Structural Basis for the Acyltransferase Activity of Lecithin:Retinol Acyltransferase-like Proteins*

Background: The enzymology of aminophospholipid-processing enzymes is not well understood. Results: Structures of HRAS-like tumor suppressors resemble those of thiol proteases with Cys-His-His catalytic triad. Conclusion: An aminophospholipid acyl group first modifies the Cys residue of HRAS-like tumor suppressor enzymes before it is transferred onto a second substrate. Significance: This study provides a structural basis for the enzymatic mechanism of HRAS-like tumor suppressors. Lecithin:retinol acyltransferase-like proteins, also referred to as HRAS-like tumor suppressors, comprise a vertebrate subfamily of papain-like or NlpC/P60 thiol proteases that function as phospholipid-metabolizing enzymes. HRAS-like tumor suppressor 3, a representative member of this group, plays a key role in regulating triglyceride accumulation and energy expenditure in adipocytes and therefore constitutes a novel pharmacological target for treatment of metabolic disorders causing obesity. Here, we delineate a catalytic mechanism common to lecithin:retinol acyltransferase-like proteins and provide evidence for their alternative robust lipid-dependent acyltransferase enzymatic activity. We also determined high resolution crystal structures of HRAS-like tumor suppressor 2 and 3 to gain insight into their active site architecture. Based on this structural analysis, two conformational states of the catalytic Cys-113 were identified that differ in reactivity and thus could define the catalytic properties of these two proteins. Finally, these structures provide a model for the topology of these enzymes and allow identification of the protein-lipid bilayer interface. This study contributes to the enzymatic and structural understanding of HRAS-like tumor suppressor enzymes.

[1]  A. Plückthun,et al.  Acyl and phosphoryl migration in lysophospholipids: importance in phospholipid synthesis and phospholipase specificity. , 1982, Biochemistry.

[2]  M. Gelb,et al.  Interfacial catalysis: the mechanism of phospholipase A2 , 1990, Science.

[3]  J. MacDonald,et al.  Phospholipid fatty acid remodeling in mammalian cells. , 1991, Biochimica et biophysica acta.

[4]  R. Huber,et al.  A rapid and efficient purification method for recombinant annexin V for biophysical studies , 1993, FEBS letters.

[5]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[6]  Ming Liu,et al.  Hydrolysis and transesterification of platelet-activating factor by lecithin-cholesterol acyltransferase. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[7]  J. Abrahams,et al.  Methods used in the structure determination of bovine mitochondrial F1 ATPase. , 1996, Acta crystallographica. Section D, Biological crystallography.

[8]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[9]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[10]  R. Williams,et al.  Structural Mapping of the Catalytic Mechanism for a Mammalian Phosphoinositide-specific Phospholipase C †, ‡ , 1996 .

[11]  G. Schneider,et al.  Crystal structure of β‐ketoacyl‐acyl carrier protein synthase II from E.coli reveals the molecular architecture of condensing enzymes , 1998, The EMBO journal.

[12]  S. White,et al.  Membrane protein folding and stability: physical principles. , 1999, Annual review of biophysics and biomolecular structure.

[13]  D. Bok,et al.  Molecular and Biochemical Characterization of Lecithin Retinol Acyltransferase* , 1999, The Journal of Biological Chemistry.

[14]  Joanne I. Yeh,et al.  Protein-sulfenic acids: diverse roles for an unlikely player in enzyme catalysis and redox regulation. , 1999, Biochemistry.

[15]  Z Dauter,et al.  Novel approach to phasing proteins: derivatization by short cryo-soaking with halides. , 2000, Acta crystallographica. Section D, Biological crystallography.

[16]  Nathan A. Baker,et al.  Electrostatics of nanosystems: Application to microtubules and the ribosome , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[17]  D. Fournand,et al.  Aliphatic and enantioselective amidases: from hydrolysis to acyl transfer activity , 2001, Journal of applied microbiology.

[18]  G N Murshudov,et al.  Use of TLS parameters to model anisotropic displacements in macromolecular refinement. , 2001, Acta crystallographica. Section D, Biological crystallography.

[19]  Irene T Weber,et al.  Geometric criteria of hydrogen bonds in proteins and identification of "bifurcated" hydrogen bonds. , 2002, Protein engineering.

[20]  H. Chahinian,et al.  Distinction between esterases and lipases , 2002 .

[21]  Vivek Anantharaman,et al.  Evolutionary history, structural features and biochemical diversity of the NlpC/P60 superfamily of enzymes , 2003, Genome Biology.

[22]  G Bricogne,et al.  Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0. , 2003, Acta crystallographica. Section D, Biological crystallography.

[23]  Martyn D Winn,et al.  Macromolecular TLS refinement in REFMAC at moderate resolutions. , 2003, Methods in enzymology.

[24]  R. V. Van Gelder,et al.  Lecithin-retinol Acyltransferase Is Essential for Accumulation of All-trans-Retinyl Esters in the Eye and in the Liver* , 2004, Journal of Biological Chemistry.

[25]  S. White,et al.  Interfacial folding and membrane insertion of a designed helical peptide. , 2004, Biochemistry.

[26]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[27]  P. Karplus,et al.  Protein sulfenic acids in redox signaling. , 2004, Annual review of pharmacology and toxicology.

[28]  K. Palczewski,et al.  Lecithin:Retinol Acyltransferase Is Responsible for Amidation of Retinylamine, a Potent Inhibitor of the Retinoid Cycle* , 2005, Journal of Biological Chemistry.

[29]  S. Gaetani,et al.  Oleoylethanolamide, an endogenous PPAR-α agonist, lowers body weight and hyperlipidemia in obese rats , 2005, Neuropharmacology.

[30]  Hua Xu,et al.  A mass accuracy sensitive probability based scoring algorithm for database searching of tandem mass spectrometry data , 2007, BMC Bioinformatics.

[31]  Martin Phillips,et al.  Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[32]  R. Capasso,et al.  Role and regulation of acylethanolamides in energy balance: focus on adipocytes and beta-cells. , 2007, British journal of pharmacology.

[33]  M. Wenk,et al.  Membrane lipids as signaling molecules , 2007, Current opinion in lipidology.

[34]  N. Ueda,et al.  Discovery and Characterization of a Ca2+-independent Phosphatidylethanolamine N-Acyltransferase Generating the Anandamide Precursor and Its Congeners* , 2006, Journal of Biological Chemistry.

[35]  Eric Blanc,et al.  Automated structure solution with autoSHARP. , 2007, Methods in molecular biology.

[36]  R. Capasso,et al.  Role and regulation of acylethanolamides in energy balance: focus on adipocytes and β‐cells , 2007 .

[37]  K. Palczewski,et al.  Topology and Membrane Association of Lecithin: Retinol Acyltransferase* , 2007, Journal of Biological Chemistry.

[38]  Jack Snoeyink,et al.  Nucleic Acids Research Advance Access published April 22, 2007 MolProbity: all-atom contacts and structure validation for proteins and nucleic acids , 2007 .

[39]  Randy J. Read,et al.  Phaser crystallographic software , 2007, Journal of applied crystallography.

[40]  Jessica Y Locke,et al.  Solution NMR structure of the NlpC/P60 domain of lipoprotein Spr from Escherichia coli: structural evidence for a novel cysteine peptidase catalytic triad. , 2008, Biochemistry.

[41]  Emma K. Farrell,et al.  Biosynthesis, degradation and pharmacological importance of the fatty acid amides. , 2008, Drug discovery today.

[42]  R. Capasso,et al.  Dysregulation of peripheral endocannabinoid levels in hyperglycemia and obesity: Effect of high fat diets , 2008, Molecular and Cellular Endocrinology.

[43]  H. Sul,et al.  Identification and Functional Characterization of Adipose-specific Phospholipase A2 (AdPLA)* , 2008, Journal of Biological Chemistry.

[44]  Yusuf A. Hannun,et al.  Principles of bioactive lipid signalling: lessons from sphingolipids , 2008, Nature Reviews Molecular Cell Biology.

[45]  G. Meer,et al.  Membrane lipids: where they are and how they behave , 2008, Nature Reviews Molecular Cell Biology.

[46]  B. Desbat,et al.  Secondary structure of a truncated form of lecithin retinol acyltransferase in solution and evidence for its binding and hydrolytic action in monolayers. , 2008, Biochimica et biophysica acta.

[47]  G. Sheldrick.  A short history of SHELX. , 2008, Acta crystallographica. Section A, Foundations of crystallography.

[48]  T. Uyama,et al.  Characterization of the human tumor suppressors TIG3 and HRASLS2 as phospholipid-metabolizing enzymes. , 2009, Biochimica et biophysica acta.

[49]  L. Sarda,et al.  Distinction between esterases and lipases: comparative biochemical properties of sequence-related carboxylesterases. , 2009, Protein and peptide letters.

[50]  G. Shulman,et al.  AdPLA ablation increases lipolysis and prevents obesity induced by high fat feeding or leptin deficiency , 2009, Nature Medicine.

[51]  V. Marzo,et al.  Anti‐inflammatory Effect of Palmitoylethanolamide on Human Adipocytes , 2009, Obesity.

[52]  Anna Szlachcic,et al.  The structural basis of tail-anchored membrane protein recognition by Get3 , 2009, Nature.

[53]  G. Wolf.  Adipose-specific phospholipase as regulator of adiposity. , 2009, Nutrition reviews.

[54]  Dale L Boger,et al.  Fatty acid amide signaling molecules. , 2010, Bioorganic & medicinal chemistry letters.

[55]  Paul D. Adams,et al.  phenix.model_vs_data: a high-level tool for the calculation of crystallographic model and data statistics , 2010, Journal of applied crystallography.

[56]  W. Kabsch.  XDS , 2010, Acta crystallographica. Section D, Biological crystallography.

[57]  Soo-Youl Kim,et al.  Expression, purification and biochemical characterization of the N-terminal regions of human TIG3 and HRASLS3 proteins. , 2010, Protein expression and purification.

[58]  K. Palczewski,et al.  An Acyl-covalent Enzyme Intermediate of Lecithin:Retinol Acyltransferase* , 2010, The Journal of Biological Chemistry.

[59]  Wolfgang Kabsch,et al.  Integration, scaling, space-group assignment and post-refinement , 2010, Acta crystallographica. Section D, Biological crystallography.

[60]  C. Jin,et al.  Solution structure of the N‐terminal catalytic domain of human H‐REV107 – A novel circular permutated NlpC/P60 domain , 2010, FEBS letters.

[61]  Randy J. Read,et al.  Acta Crystallographica Section D Biological , 2003 .

[62]  H. Arai,et al.  Regulation of Peroxisomal Lipid Metabolism by Catalytic Activity of Tumor Suppressor H-rev107* , 2011, The Journal of Biological Chemistry.

[63]  K. Verhoeckx,et al.  Presence, formation and putative biological activities of N-acyl serotonins, a novel class of fatty-acid derived mediators, in the intestinal tract. , 2011, Biochimica et biophysica acta.

[64]  T. Uyama,et al.  Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes , 2011, Journal of Lipid Research.

[65]  J. Schrag,et al.  Different Active‐Site Loop Orientation in Serine Hydrolases versus Acyltransferases , 2011, Chembiochem : a European journal of chemical biology.

[66]  Andrei L. Lomize,et al.  Anisotropic Solvent Model of the Lipid Bilayer. 2. Energetics of Insertion of Small Molecules, Peptides, and Proteins in Membranes , 2011, J. Chem. Inf. Model..

[67]  M. Lewis,et al.  Altered hepatic lipid metabolism in C57BL/6 mice fed alcohol: a targeted lipidomic and gene expression study[S] , 2011, Journal of Lipid Research.

[68]  S. S. Veiga,et al.  Structure of a novel class II phospholipase D: catalytic cleft is modified by a disulphide bridge. , 2011, Biochemical and biophysical research communications.

[69]  Andrei L. Lomize,et al.  Anisotropic Solvent Model of the Lipid Bilayer. 1. Parameterization of Long-Range Electrostatics and First Solvation Shell Effects , 2011, J. Chem. Inf. Model..

[70]  B. Desbat,et al.  Binding of a truncated form of lecithin:retinol acyltransferase and its N- and C-terminal peptides to lipid monolayers. , 2012, Langmuir : the ACS journal of surfaces and colloids.

[71]  K. Palczewski,et al.  Key enzymes of the retinoid (visual) cycle in vertebrate retina. , 2012, Biochimica et biophysica acta.

[72]  D. Otzen.  Membrane protein folding and stability. , 2014, Archives of biochemistry and biophysics.