Molecular Recognition and Interfacial Catalysis by the Essential Phosphatidylinositol Mannosyltransferase PimA from Mycobacteria

Abstract

Mycobacterial phosphatidylinositol mannosides (PIMs) and metabolically derived cell wall lipoglycans play important roles in host-pathogen interactions, but their biosynthetic pathways are poorly understood. Here we focus on Mycobacterium smegmatis PimA, an essential enzyme responsible for the initial mannosylation of phosphatidylinositol. The structure of PimA in complex with GDP-mannose shows the two-domain organization and the catalytic machinery typical of GT-B glycosyltransferases. PimA is an amphitrophic enzyme that binds mono-disperse phosphatidylinositol, but its transferase activity is stimulated by high concentrations of non-substrate anionic surfactants, indicating that the early stages of PIM biosynthesis involve lipid-water interfacial catalysis. Based on structural, calorimetric, and mutagenesis studies, we propose a model wherein PimA attaches to the membrane through its N-terminal domain, and this association leads to enzyme activation. Our results reveal a novel mode of phosphatidylinositol recognition and provide a template for the development of potential antimycobacterial compounds. Mycobacterial phosphatidylinositol mannosides (PIMs) and metabolically derived cell wall lipoglycans play important roles in host-pathogen interactions, but their biosynthetic pathways are poorly understood. Here we focus on Mycobacterium smegmatis PimA, an essential enzyme responsible for the initial mannosylation of phosphatidylinositol. The structure of PimA in complex with GDP-mannose shows the two-domain organization and the catalytic machinery typical of GT-B glycosyltransferases. PimA is an amphitrophic enzyme that binds mono-disperse phosphatidylinositol, but its transferase activity is stimulated by high concentrations of non-substrate anionic surfactants, indicating that the early stages of PIM biosynthesis involve lipid-water interfacial catalysis. Based on structural, calorimetric, and mutagenesis studies, we propose a model wherein PimA attaches to the membrane through its N-terminal domain, and this association leads to enzyme activation. Our results reveal a novel mode of phosphatidylinositol recognition and provide a template for the development of potential antimycobacterial compounds. Phosphatidylinositol (PI) 2The abbreviations used are: PI, phosphatidylinositol; PIM, PI mannoside; LAM, lipoarabinomannan; LM, lipomannan; GDP, guanosine diphosphate; GDP-Man, guanosinediphosphomannose; ITC, isothermal titration calorimetry; GPI, glycerophosphoryl-myo-inositol; GT, glycosyltransferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; r.m.s.d., root mean square deviation. and its metabolites play a myriad of diverse cellular roles in eukaryotic cells. In addition to its role in membrane structure, mammalian cells use PI to synthesize phosphoinositides, inositol polyphosphates, and complex sphingolipids, which together regulate several cellular processes such as glycolipid anchoring of proteins (1Shields P. Arvan P. Curr. Opin. Cell Biol. 1999; 11: 489-494Crossref PubMed Scopus (21) Google Scholar), signal transduction (2Divecha N. Irvine R.F. Cell. 1995; 80: 269-278Abstract Full Text PDF PubMed Scopus (590) Google Scholar), or vesicle trafficking (3Czech M.P. Cell. 2000; 100: 603-606Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar, 4Martin T.F. Curr. Opin. Cell Biol. 2001; 13: 493-499Crossref PubMed Scopus (329) Google Scholar) and also serve as reservoirs of second messengers (5Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 261-399Crossref PubMed Scopus (264) Google Scholar, 6Greenberg M.L. Lopes J.M. Microbiol. Rev. 1996; 60: 1-20Crossref PubMed Google Scholar, 7Henry S.A. Patton-Vogt J.L. Prog. Nucleic Acid Res. Mol. Biol. 1998; 61: 133-179Crossref PubMed Google Scholar). In contrast to eukaryotic cells, PI has seldom been found in prokaryotes where it seems to be confined to some actinomycetes, myxobacteria, and Treponema (8Jackson M. Crick D.C. Brennan P.J. J. Biol. Chem. 2000; 275: 30092-30099Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). In mycobacteria, PI is the precursor for the synthesis of phosphatidylinositol mannosides (PIMs) and higher order glycolipids and lipoglycans such as lipomannan (LM) and lipoarabinomannan (LAM) (9Khoo K.H. Dell A. Morris H.R. Brennan P.J. Chatterjee D. Glycobiology. 1995; 5: 117-127Crossref PubMed Scopus (117) Google Scholar, 10Nigou J. Gilleron M. Puzo G. Biochimie (Paris). 2003; 85: 153-166Crossref PubMed Scopus (220) Google Scholar, 11Briken V. Porcelli S.A. Besra G.S. Kremer L. Mol. Microbiol. 2004; 53: 391-403Crossref PubMed Scopus (357) Google Scholar) (see Fig. 1). PIM, LM, and LAM are interspersed within the covalent mycolyl-arabinogalactan-peptidoglycan complex that makes up the core of the mycobacterial cell envelope (12Brennan P.J. Nikaido H. Annu. Rev. Biochem. 1995; 64: 29-63Crossref PubMed Scopus (1572) Google Scholar, 13Chatterjee D. Curr. Opin. Chem. Biol. 1997; 1: 579-588Crossref PubMed Scopus (167) Google Scholar) and play important roles in mycobacterial physiology as well as in the immunopathogenesis of tuberculosis and leprosy (10Nigou J. Gilleron M. Puzo G. Biochimie (Paris). 2003; 85: 153-166Crossref PubMed Scopus (220) Google Scholar, 11Briken V. Porcelli S.A. Besra G.S. Kremer L. Mol. Microbiol. 2004; 53: 391-403Crossref PubMed Scopus (357) Google Scholar). The availability of mycobacterial genome sequences together with advances in the genetic manipulation of mycobacteria has led to the identification of some of the biosynthetic enzymes involved in the early stages of PIM, LM, and LAM synthesis. The phosphatidyl-myo-inositol mannosyltransferase (PimA, E.C. 2.4.1.57) attaches the first mannosyl residue to the 2-position of the PI inositol moiety, yielding phosphatidylinositol monomannoside (PIM1) (14Kordulakova J. Gilleron M. Puzo G. Brennan P.J. Gicquel B. Mikusova K. Jackson M. J. Biol. Chem. 2003; 278: 36285-36295Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), and additional mannosyl- and acyltransferases catalyze the subsequent steps (14Kordulakova J. Gilleron M. Puzo G. Brennan P.J. Gicquel B. Mikusova K. Jackson M. J. Biol. Chem. 2003; 278: 36285-36295Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 15Schaeffer M.L. Khoo K.H. Besra G.S. Chatterjee D. Brennan P.J. Belisle J.T. Inamine J.M. J. Biol. Chem. 1999; 274: 31625-31631Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 16Kremer L. Gurcha S.S. Bifani P. Hitchen P.G. Baulard A. Morris H.R. Dell A. Brennan P.J. Besra G.S. Biochem. J. 2002; 363: 437-447Crossref PubMed Google Scholar, 17Morita Y.S. Sena C.B. Waller R.F. Kurokawa K. Sernee M.F. Nakatani F. Haites R.E. Billman-Jacobe H. McConville M.J. Maeda Y. Kinoshita T. J. Biol. Chem. 2006; 281: 25143-25155Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) (Fig. 1). The PI synthase (8Jackson M. Crick D.C. Brennan P.J. J. Biol. Chem. 2000; 275: 30092-30099Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar), the mannosyltransferase PimA (18Kordulakova J. Gilleron M. Mikusova K. Puzo G. Brennan P.J. Gicquel B. Jackson M. J. Biol. Chem. 2002; 277: 31335-31344Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), and the acyltransferase encoded by Rv2611c 3G. Stadthagen and M. Jackson, unpublished results. appear to be essential for mycobacterial growth. On the other hand, the mannosyltransferases PimB and PimC that transfer the second and third mannose residues, respectively, were found to be nonessential genes, suggesting the existence of alternative downstream synthetic pathways (16Kremer L. Gurcha S.S. Bifani P. Hitchen P.G. Baulard A. Morris H.R. Dell A. Brennan P.J. Besra G.S. Biochem. J. 2002; 363: 437-447Crossref PubMed Google Scholar). Both α-mannosyltransferases PimA and PimB use GDP-Man as a sugar donor and co-localize to a distinct subfraction of the plasma membrane, suggesting that the initial stages of apolar PIM synthesis take place on the cytoplasmic face of the membrane. In contrast, downstream enzymes catalyzing the synthesis of polar PIMs, and LM/LAM use polyprenol-phosphate-Man as a lipid-linked sugar donor (17Morita Y.S. Sena C.B. Waller R.F. Kurokawa K. Sernee M.F. Nakatani F. Haites R.E. Billman-Jacobe H. McConville M.J. Maeda Y. Kinoshita T. J. Biol. Chem. 2006; 281: 25143-25155Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 19Besra G.S. Morehouse C.B. Rittner C.M. Waechter C.J. Brennan P.J. J. Biol. Chem. 1997; 272: 18460-18466Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Furthermore, these enzymes co-localize with the plasma membrane and cell wall markers, indicating that the later stages of biosynthesis likely occur in the periplasmic space and/or the cell wall (20Morita Y.S. Velasquez R. Taig E. Waller R.F. Patterson J.H. Tull D. Williams S.J. Billman-Jacobe H. McConville M.J. J. Biol. Chem. 2005; 280: 21645-21652Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). To gain insight into the molecular mechanisms that govern the early stages of PIM biosynthesis, we report here molecular and structural studies of PimA. On the basis of these data, we propose a model of interfacial catalysis wherein association of the protein with anionic membrane lipids stimulates the mannosyl transfer reaction either by facilitating substrate diffusion to the catalytic site or by inducing an allosteric change in the enzyme structure. Protein Production and Crystallization—Recombinant PimA from Mycobacterium smegmatis was produced in Escherichia coli, purified to homogeneity, and crystallized as described (21Guerin M.E. Buschiazzo A. Kordulakova J. Jackson M. Alzari P.M. Acta Crystallogr. F Struct. Biol. Cryst. Commun. 2005; 61: 518-520Crossref PubMed Scopus (11) Google Scholar). The selenomethionine-labeled protein was expressed in the E. coli strain BL21(DE3) pLysS (Novagen) in M9 medium containing 0.2 g/liter selenomethionine and purified using the same procedure as described for the non-labeled protein. Crystals of PimA (10 mg/ml) in complex with GDP or GDP-Man (1 mm) were obtained by co-crystallization in 50 mm Hepes, pH 7.5, 200 mm Ca(Ac)2, 14% polyethylene glycol 8000. Rod-like crystals appeared after 2 days and grew to a maximum size of 0.5 × 0.06 × 0.06 mm. Structure Determination and Refinement—A three-wavelength MAD dataset (peak, inflection point, and high remote) was collected to 3.5 Å of resolution from a single crystal of selenomethionine-labeled protein in complex with GDP on the PXI beamline at the Swiss Light Source (SLS) (Villigen, Suisse). Single-wavelength datasets of the PimA-GDP and PimA-GDP-Man complexes were collected on beamlines ID14.1 and ID29 at the European Synchrotron Radiation Facility (Grenoble, France). All data were processed with programs MOSFLM, SCALA, and TRUNCATE from the CCP4 program suite (22P4 CC Acta Crystallogr. D. Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). Eight selenium sites could be identified using the program SHELXD (23Uson I. Sheldrick G.M. Curr. Opin. Struct. Biol. 1999; 9: 643-648Crossref PubMed Scopus (261) Google Scholar) with standard settings and optimized with the program SHARP (24Bricogne G. M. Acta Crystallogr. D. Biol. Crystallogr. 2003; PubMed Scopus Google Scholar). The with MAD the of of the for a protein that were are in data and for the resolution are in for the resolution are in for the resolution are in for the resolution are in for the resolution are in in a The initial model was with G.M. P. J. M. T. Acta Crystallogr. D. Biol. Crystallogr. 1998; PubMed Scopus Google Scholar), and subsequent were with Acta Crystallogr. D. Biol. Crystallogr. 1997; 53: PubMed Scopus Google Scholar). was with the program M. Acta Crystallogr. A. PubMed Scopus Google Scholar). The GDP and GDP-Man were from the initial and are in and were by the using and as The was obtained by the The proteins were expressed in E. coli cells and purified as described for PimA (21Guerin M.E. Buschiazzo A. Kordulakova J. Jackson M. Alzari P.M. Acta Crystallogr. F Struct. Biol. Cryst. Commun. 2005; 61: 518-520Crossref PubMed Scopus (11) Google Scholar). activity was as described (18Kordulakova J. Gilleron M. Mikusova K. Puzo G. Brennan P.J. Gicquel B. Jackson M. J. Biol. Chem. 2002; 277: 31335-31344Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) with the reaction of the membrane of M. smegmatis of a of PI, 50 of purified PimA or of PimA, and mm pH 7.5, in a mycobacterial were by of PI and/or of or of The of non-substrate was in the reaction containing were 2 at and with of by The were at and at × for and the was by as described (18Kordulakova J. Gilleron M. Mikusova K. Puzo G. Brennan P.J. Gicquel B. Jackson M. J. Biol. Chem. 2002; 277: 31335-31344Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) or by of PimA activity The standard PimA reaction 50 of GDP-Man of PI in 2 of 50 of PimA, and 50 mm pH in the of The was for 2 at and to the containing of of of in of and 50 mm pH in a of 200 in the at was the first after the addition of the PimA reaction of PimA by was in the in which the reaction was with of was also as the substrate of PimA by PI with the same of in the reaction was using a The enzyme and the were into the same of containing mm pH and and were were by (10 of GDP, PI, or mm) into the cell containing PimA or (10 at data were for of of and and were by data to a model using program was used for were using a procedure with a steps and the of the space of and its in the were obtained and to the The and were optimized by using The with the is in Fig. The Structure of crystal of PimA in complex with GDP and GDP-Man were using at and Å of 1). The which to the of enzyme data the typical of P.M. E. B. J. Mol. Biol. 2003; PubMed Scopus Google Scholar), of with a at the that the catalytic (Fig. The N-terminal and the The core of is of with of the structure or indicating the within the N-terminal and the of the protein that is in other mycobacterial PimA (Fig. of mycobacterial PimA. of Mycobacterium Mycobacterium M. smegmatis and M. tuberculosis PimA sequences is structure of PimA are the protein are in a in the crystal structure. The in that is to with membrane is involved in GDP-Man are with a using L. J. Mol. Biol. PubMed Scopus Google Scholar) a structural of PimA with several GT-B enzymes to P.M. E. B. J. Mol. Biol. 2003; PubMed Scopus Google Scholar). The structural is the synthase of of Å and by the the 14% and the synthase from structural are also found with enzymes from other such as and the of the The structure of the of PimA, containing the is to of other for which a crystal structure is M. Chem. Biol. 2006; 13: Full Text Full Text PDF PubMed Scopus Google Scholar), with of important structural are for the N-terminal domain, which is involved in with the the with the the structural of the PimA N-terminal are of the and the E. coli The potential of PimA (Fig. a polar protein with a N-terminal and a the of the N-terminal domain, a of and on the face of is to a and to a that is in mycobacterial (Fig. suggesting the of this in membrane to the site the N-terminal a or (Fig. that is by from and play some role in the of the The substrate GDP-Man is in the (Fig. The binds to a by the from the N-terminal the of and and the from the the donor substrate into the (Fig. In the of the with the of and the of for the of this of enzymes J. Biol. Chem. Full Text PDF PubMed Google Scholar). and with the of a residue in the J. Mol. Biol. 2001; PubMed Scopus Google Scholar). The with and and with the of from the is to in other GT-B A. M.E. Alzari P.M. J. 2004; PubMed Scopus Google Scholar, Y. L. B. B. D. M. A. 2003; 100: PubMed Scopus Google Scholar). The mannosyl is also within the site through several of the sugar with protein and the of (Fig. we were to PimA in complex with PI or their the structure into the of the polar of the substrate within the the inositol of PI with the of with its to the mannosyl residue from GDP-Man (Fig. The model an important role of in substrate its is well to with the PI and/or the also that the in the N-terminal (Fig. could be to at in the substrate model were by The role of and in substrate was their by in enzyme (Fig. and In contrast, a in which was by a to the its the to (Fig. Furthermore, the activity of this using PI as substrate was to that of the protein (Fig. to be important for catalysis in GT-B are and The is of a of the J. Mol. Biol. 2001; PubMed Scopus Google Scholar) and makes with the from the mannosyl (Fig. is residue in the is to the sugar and is involved in catalysis P. 1996; PubMed Scopus Google Scholar). in other GT-B these also a role in PimA catalysis their by the catalytic activity The initial mannosylation of PI in mycobacteria is to occur at the of the inositol J. Biol. Chem. Full Text PDF PubMed Google Scholar, K. PubMed Scopus Google Scholar). The model described for this of the of with the face of Furthermore, it that the involved in the subsequent mannosylation of PIM (Fig. a of their substrate to the of the seems to be the for which the transfer of the second mannosyl residue at of the inositol of M.L. Khoo K.H. Besra G.S. Chatterjee D. Brennan P.J. Belisle J.T. Inamine J.M. J. Biol. Chem. 1999; 274: 31625-31631Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). PimA and PimB to the same and some of the in PI are the In PimB in the as well as in the containing the which in PimA. The results a site for the polar of the the of PI containing as a substrate for PimA, the was a of the substrate (Fig. studies using isothermal titration (Fig. are with these GDP-Man and GDP were to PimA in with of and On the other hand, the substrate PI with the enzyme in and to the PimA-GDP complex to PimA of could be either to PimA or to the PimA-GDP complex 50 the same (Fig. results the of the for substrate and catalysis. and the of PI to PimA at substrate the of the complex was to on PI (Fig. The protein is to with mono-disperse PI through its site but also with or through a of the protein. Furthermore, these were to the catalytic as by a in enzyme activity at higher PI concentrations (Fig. The same was these were for a PI with for which the has been as M.J. R. T. R. J. Biol. Chem. Full Text PDF PubMed Google Scholar) (Fig. In this the enzyme activity at a substrate of the was higher the activity at the in activity was also concentrations of the substrate were with high concentrations of anionic non-substrate surfactants, such as or but with the or (Fig. that of PimA catalysis on the association of the protein with anionic in with the the protein was found in membrane and of a M. smegmatis strain PimA, the mannosyltransferase activity was with the membrane (18Kordulakova J. Gilleron M. Mikusova K. Puzo G. Brennan P.J. Gicquel B. Jackson M. J. Biol. Chem. 2002; 277: 31335-31344Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). structural, and studies the of and as for membrane association of to membrane from the of protein and membrane as and from the protein and membrane D. A. B. Curr. 2002; Google Scholar). of the PimA structure that the molecular of the N-terminal domain, to the substrate and in mycobacterial (Fig. could these to The interfacial an to a of in 2 and to the which is in the structure and in J. M. R. 2005; PubMed Scopus Google Scholar) this to the of the protein (Fig. the identification of the as the interfacial was by the of PimA in which the was by mutagenesis or the and on 2 were by are from the catalytic and to (Fig. and are to with the catalytic the were to GDP with in the suggesting that the of the protein was the PimA (Fig. and the of the protein to (Fig. with Fig. for the The of the of in is with the activity of PimA in the of but (Fig. also results that of mycobacterial plasma the synthesis of (20Morita Y.S. Velasquez R. Taig E. Waller R.F. Patterson J.H. Tull D. Williams S.J. Billman-Jacobe H. McConville M.J. J. Biol. 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K. 1998; PubMed Scopus Google Scholar), of which are to enzymes that with the PI synthase and or are in the other the acyltransferase Rv2611c and and (8Jackson M. Crick D.C. Brennan P.J. J. Biol. Chem. 2000; 275: 30092-30099Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, V. P.M. Biochem. 2005; PubMed Scopus Google Scholar, 2002; PubMed Scopus Google Scholar). the mannosyltransferase which a second mannose residue was also found to co-localize with this GT-B enzyme a as M.L. Khoo K.H. Besra G.S. Chatterjee D. Brennan P.J. Belisle J.T. Inamine J.M. J. Biol. Chem. 1999; 274: 31625-31631Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). its and structural with PimA that PimB could use a for membrane interfacial catalytic machinery for the synthesis of apolar is to the association of and with the facilitating the of the the In results a model of interfacial catalysis for PimA, to which the enzyme the substrate with its polar within the catalytic and the from the is by an interfacial on the N-terminal of the which likely a of and the catalysis by facilitating substrate diffusion from the to the catalytic site and/or by inducing allosteric in the protein. The mycobacterial cell wall is the site of of antimycobacterial the of is for cell and it is to mycobacteria, the here provide a template for the of novel M. J. A. and for with protein and data and for with the use of