24 research outputs found
Biochemistry of the Mixed Lineage Leukemia 1 (MLL1) Protein and Targeted Therapies for Associated Leukemia
A novel non-SET domain multi-subunit methyltransferase required for sequential nucleosomal histone H3 methylation by the mixed lineage leukemia protein-1 (MLL1) core complex.
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A Steric āBall-and-Chainā Mechanism for pH-Mediated Regulation of Gap Junction Channels
Gap junction channels (GJCs) mediate intercellular communication and are gated by numerous conditions such as pH. The electron cryomicroscopy (cryo-EM) structure of Cx26 GJC at physiological pH recapitulates previous GJC structures in lipid bilayers. At pH 6.4, we identify two conformational states, one resembling the open physiological-pH structure and a closed conformation that displays six threads of density, that join to form a pore-occluding density. Crosslinking and hydrogen-deuterium exchange mass spectrometry reveal closer association between the N-terminal (NT) domains and the cytoplasmic loops (CL) at acidic pH. Previous electrophysiologic studies suggest an association between NT residue N14 and H100 near M2, which may trigger the observed movement of M2 toward M1 in our cryo-EM maps, thereby accounting for additional NT-CL crosslinks at acidic pH. We propose that these pH-induced interactions and conformational changes result in extension, ordering, and association of the acetylated NT domains to form a hexameric āball-and-chainā gating particle.
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ā¢pH gating of human Cx26 gap junction channels occurs through a āball-and-chainā mechanismā¢Gating involves cooperation of all subunits within each hexamer of the junctional channelā¢In acidic pH, acetylated N termini favor an extended conformation that occludes the pore
During tissue injury, acidic pH blocks gap junction channel intercellular communication. Khan etĀ al. use cryo-EM and mass spectrometry to show that acidification causes extension, ordering, and association of the N-terminal domains to form a hexameric gating particle that sterically occludes the human Cx26 gap junction channel pore
Inhibiting AMPylation: a novel screen to identify the first small molecule inhibitors of protein AMPylation
Enzymatic transfer of the AMP portion of ATP to substrate proteins has recently been described as an essential mechanism of bacterial infection for several pathogens. The first AMPylator to be discovered, VopS from Vibrio parahemolyticus, catalyzes the transfer of AMP onto the host GTPases Cdc42 and Rac1. Modification of these proteins disrupts downstream signaling events, contributing to cell rounding and apoptosis, and recent studies have suggested that blocking AMPylation may be an effective route to stop infection. To date, however, no small molecule inhibitors have been discovered for any of the AMPylators. Therefore, we developed a fluorescence-polarization-based high-throughput screening assay and used it to discover the first inhibitors of protein AMPylation. Herein we report the discovery of the first small molecule VopS inhibitors (e.g., calmidazolium, GW7647, and MK886) with Ki\u27s ranging from 6 to 50 muM and upward of 30-fold selectivity versus HYPE, the only known human AMPylator
Structure and dynamics of the liver receptor homolog 1āPGC1Ī± complex
Peroxisome proliferator-activated gamma coactivator 1-Ī± (PGC1Ī±) regulates energy metabolism by directly interacting with transcription factors to modulate gene expression. Among the PGC1Ī± binding partners is liver receptor homolog 1 (LRH-1; NR5A2), an orphan nuclear hormone receptor that controls lipid and glucose homeostasis. Although PGC1Ī± is known to bind and activate LRH-1, mechanisms through which PGC1Ī± changes LRH-1 conformation to drive transcription are unknown. Here, we used biochemical and structural methods to interrogate the LRH-1āPGC1Ī± complex. Purified, full-length LRH-1, as well as isolated ligand binding domain, bound to PGC1Ī± with higher affinity than to the coactivator, nuclear receptor coactivator-2 (Tif2), in coregulator peptide recruitment assays. We present the first crystal structure of the LRH-1āPGC1Ī± complex, which depicts several hydrophobic contacts and a strong charge clamp at the interface between these partners. In molecular dynamics simulations, PGC1Ī± induced correlated atomic motion throughout the entire LRH-1 activation function surface, which was dependent on charge-clamp formation. In contrast, Tif2 induced weaker signaling at the activation function surface than PGC1Ī± but promoted allosteric signaling from the helix 6/Ī²-sheet region of LRH-1 to the activation function surface. These studies are the first to probe mechanisms underlying the LRH-1āPGC1Ī± interaction and may illuminate strategies for selective therapeutic targeting of PGC1Ī±-dependent LRH-1 signaling pathways
Histone H3 binding to the PHD1 domain of histone demethylase KDM5A enables active site remodeling.
Histone demethylase KDM5A removes methyl marks from lysine 4 of histone H3 and is often overexpressed in cancer. The in vitro demethylase activity of KDM5A is allosterically enhanced by binding of its product, unmodified H3 peptides, to its PHD1 reader domain. However, the molecular basis of this allosteric enhancement is unclear. Here we show that saturation of the PHD1 domain by the H3 N-terminal tail peptides stabilizes binding of the substrate to the catalytic domain and improves the catalytic efficiency of demethylation. When present in saturating concentrations, differently modified H3 N-terminal tail peptides have a similar effect on demethylation. However, they vary greatly in their affinity towards the PHD1 domain, suggesting that H3 modifications can tune KDM5A activity. Furthermore, hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) experiments reveal conformational changes in the allosterically enhanced state. Our findings may enable future development of anti-cancer therapies targeting regions involved in allosteric regulation
Inhibiting AMPylation: A Novel Screen To Identify the First Small Molecule Inhibitors of Protein AMPylation
Enzymatic
transfer of the AMP portion of ATP to substrate proteins has recently
been described as an essential mechanism of bacterial infection for
several pathogens. The first AMPylator to be discovered, VopS from <i>Vibrio parahemolyticus</i>, catalyzes the transfer of AMP onto
the host GTPases Cdc42 and Rac1. Modification of these proteins disrupts
downstream signaling events, contributing to cell rounding and apoptosis,
and recent studies have suggested that blocking AMPylation may be
an effective route to stop infection. To date, however, no small molecule
inhibitors have been discovered for any of the AMPylators. Therefore,
we developed a fluorescence-polarization-based high-throughput screening
assay and used it to discover the first inhibitors of protein AMPylation.
Herein we report the discovery of the first small molecule VopS inhibitors
(e.g., calmidazolium, GW7647, and MK886) with <i>K</i><sub>i</sub>ās ranging from 6 to 50 Ī¼M and upward of 30-fold
selectivity versus HYPE, the only known human AMPylator