Chapter 8
Structural Analysis of the Catalysis and Membrane Association of PLC-δ 1
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L.-O. Essen , P. D.Brownlie ,O.Perisic ,M . Katan , and R. L. Williams 1
Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge CB2 2QH, United Kingdom Department of Membrane Biochemistry, Max-Planck-Institute for Biochemistry, Am Klopferspitz 18a, D-82152 Martinsried, Germany CRC Centre for Cell and Molecular Biology, Chester Beatty Laboratories, Fulham Road, London SW3 6JB, United Kingdom
Downloaded by UNIV OF PITTSBURGH on May 3, 2015 | http://pubs.acs.org Publication Date: December 15, 1998 | doi: 10.1021/bk-1999-0718.ch008
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The structural analysis of the phosphoinositide-specific phospholipase C δ showed that a catalytic TIM-barrel-like domain is accompanied by three accessory domains: a P H domain, a calmodulin-like EF-hand domain and a C-terminal C2 domain. Crystallographic studies on complexes with substrate analogues combined with mutagenesis data established a reaction pathway where calcium is intimately involved in substrate binding and catalysis. The new 2.4 Å structure of PLC-δ complexed with glycero-phosphoinositol-4-phosphate, together with previously published complex structures, gives a structural basis for the substrate preference PIP2 > PIP >> PI: The 4-phosphoryl accomplishes a defined binding mode of the inositol head group, whereas the 5-phosphoryl determines its precise orientation in the active site. The refined 2.5 Å structure of the PLC-δ /CHAPSO complex identifies a hydrophobic ridge on the catalytic domain as a putative membrane penetration site. These structural features suggest a framework for the future design of PI-PLC specific drugs. 1
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Mammalian phosphoinositide-specific phospholipases C (PI-PLC) catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP ), a minor phospholipid component of biological membranes, to the second messengers diacylglycerol (DAG) andD-myo-inositol-l,4,5-trisphosphate (IP ). This reaction is a highly regulated step of numerous signal transduction cascades (i-3); the soluble product, IP , triggers the influx of calcium from intracellular stores into the cytosol, whereas the membraneresident diacylglycerol controls cellular protein phosphorylation states by stimulating protein kinase C isozymes. Another important aspect of eukaryotic PI-PLC activity is the regulation of the cellular PIP content, since many cellular functions like the cytoskeletal organisation depend on proteins which are regulated by interaction with PIP (4,5). Ten different isozymes of mammalian PI-PLCs which cluster in three major classes are currently known: β,-β , γ,-γ and δ,-δ . The β- and γ-isozymes (MW 145150 kDa) are regulated by G-protein-coupled and tyrosine-kinase-hnked receptors, respectively. The δ-isozymes are smaller (85 kDa) than PLC-β and -γ and their widespread occurrence among other eukaryotes such as plants, yeasts or slime molds 2
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©1999 American Chemical Society
In Phosphoinositides; Bruzik, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.
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122 indicates that this archetypal enzyme class evolved rather early in eukaryotes. A l l mammalian isozymes are apparentiy strictiy regulated in vivo (2). PLC-β and -γ contain regulatory domain modules in addition to those found in PLC-δ: PLC-β carries a C-terrninal domain that mediates activation by G
