Polycomb repressive complex 2 (PRC2) trimethylates histone H3 on K27 (H3K27me3) leading to gene silencing that is essential for embryonic development and maintenance of cell identity. PRC2 is regulated by protein cofactors and their crosstalk with histone modifications. Trimethylated histone H3 on K4 (H3K4me3) and K36 (H3K36me3) localize to sites of active transcription and inhibit PRC2 activity through unknown mechanisms. Using cryo-electron microscopy, we reveal that histone H3 tails containing H3K36me3 engage poorly with PRC2 and preclude its effective interaction with chromatin, while H3K4me3 binds to the allosteric site in the EED subunit, acting as an antagonist that competes with activators required for spreading of the H3K27me3 repressive mark. Thus, the location of the H3K4me3 and H3K36me3 modifications along the H3 tail allows them to target two requirements for efficient trimethylation of H3K27 by PRC2. We further show that the JARID2 cofactor modulates PRC2 activity in the presence of these histone modifications.

Polycomb repressive complex 2 (PRC2) is an epigenetic regulator that trimethylates lysine 27 of histone 3 (H3K27me3) and is essential for embryonic development and cellular differentiation. H3K27me3 is associated with transcriptionally repressed chromatin and is established when PRC2 is allosterically activated upon methyl-lysine binding by the regulatory subunit EED. Automethylation of the catalytic subunit enhancer of zeste homolog 2 (EZH2) stimulates its activity by an unknown mechanism. Here, we show that human PRC2 forms a dimer on chromatin in which an inactive, automethylated PRC2 protomer is the allosteric activator of a second PRC2 that is poised to methylate H3 of a substrate nucleosome. Functional assays support our model of allosteric trans-autoactivation via EED, suggesting a previously unknown mechanism mediating context-dependent activation of PRC2. Our work showcases the molecular mechanism of auto-modification-coupled dimerization in the regulation of chromatin-modifying complexes.

Streptavidin affinity grids provide strategies to overcome many commonly encountered cryo-electron microscopy (cryo-EM) sample preparation challenges, including sample denaturation and preferential orientations that can occur due to the air-water interface. Streptavidin affinity grids, however, are currently utilized by few cryo-EM labs because they are not commercially available and require a careful fabrication process. Two-dimensional streptavidin crystals are grown onto a biotinylated lipid monolayer that is applied directly to standard holey-carbon cryo-EM grids. The high-affinity interaction between streptavidin and biotin allows for the subsequent binding of biotinylated samples that are protected from the air-water interface during cryo-EM sample preparation. Additionally, these grids provide a strategy for concentrating samples available in limited quantities and purifying protein complexes of interest directly on the grids. Here, a step-by-step, optimized protocol is provided for the robust fabrication of streptavidin affinity grids for use in cryo-EM and negative-stain experiments. Additionally, a trouble-shooting guide is included for commonly experienced challenges to make the use of streptavidin affinity grids more accessible to the larger cryo-EM community.

Significance Ras GTPases, KRas, HRas, and NRas control cell proliferation via the Ras/Raf/MEK/ERK pathway through a process in which Ras dimerization is required but not mechanistically understood. Here, we show that Raf-RBD promotes Ras dimerization resulting in strong allosteric linkages between Galectin-binding residues on Raf-RBD at the two extreme ends of the dimer. This suggests that the dimer of the Ras/Raf–RBD complex couples to Galectin dimers to form a multivalent signaling complex for synchronized activation of Raf kinase, providing a new framework to be tested in the mechanistic understanding of Ras/Raf signaling.

Ras and Raf-kinase interact through the Ras-binding (RBD) and cysteine-rich domains (CRD) of Raf to signal through the mitogen-activated protein kinase pathway, yet the molecular mechanism leading to Raf activation has remained elusive. We present the 2.8 Å crystal structure of the HRas–CRaf-RBD_CRD complex showing the Ras–Raf interface as a continuous surface on Ras, as seen in the KRas–CRaf-RBD_CRD structure. In molecular dynamics simulations of a Ras dimer model formed through the α4–α5 interface, the CRD is dynamic and located between the two Ras protomers, poised for direct or allosteric modulation of functionally relevant regions of Ras and Raf. We propose a molecular model in which Ras binding is involved in the release of Raf autoinhibition while the Ras–Raf complex dimerizes to promote a platform for signal amplification, with Raf-CRD centrally located to impact regulation and function.

The Ras/Raf/MEK/ERK pathway is a major regulator of cell proliferation and is highly implicated in human cancers. Ras dimerization was first proposed thirty years ago, and it is known that Ras dimerization is essential for Raf activation, yet the mechanism through which this occurs has remained elusive. Attempts to detect Ras dimers on supported membranes have led to the conclusion that Ras does not dimerize in the absence of other factors. Here we show that the Ras binding domain of Raf (Raf‐RBD) is the key element prompting Ras dimerization. A small amount of Ras/Raf‐RBD dimer is seen in solution, while the addition of Raf‐RBD to Ras on supported lipid membranes produces robust levels of dimers. On the membrane, Ras/Raf‐RBD binding and Ras dimerization appear to be concerted events that lead to a high‐affinity signaling complex. Molecular dynamics simulations of Ras and Raf‐RBD alone, in a complex, and in the dimer reveal that the proteins become increasingly connected with higher levels of allosteric networks. This is apparent in the increasing connectivity between the helices involved in dimerization, helices 4 and 5, developing more consistent communication with the phosphate binding loop in the Ras active site. These data reveal a novel mode of regulation of the mitogenic Ras/Raf/MEK/ERK pathway.Support or Funding InformationNSF‐MCB‐1517295

The interaction between Ras and Raf kinase at the membrane promotes cell proliferation through the mitogen activated protein kinase (MAPK) pathway. Ras mutations drive 20% of all human cancers and despite great efforts, there are currently no drugs targeting Ras. Raf interacts with Ras via its two N‐terminal Ras‐binding domains: the Ras‐binding domain (RBD) and the cysteine‐rich domain. Binding of both the Raf‐RBD and CRD are required for Ras‐mediated activation of Raf kinase, however, the mechanism that results in the activation of the C‐terminal Raf kinase domain remains unknown. Here we present the 2.8 Å crystal structure of Ras in complex with a Raf construct containing both the RBD and CRD, revealing the interface for Raf‐CRD binding. In combination with molecular dynamic simulations, we identify allosteric effects induced by Raf‐CRD binding that stabilize the Ras active site to promote a Ras conformation poised for intrinsic hydrolysis.Support or Funding InformationNSF, MCB‐1517295. Northeastern Office of Undergraduate Research and Fellowships