title: Irreversible binding and activity control of the 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii at an anionic lipid bilayer surface.
abstract: 1,2-Diacylglycerol 3-glucosyltransferase is associated with the membrane surface catalyzing the synthesis of the major nonbilayer-prone lipid alpha-monoglucosyl diacylglycerol (MGlcDAG) from 1,2-DAG in the cell wall-less Acholeplasma laidlawii. Phosphatidylglycerol (PG), but not neutral or zwitterionic lipids, seems to be essential for an active conformation and function of the enzyme. Surface plasmon resonance analysis was employed to study association of the enzyme with lipid bilayers. Binding kinetics could be well fitted only to a two-state model, implying also a (second) conformational step. The enzyme bound less efficiently to liposomes containing only zwitterionic lipids, whereas increasing molar fractions of the anionic PG or cardiolipin (CL) strongly promoted binding by improved association (k(a1)), and especially a decreased rate of return (k(d2)) from the second state. This yielded a very low overall dissociation constant (K(D)), corresponding to an essentially irreversible membrane association. Both liposome binding and consecutive activity of the enzyme correlated with the PG concentration. The importance of the electrostatic interactions with anionic lipids was shown by quenching of both binding and activity with increasing NaCl concentrations, and corroborated in vivo for an active enzyme-green fluorescent protein hybrid in Escherichia coli. Nonbilayer-prone lipids substantially enhanced enzyme-liposome binding by promoting a changed conformation (decreasing k(d2)), similar to the anionic lipids, indicating the importance of hydrophobic interactions and a curvature packing stress. For CL and the nonbilayer lipids, effects on enzyme binding and consecutive activity were not correlated, suggesting a separate lipid control of activity. Similar features were recorded with polylysine (cationic) and polyglutamate (anionic) peptides present, but here probably dependent on the selective charge interactions with the enzyme N- and C-domains, respectively. A lipid-dependent conformational change and PG association of the enzyme were verified by circular dichroism, intrinsic tryptophan, and pyrene-probe fluorescence analyses, respectively. It is concluded that an electrostatic association of the enzyme with the membrane surface is accompanied by hydrophobic interactions and a conformational change. However, specific lipids, the curvature packing stress, and proteins or small molecules bound to the enzyme can modulate the activity of the bound A. laidlawii MGlcDAG synthase.
authors.affiliation: Department of Biochemistry, Umeå University, Umeå, Sweden.
authors.forename: Lu
authors.initials: L
authors.lastname: Li
authors.forename: Patrik
authors.initials: P
authors.lastname: Storm
authors.forename: Olof P
authors.initials: OP
authors.lastname: Karlsson
authors.forename: Stefan
authors.initials: S
authors.lastname: Berg
authors.forename: Ake
authors.initials: A
authors.lastname: Wieslander
mesh_terms: D000128:Acholeplasma laidlawii / Q000201:enzymology*; D000818:Animals; D000838:Anions; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D005964:Glucosyltransferases / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008081:Liposomes / Q000737:chemistry; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism; D008555:Melitten / Q000737:chemistry / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D010715:Phosphatidylglycerols / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011721:Pyrenes / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D013499:Surface Properties
chemical_list: D000838:Anions; D008051:Lipid Bilayers; D008081:Liposomes; D008164:Luminescent Proteins; D010715:Phosphatidylglycerols; D011721:Pyrenes; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D008555:Melitten; D005964:Glucosyltransferases; C103660:1,2-diacylglycerol 3-glucosyltransferase; D003545:Cysteine