstrate the power of using cross-linking mass spectrometry (XL-MS) for the high-throughput experimental assessment of the structures of proteins and protein complexes. This included those produced by high-resolution but in vitro experimental data, as well as in silico predictions based on amino acid sequence alone. We present the largest XL-MS dataset to date, describing 28,910 unique residue pairs captured across 4,084 unique human proteins and 2,110 unique protein–protein interactions. We show that models of proteins and their complexes predicted by AlphaFold2, and inspired and corroborated by the XL-MS data, offer opportunities to deeply mine the structural proteome and interactome and reveal mechanisms underlying protein structure and function.

Isoprene was recently shown to act as an electron acceptor for a homoacetogenic bacterium. The focus of this study is the molecular basis for isoprene reduction.