Structure and Conformational Dynamics of a COMPASS Histone H3K4 Methyltransferase Complex.

The methylation of histone 3 lysine 4 (H3K4) is carried out by an evolutionarily conserved family of methyltransferases referred to as complex of proteins associated with Set1 (COMPASS). The activity of the catalytic SET domain (su(var)3-9, enhancer-of-zeste, and trithorax) is endowed through forming a complex with a set of core proteins that are widely shared from yeast to humans. We obtained cryo-electron microscopy (cryo-EM) maps of the yeast Set1/COMPASS core complex at overall 4.0- to 4.4-Å resolution, providing insights into its structural organization and conformational dynamics. The Cps50 C-terminal tail weaves within the complex to provide a central scaffold for assembly. The SET domain, snugly positioned at the junction of the Y-shaped complex, is extensively contacted by Cps60 (Bre2), Cps50 (Swd1), and Cps30 (Swd3). The mobile SET-I motif of the SET domain is engaged by Cps30, explaining its key role in COMPASS catalytic activity toward higher H3K4 methylation states.

Acetylation of PCNA Sliding Surface by Eco1 Promotes Genome Stability through Homologous Recombination.

During DNA replication, proliferating cell nuclear antigen (PCNA) adopts a ring-shaped structure to promote processive DNA synthesis, acting as a sliding clamp for polymerases. Known posttranslational modifications function at the outer surface of the PCNA ring to favor DNA damage bypass. Here, we demonstrate that acetylation of lysine residues at the inner surface of PCNA is induced by DNA lesions. We show that cohesin acetyltransferase Eco1 targets lysine 20 at the sliding surface of the PCNA ring in vitro and in vivo in response to DNA damage. Mimicking constitutive acetylation stimulates homologous recombination and robustly suppresses the DNA damage sensitivity of mutations in damage tolerance pathways. In comparison to the unmodified trimer, structural differences are observed at the interface between protomers in the crystal structure of the PCNA-K20ac ring. Thus, acetylation regulates PCNA sliding on DNA in the presence of DNA damage, favoring homologous recombination linked to sister-chromatid cohesion.

A phosphorylation switch on RbBP5 regulates histone H3 Lys4 methylation.

The methyltransferase activity of the trithorax group (TrxG) protein MLL1 found within its COMPASS (complex associated with SET1)-like complex is allosterically regulated by a four-subunit complex composed of WDR5, RbBP5, Ash2L, and DPY30 (also referred to as WRAD). We report structural evidence showing that in WRAD, a concave surface of the Ash2L SPIa and ryanodine receptor (SPRY) domain binds to a cluster of acidic residues, referred to as the D/E box, in RbBP5. Mutational analysis shows that residues forming the Ash2L/RbBP5 interface are important for heterodimer formation, stimulation of MLL1 catalytic activity, and erythroid cell terminal differentiation. We also demonstrate that a phosphorylation switch on RbBP5 stimulates WRAD complex formation and significantly increases KMT2 (lysine [K] methyltransferase 2) enzyme methylation rates. Overall, our findings provide structural insights into the assembly of the WRAD complex and point to a novel regulatory mechanism controlling the activity of the KMT2/COMPASS family of lysine methyltransferases.

Functional and Structural Characterization of OXA-935, a Novel OXA-10-Family ß-Lactamase from Pseudomonas aeruginosa.

Resistance to antipseudomonal penicillins and cephalosporins is often driven by the overproduction of the intrinsic ß-lactamase AmpC. However, OXA-10-family ß-lactamases are a rich source of resistance in Pseudomonas aeruginosa. OXA ß-lactamases have a propensity for mutation that leads to extended spectrum cephalosporinase and carbapenemase activity. In this study, we identified isolates from a subclade of the multidrug-resistant (MDR) high risk P. aeruginosa clonal complex CC446 with a resistance to ceftazidime. A genomic analysis revealed that these isolates harbored a plasmid containing a novel allele of blaOXA-10, named blaOXA-935, which was predicted to produce an OXA-10 variant with two amino acid substitutions: an aspartic acid instead of a glycine at position 157 and a serine instead of a phenylalanine at position 153. The G157D mutation, present in OXA-14, is associated with the resistance of P. aeruginosa to ceftazidime. Compared to OXA-14, OXA-935 showed increased catalytic efficiency for ceftazidime. The deletion of blaOXA-935 restored the sensitivity to ceftazidime, and susceptibility profiling of P. aeruginosa laboratory strains expressing blaOXA-935 revealed that OXA-935 conferred ceftazidime resistance. To better understand the impacts of the variant amino acids, we determined the crystal structures of OXA-14 and OXA-935. Compared to OXA-14, the F153S mutation in OXA-935 conferred increased flexibility in the omega (Ω) loop. Amino acid changes that confer extended spectrum cephalosporinase activity to OXA-10-family ß-lactamases are concerning, given the rising reliance on novel ß-lactam/ß-lactamase inhibitor combinations, such as ceftolozane-tazobactam and ceftazidime-avibactam, to treat MDR P. aeruginosa infections.

Structural basis of Fusarium myosin I inhibition by phenamacril.

Fusarium is a genus of filamentous fungi that includes species that cause devastating diseases in major staple crops, such as wheat, maize, rice, and barley, resulting in severe yield losses and mycotoxin contamination of infected grains. Phenamacril is a novel fungicide that is considered environmentally benign due to its exceptional specificity; it inhibits the ATPase activity of the sole class I myosin of only a subset of Fusarium species including the major plant pathogens F. graminearum, F. asiaticum and F. fujikuroi. To understand the underlying mechanisms of inhibition, species specificity, and resistance mutations, we have determined the crystal structure of phenamacril-bound F. graminearum myosin I. Phenamacril binds in the actin-binding cleft in a new allosteric pocket that contains the central residue of the regulatory Switch 2 loop and that is collapsed in the structure of a myosin with closed actin-binding cleft, suggesting that pocket occupancy blocks cleft closure. We have further identified a single, transferable phenamacril-binding residue found exclusively in phenamacril-sensitive myosins to confer phenamacril selectivity.

Structural basis of binding and inhibition of ornithine decarboxylase by 1-amino-oxy-3-aminopropane.

Ornithine decarboxylase (ODC) is the rate-limiting enzyme for the synthesis of polyamines (PAs). PAs are oncometabolites that are required for proliferation, and pharmaceutical ODC inhibition is pursued for the treatment of hyperproliferative diseases, including cancer and infectious diseases. The most potent ODC inhibitor is 1-amino-oxy-3-aminopropane (APA). A previous crystal structure of an ODC-APA complex indicated that APA non-covalently binds ODC and its cofactor pyridoxal 5-phosphate (PLP) and functions by competing with the ODC substrate ornithine for binding to the catalytic site. We have revisited the mechanism of APA binding and ODC inhibition through a new crystal structure of APA-bound ODC, which we solved at 2.49 Šresolution. The structure unambiguously shows the presence of a covalent oxime between APA and PLP in the catalytic site, which we confirmed in solution by mass spectrometry. The stable oxime makes extensive interactions with ODC but cannot be catabolized, explaining APA's high potency in ODC inhibition. In addition, we solved an ODC/PLP complex structure with citrate bound at the substrate-binding pocket. These two structures provide new structural scaffolds for developing more efficient pharmaceutical ODC inhibitors.

A non-canonical monovalent zinc finger stabilizes the integration of Cfp1 into the H3K4 methyltransferase complex COMPASS.

COMPlex ASsociating with SET1 (COMPASS) is a histone H3 Lys-4 methyltransferase that typically marks the promoter region of actively transcribed genes. COMPASS is a multi-subunit complex in which the catalytic unit, SET1, is required for H3K4 methylation. An important subunit known to regulate SET1 methyltransferase activity is the CxxC zinc finger protein 1 (Cfp1). Cfp1 binds to COMPASS and is critical to maintain high level of H3K4me3 in cells but the mechanisms underlying its stimulatory activity is poorly understood. In this study, we show that Cfp1 only modestly activates COMPASS methyltransferase activity in vitro. Binding of Cfp1 to COMPASS is in part mediated by a new type of monovalent zinc finger (ZnF). This ZnF interacts with the COMPASS's subunits RbBP5 and disruption of this interaction blunts its methyltransferase activity in cells and in vivo. Collectively, our studies reveal that a novel form of ZnF on Cfp1 enables its integration into COMPASS and contributes to epigenetic signaling.

Structures of AMP-activated protein kinase bound to novel pharmacological activators in phosphorylated, non-phosphorylated, and nucleotide-free states.

AMP-activated protein kinase (AMPK) is an attractive therapeutic target for managing metabolic diseases. A class of pharmacological activators, including Merck 991, binds the AMPK ADaM site, which forms the interaction surface between the kinase domain (KD) of the α-subunit and the carbohydrate-binding module (CBM) of the ß-subunit. Here, we report the development of two new 991-derivative compounds, R734 and R739, which potently activate AMPK in a variety of cell types, including ß2-specific skeletal muscle cells. Surprisingly, we found that they have only minor effects on direct kinase activity of the recombinant α1ß2γ1 isoform yet robustly enhance protection against activation loop dephosphorylation. This mode of activation is reminiscent of that of ADP, which activates AMPK by binding to the nucleotide-binding sites in the γ-subunit, more than 60 Å away from the ADaM site. To understand the mechanisms of full and partial AMPK activation, we determined the crystal structures of fully active phosphorylated AMPK α1ß1γ1 bound to AMP and R734/R739 as well as partially active nonphosphorylated AMPK bound to R734 and AMP and phosphorylated AMPK bound to R734 in the absence of added nucleotides at <3-Å resolution. These structures and associated analyses identified a novel conformational state of the AMPK autoinhibitory domain associated with partial kinase activity and provide new insights into phosphorylation-dependent activation loop stabilization in AMPK.

Serine-47 phosphorylation of cytochrome c in the mammalian brain regulates cytochrome c oxidase and caspase-3 activity.

Cytochrome c (Cytc) is a multifunctional protein that operates as an electron carrier in the mitochondrial electron transport chain and plays a key role in apoptosis. We have previously shown that tissue-specific phosphorylations of Cytc in the heart, liver, and kidney play an important role in the regulation of cellular respiration and cell death. Here, we report that Cytc purified from mammalian brain is phosphorylated on S47 and that this phosphorylation is lost during ischemia. We have characterized the functional effects in vitro using phosphorylated Cytc purified from pig brain tissue and a recombinant phosphomimetic mutant (S47E). We crystallized S47E phosphomimetic Cytc at 1.55 Å and suggest that it spatially matches S47-phosphorylated Cytc, making it a good model system. Both S47-phosphorylated and phosphomimetic Cytc showed a lower oxygen consumption rate in reaction with isolated Cytc oxidase, which we propose maintains intermediate mitochondrial membrane potentials under physiologic conditions, thus minimizing production of reactive oxygen species. S47-phosphorylated and phosphomimetic Cytc showed lower caspase-3 activity. Furthermore, phosphomimetic Cytc had decreased cardiolipin peroxidase activity and is more stable in the presence of H2O2. Our data suggest that S47 phosphorylation of Cytc is tissue protective and promotes cell survival in the brain.-Kalpage, H. A., Vaishnav, A., Liu, J., Varughese, A., Wan, J., Turner, A. A., Ji, Q., Zurek, M. P., Kapralov, A. A., Kagan, V. E., Brunzelle, J. S., Recanati, M.-A., Grossman, L. I., Sanderson, T. H., Lee, I., Salomon, A. R., Edwards, B. F. P, Hüttemann, M. Serine-47 phosphorylation of cytochrome c in the mammalian brain regulates cytochrome c oxidase and caspase-3 activity.

Structural basis for molecular recognition of folic acid by folate receptors.

Folate receptors (FRα, FRß and FRγ) are cysteine-rich cell-surface glycoproteins that bind folate with high affinity to mediate cellular uptake of folate. Although expressed at very low levels in most tissues, folate receptors, especially FRα, are expressed at high levels in numerous cancers to meet the folate demand of rapidly dividing cells under low folate conditions. The folate dependency of many tumours has been therapeutically and diagnostically exploited by administration of anti-FRα antibodies, high-affinity antifolates, folate-based imaging agents and folate-conjugated drugs and toxins. To understand how folate binds its receptors, we determined the crystal structure of human FRα in complex with folic acid at 2.8 Å resolution. FRα has a globular structure stabilized by eight disulphide bonds and contains a deep open folate-binding pocket comprised of residues that are conserved in all receptor subtypes. The folate pteroate moiety is buried inside the receptor, whereas its glutamate moiety is solvent-exposed and sticks out of the pocket entrance, allowing it to be conjugated to drugs without adversely affecting FRα binding. The extensive interactions between the receptor and ligand readily explain the high folate-binding affinity of folate receptors and provide a template for designing more specific drugs targeting the folate receptor system.

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.

Structural Determinants of the Stereoinverting Activity of Pseudomonas stutzeri d-Phenylglycine Aminotransferase.

Aromatic d-amino acids are key precursors for the production of many small molecule therapeutics. Therefore, the development of biocatalytic methods for their synthesis is of great interest. An enzyme that has great potential as a biocatalyst for the synthesis of d-amino acids is the stereoinverting d-phenylglycine aminotransferase (DPAT) from Pseudomonas stutzeri ST-201. This enzyme catalyzes a unique l to d transamination reaction that produces d-phenylglycine and α-ketoglutarate from benzoylformate and l-glutamate, via a mechanism that is poorly understood. Here, we present the crystal structure of DPAT, which shows that the enzyme folds into a two-domain structure representative of class III aminotransferases. Guided by the crystal structure, we performed saturation mutagenesis to probe the substrate binding pockets of the enzyme. These experiments helped us identify two arginine residues (R34 and R407), one in each binding pocket, that are essential to catalysis. Together with kinetic analyses using a library of amino acid substrates, our mutagenesis and structural studies allow us to propose a binding model that explains the dual l/d specificity of DPAT. Our kinetic analyses also demonstrate that DPAT can catalyze the transamination of ß- and γ-amino acids, reclassifying this enzyme as an ω-aminotransferase. Collectively, our studies highlight that the DPAT active site is amenable to protein engineering for expansion of its substrate scope, which offers the opportunity to generate new biocatalysts for the synthesis of a variety of valuable optically pure d-amino acids from inexpensive and abundant l-amino acids.

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE.

Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr28, leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨm), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr28 in vivo We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via "controlled respiration," preventing ΔΨm hyperpolarization, a known cause of ROS and trigger of apoptosis.

Ligand Binding Enhances Millisecond Conformational Exchange in Xylanase B2 from Streptomyces lividans.

Xylanases catalyze the hydrolysis of xylan, an abundant carbon and energy source with important commercial ramifications. Despite tremendous efforts devoted to the catalytic improvement of xylanases, success remains limited because of our relatively poor understanding of their molecular properties. Previous reports suggested the potential role of atomic-scale residue dynamics in modulating the catalytic activity of GH11 xylanases; however, dynamics in these studies was probed on time scales orders of magnitude faster than the catalytic time frame. Here, we used nuclear magnetic resonance titration and relaxation dispersion experiments ((15)N-CPMG) in combination with X-ray crystallography and computational simulations to probe conformational motions occurring on the catalytically relevant millisecond time frame in xylanase B2 (XlnB2) and its catalytically impaired mutant E87A from Streptomyces lividans 66. Our results show distinct dynamical properties for the apo and ligand-bound states of the enzymes. The apo form of XlnB2 experiences conformational exchange for residues in the fingers and palm regions of the catalytic cleft, while the catalytically impaired E87A variant displays millisecond dynamics only in the fingers, demonstrating the long-range effect of the mutation on flexibility. Ligand binding induces enhanced conformational exchange of residues interacting with the ligand in the fingers and thumb loop regions, emphasizing the potential role of residue motions in the fingers and thumb loop regions for recognition, positioning, processivity, and/or stabilization of ligands in XlnB2. To the best of our knowledge, this work represents the first experimental characterization of millisecond dynamics in a GH11 xylanase family member. These results offer new insights into the potential role of conformational exchange in GH11 enzymes, providing essential dynamic information to help improve protein engineering and design applications.

Structural and functional study of D-glucuronyl C5-epimerase.

Heparan sulfate (HS) is a glycosaminoglycan present on the cell surface and in the extracellular matrix, which interacts with diverse signal molecules and is essential for many physiological processes including embryonic development, cell growth, inflammation, and blood coagulation. D-glucuronyl C5-epimerase (Glce) is a crucial enzyme in HS synthesis, converting D-glucuronic acid to L-iduronic acid to increase HS flexibility. This modification of HS is important for protein ligand recognition. We have determined the crystal structures of Glce in apo-form (unliganded) and in complex with heparin hexasaccharide (product of Glce following O-sulfation), both in a stable dimer conformation. A Glce dimer contains two catalytic sites, each at a positively charged cleft in C-terminal α-helical domains binding one negatively charged hexasaccharide. Based on the structural and mutagenesis studies, three tyrosine residues, Tyr(468), Tyr(528), and Tyr(546), in the active site were found to be crucial for the enzymatic activity. The complex structure also reveals the mechanism of product inhibition (i.e. 2-O- and 6-O-sulfation of HS keeps the C5 carbon of L-iduronic acid away from the active-site tyrosine residues). Our structural and functional data advance understanding of the key modification in HS biosynthesis.

Agonist ligands mediate the transcriptional response of nuclear receptor heterodimers through distinct stoichiometric assemblies with coactivators.

The constitutive androstane (CAR) and retinoid X receptors (RXR) are ligand-mediated transcription factors of the nuclear receptor protein superfamily. Functional CAR:RXR heterodimers recruit coactivator proteins, such as the steroid receptor coactivator-1 (SRC1). Here, we show that agonist ligands can potentiate transactivation through both coactivator binding sites on CAR:RXR, which distinctly bind two SRC1 molecules. We also observe that SRC1 transitions from a structurally plastic to a compact form upon binding CAR:RXR. Using small angle x-ray scattering (SAXS) we show that the CAR(tcp):RXR(9c)·SRC1 complex can encompass two SRC1 molecules compared with the CAR(tcp):RXR·SRC1, which binds only a single SRC1. Moreover, sedimentation coefficients and molecular weights determined by analytical ultracentrifugation confirm the SAXS model. Cell-based transcription assays show that disrupting the SRC1 binding site on RXR alters the transactivation by CAR:RXR. These data suggest a broader role for RXR within heterodimers, whereas offering multiple strategies for the assembly of the transcription complex.

Structure and regulon of Campylobacter jejuni ferric uptake regulator Fur define apo-Fur regulation.

The full regulatory potential of the ferric uptake regulator (Fur) family of proteins remains undefined despite over 20 years of study. We report herein an integrated approach that combines both genome-wide technologies and structural studies to define the role of Fur in Campylobacter jejuni (Cj). CjFur ChIP-chip assays identified 95 genomic loci bound by CjFur associated with functions as diverse as iron acquisition, flagellar biogenesis, and non-iron ion transport. Comparative analysis with transcriptomic data revealed that CjFur regulation extends beyond solely repression and also includes both gene activation and iron-independent regulation. Computational analysis revealed the presence of an elongated holo-Fur repression motif along with a divergent holo-Fur activation motif. This diversity of CjFur DNA-binding elements is supported by the crystal structure of CjFur, which revealed a unique conformation of its DNA-binding domain and the absence of metal in the regulatory site. Strikingly, our results indicate that the apo-CjFur structure retains the canonical V-shaped dimer reminiscent of previously characterized holo-Fur proteins enabling DNA interaction. This conformation stems from a structurally unique hinge domain that is poised to further contribute to CjFur's regulatory functions by modulating the orientation of the DNA-binding domain upon binding of iron. The unique features of the CjFur crystal structure rationalize the binding sequence diversity that was uncovered during ChIP-chip analysis and defines apo-Fur regulation.

Structural basis for negative cooperativity within agonist-bound TR:RXR heterodimers.

Thyroid hormones such as 3,3',5 triiodo-L-thyronine (T3) control numerous aspects of mammalian development and metabolism. The actions of such hormones are mediated by specific thyroid hormone receptors (TRs). TR belongs to the nuclear receptor family of modular transcription factors that binds to specific DNA-response elements within target promoters. These receptors can function as homo- or heterodimers such as TR:9-cis retinoic acid receptor (RXR). Here, we present the atomic resolution structure of the TRα•T3:RXRα•9-cis retinoic acid (9c) ligand binding domain heterodimer complex at 2.95 Å along with T3 hormone binding and dissociation and coactivator binding studies. Our data provide a structural basis for allosteric communication between T3 and 9c and negative cooperativity between their binding pockets. In this structure, both TR and RXR are in the active state conformation for optimal binding to coactivator proteins. However, the structure of TR•T3 within TR•T3:RXR•9c is in a relative state of disorder, and the observed kinetics of binding show that T3 dissociates more rapidly from TR•T3:RXR•9c than from TR•T3:RXR. Also, coactivator binding studies with a steroid receptor coactivator-1 (receptor interaction domains 1-3) fragment show lower affinities (K(a)) for TR•T3:RXR•9c than TR•T3:RXR. Our study corroborates previously reported observations from cell-based and binding studies and offers a structural mechanism for the repression of TR•T3:RXR transactivation by RXR agonists. Furthermore, the recent discoveries of multiple endogenous RXR agonists that mediate physiological tasks such as lipid biosynthesis underscore the pharmacological importance of negative cooperativity in ligand binding within TR:RXR heterodimers.

The plasticity of WDR5 peptide-binding cleft enables the binding of the SET1 family of histone methyltransferases.

In mammals, the SET1 family of lysine methyltransferases (KMTs), which includes MLL1-5, SET1A and SET1B, catalyzes the methylation of lysine-4 (Lys-4) on histone H3. Recent reports have demonstrated that a three-subunit complex composed of WD-repeat protein-5 (WDR5), retinoblastoma-binding protein-5 (RbBP5) and absent, small, homeotic disks-2-like (ASH2L) stimulates the methyltransferase activity of MLL1. On the basis of studies showing that this stimulation is in part controlled by an interaction between WDR5 and a small region located in close proximity of the MLL1 catalytic domain [referred to as the WDR5-interacting motif (Win)], it has been suggested that WDR5 might play an analogous role in scaffolding the other SET1 complexes. We herein provide biochemical and structural evidence showing that WDR5 binds the Win motifs of MLL2-4, SET1A and SET1B. Comparative analysis of WDR5-Win complexes reveals that binding of the Win motifs is achieved by the plasticity of WDR5 peptidyl-arginine-binding cleft allowing the C-terminal ends of the Win motifs to be maintained in structurally divergent conformations. Consistently, enzymatic assays reveal that WDR5 plays an important role in the optimal stimulation of MLL2-4, SET1A and SET1B methyltransferase activity by the RbBP5-ASH2L heterodimer. Overall, our findings illustrate the function of WDR5 in scaffolding the SET1 family of KMTs and further emphasize on the important role of WDR5 in regulating global histone H3 Lys-4 methylation.

Structural insights into an atypical histone binding mechanism by a PHD finger.

Complex associating with SET1 (COMPASS) is a histone H3K4 tri-methyltransferase controlled by several regulatory subunits including CXXC zinc finger protein 1 (Cfp1). Prior studies established the structural underpinnings controlling H3K4me3 recognition by the PHD domain of Cfp1's yeast homolog (Spp1). However, metazoans Cfp1PHD lacks structural elements important for H3K4me3 stabilization in Spp1, suggesting that in metazoans, Cfp1PHD domain binds H3K4me3 differently. The structure of Cfp1PHD in complex with H3K4me3 shows unique features such as non-canonical coordination of the first zinc atom and a disulfide bond forcing the reorientation of Cfp1PHD N-terminus, thereby leading to an atypical H3K4me3 binding pocket. This configuration minimizes Cfp1PHD reliance on canonical residues important for histone binding functions of other PHD domains. Cancer-related mutations in Cfp1PHD impair H3K4me3 binding, implying a potential impact on epigenetic signaling. Our work highlights a potential diversification of PHD histone binding modes and the impact of cancer mutations on Cfp1 functions.