Raczkowski, A. | Search bioRxiv

bioRxiv2024

Structure of the Ion Channel Kir7.1 and Implications for its Function in Normal and Pathophysiologic States

Hereditary defects in the function of the Kir7.1 in the retinal pigment epithelium are associated with the ocular diseases retinitis pigmentosa, Leber congenital amaurosis, and snowflake vitreal degeneration. Studies also suggest that Kir7.1 may be regulated by a GPCR, the melanocortin-4 receptor, in certain hypothalamic neurons. We present the first structures of human Kir7.1 and describe the conformational bias displayed by two pathogenic mutations, R162Q and E276A, to provide an explanation for the basis of disease and illuminate the gating pathway. We also demonstrate the structural basis for the blockade of the channel by a small molecule ML418 and demonstrate that channel blockade in vivo activates MC4R neurons in the paraventricular nucleus of the hypothalamus (PVH), inhibiting food intake and inducing weight loss. Preliminary purification, and structural and pharmacological characterization of an in tandem construct of MC4R and Kir7.1 suggests that the fusion protein forms a homotetrameric channel that retains regulation by liganded MC4R molecules.

neuroscience↗

bioRxiv2019

Structural Basis of Protein Arginine Methyltransferase Activation by a Catalytically Dead Homolog (Prozyme)

Prozymes are pseudoenzymes that stimulate the function of weakly active enzymes through complex formation. The major Trypanosoma brucei protein arginine methyltransferase, TbPRMT1 enzyme (ENZ), requires TbPRMT1 prozyme (PRO) to form an active heterotetrameric complex. Here we present the x-ray crystal structure of the ENZ-{Delta}52PRO tetrameric complex with the cofactor product S-adenosyl-L-homocysteine (AdoHcy) at 2.4 [A] resolution. The individual ENZ and PRO units adopt the highly-conserved PRMT domain architecture and form an antiparallel heterodimer that corresponds to the canonical homodimer observed in all previously reported PRMTs. In turn, two such heterodimers assemble into a tetramer both in the crystal and in solution with twofold rotational symmetry. ENZ is unstable in absence of PRO and incapable of forming a homodimer due to a steric clash of a non-conserved tyrosine within the dimerization arm, rationalizing why PRO is required to complement ENZ to form a PRMT dimer that is necessary, but not sufficient for PRMT activity. The PRO structure deviates from other, active PRTMs in that it lacks the conserved {eta}2 310-helix within the Rossmann fold, abolishing co-factor binding. In addition to its chaperone function for ENZ, PRO substantially contributes to substrate binding. Heterotetramerization is required for catalysis, since heterodimeric ENZ-PRO mutants lack binding affinity and methyltransferase activity towards the substrate protein TbRGG1. Together, we provide a structural basis for PRMT1 ENZ activation by PRO heterotetramer formation, which is conserved across all kinetoplastids, and describe a chaperone function of the PRMT1 prozyme, which represents a novel mode of PRMT regulation.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC=\"FIGDIR/small/776112v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (56K):\norg.highwire.dtl.DTLVardef@3d970dorg.highwire.dtl.DTLVardef@187f03forg.highwire.dtl.DTLVardef@1adee39org.highwire.dtl.DTLVardef@1f30f34_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG HighlightsO_LIThe crystal structure of Trypanosoma brucei protein arginine methyltransferase 1 (PRMT1) is reported\nC_LIO_LITwo enzyme-prozyme heterodimers form a stable heterotetramer essential for catalysis\nC_LIO_LIThe catalytically dead prozyme lacks elements essential for AdoMet binding\nC_LIO_LIThe enzyme alone is unstable and cannot form a canonical dimer due to steric clashes\nC_LIO_LIT. brucei PRMT1 prozyme adopts a chaperone function conserved across kinetoplastids\nC_LI

biochemistry↗

bioRxiv2018

Cryo-EM structures reveal bilayer remodeling during Ca2+ activation of a TMEM16 scramblase

The lipid distribution of plasma membranes of eukaryotic cells is asymmetric and phospholipid scramblases disrupt this asymmetry by mediating the rapid nonselective transport of lipids down their concentration gradients. As a result, phosphatidylserine is exposed to the outer leaflet of membrane, an important step in extracellular signaling networks controlling processes such as apoptosis, blood coagulation, membrane fusion and repair. Several members of the TMEM16 family have been identified as Ca2+-activated scramblases but the mechanisms underlying their Ca2+-dependent gating and their effects on the surrounding lipid bilayer remain poorly understood. Here we describe three high-resolution cryo-electron microscopy structures of a fungal scramblase from Aspergillus fumigatus, afTMEM16, reconstituted in lipid nanodiscs, revealing large Ca2+-dependent conformational changes of the protein as well as significant, function dependent membrane reorganization.

biochemistry↗