Molecular basis for the methylation specificity of ATXR5 for histone H3.

In plants, the histone H3.1 lysine 27 (H3K27) mono-methyltransferases ARABIDOPSIS TRITHORAX RELATED PROTEIN 5 and 6 (ATXR5/6) regulate heterochromatic DNA replication and genome stability. Our initial studies showed that ATXR5/6 discriminate between histone H3 variants and preferentially methylate K27 on H3.1. In this study, we report three regulatory mechanisms contributing to the specificity of ATXR5/6. First, we show that ATXR5 preferentially methylates the R/F-K*-S/C-G/A-P/C motif with striking preference for hydrophobic and aromatic residues in positions flanking this core of five amino acids. Second, we demonstrate that post-transcriptional modifications of residues neighboring K27 that are typically associated with actively transcribed chromatin are detrimental to ATXR5 activity. Third, we show that ATXR5 PHD domain employs a narrow binding pocket to selectively recognize unmethylated K4 of histone H3. Finally, we demonstrate that deletion or mutation of the PHD domain reduces the catalytic efficiency (kcat/Km of AdoMet) of ATXR5 up to 58-fold, highlighting the multifunctional nature of ATXR5 PHD domain. Overall, our results suggest that several molecular determinants regulate ATXR5/6 methyltransferase activity and epigenetic inheritance of H3.1 K27me1 mark in plants.

Keeping them all together: ß-propeller domains in histone methyltransferase complexes.

Histone methyltransferases (HKMTs) residing in multi-subunit protein complexes frequently require the presence of ß-propeller proteins to achieve their biological functions. Recent biochemical studies have highlighted the functional diversity of these scaffolding proteins in maintaining the integrity of the complexes, allosterically regulating HKMT enzymatic activity and acting as "histone tethering devices" to facilitate the interaction between HKMTs and their substrates. Structural studies have revealed that, while ß-propeller domain proteins share structural similarity, they employ divergent mechanisms to achieve their functions. This review focuses on the progress made in the last decade to identify the biochemical determinants underlying the functions of these important proteins.

Selective methylation of histone H3 variant H3.1 regulates heterochromatin replication.

Histone variants have been proposed to act as determinants for posttranslational modifications with widespread regulatory functions. We identify a histone-modifying enzyme that selectively methylates the replication-dependent histone H3 variant H3.1. The crystal structure of the SET domain of the histone H3 lysine-27 (H3K27) methyltransferase ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) in complex with a H3.1 peptide shows that ATXR5 contains a bipartite catalytic domain that specifically "reads" alanine-31 of H3.1. Variation at position 31 between H3.1 and replication-independent H3.3 is conserved in plants and animals, and threonine-31 in H3.3 is responsible for inhibiting the activity of ATXR5 and its paralog, ATXR6. Our results suggest a simple model for the mitotic inheritance of the heterochromatic mark H3K27me1 and the protection of H3.3-enriched genes against heterochromatization during DNA replication.

The many facets of MLL1 regulation.

In the last 20 years, we have witnessed an exponential number of evidences linking the human mixed lineage leukemia-1 (MLL1) gene to several acute and myelogenous leukemias. MLL1 is one of the founding members of the SET1 family of lysine methyltransferases and is key for the proper control of developmentally regulated gene expression. MLL1 is a structurally complex protein composed of several functional domains. These domains play pivotal roles for the recruitment of regulatory proteins. These MLL1 regulatory proteins (MRPs) dynamically interact with MLL1 and consequently control gene expression. In this review, we summarize recent structural and functional studies of MRPs and discuss emergent structural paradigms for the control of MLL1 activity.

Structural insights into the assembly of large oligomeric signalosomes in the Toll-like receptor-interleukin-1 receptor superfamily.

The Toll-like receptor (TLR)-interleukin 1 receptor (IL-1R) superfamily plays fundamentally important roles in innate immune and inflammatory responses. Structural studies have begun to show that upon ligand stimulation, TLRs and IL-1Rs assemble large oligomeric intracellular signaling complexes, or "signalosomes," to induce the activation of kinases and E3 ubiquitin ligases, leading eventually to the activation of the transcription factors that are responsible for the expression of genes whose products mediate immune and inflammatory responses. The different scaffolds identified by these structural studies provide a molecular foundation for understanding the formation of microscopically visible signaling clusters that have long been known to cell biologists. Here, we illustrate the potential mechanisms of step-by-step assembly from the membrane-proximal interactions to the more downstream events. Formation of large oligomeric signalosomes may help to establish a digital threshold response in TLR and IL-1R signaling.

The cytoplasmic adaptor protein Dok7 activates the receptor tyrosine kinase MuSK via dimerization.

Formation of the vertebrate neuromuscular junction requires, among others proteins, Agrin, a neuronally derived ligand, and the following muscle proteins: LRP4, the receptor for Agrin; MuSK, a receptor tyrosine kinase (RTK); and Dok7 (or Dok-7), a cytoplasmic adaptor protein. Dok7 comprises a pleckstrin-homology (PH) domain, a phosphotyrosine-binding (PTB) domain, and C-terminal sites of tyrosine phosphorylation. Unique among adaptor proteins recruited to RTKs, Dok7 is not only a substrate of MuSK, but also an activator of MuSK's kinase activity. Here, we present the crystal structure of the Dok7 PH-PTB domains in complex with a phosphopeptide representing the Dok7-binding site on MuSK. The structure and biochemical data reveal a dimeric arrangement of Dok7 PH-PTB that facilitates trans-autophosphorylation of the kinase activation loop. The structure provides the molecular basis for MuSK activation by Dok7 and for rationalizing several Dok7 loss-of-function mutations found in patients with congenital myasthenic syndromes.

Structural basis for phosphotyrosine recognition by suppressor of cytokine signaling-3.

Suppressor of cytokine signaling (SOCS) proteins are indispensable negative regulators of cytokine-stimulated Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathways. SOCS proteins (SOCS1-7 and CIS) consist of a variable N-terminal region, a central Src homology-2 (SH2) domain, and a C-terminal SOCS box. The N-terminal region in SOCS1 and SOCS3 includes the so-called kinase inhibitory region that has been shown to inhibit the catalytic activity of JAK2. Here, we present a crystal structure at 2.0 A resolution of the N-terminally extended SH2 domain of SOCS3 in complex with its phosphopeptide target on the cytokine receptor gp130. The structure reveals that major insertions in the EF and BG loops of the SOCS3 SH2 domain are responsible for binding to gp130 with high affinity and specificity. In addition, the structure provides insights into the possible mechanisms by which SOCS3 and SOCS1 inhibit JAK2 kinase activity.