title: Conformational plasticity of the essential membrane-associated mannosyltransferase PimA from mycobacteria.
abstract: Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase (GT) that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides, lipomannan, and lipoarabinomannan, which are key glycolipids/lipoglycans of the mycobacterial cell envelope. PimA belongs to a large family of peripheral membrane-associated GTs for which the understanding of the molecular mechanism and conformational changes that govern substrate/membrane recognition and catalysis remains a major challenge. Here we used single molecule force spectroscopy techniques to study the mechanical and conformational properties of PimA. In our studies, we engineered a polyprotein containing PimA flanked by four copies of the well characterized I27 protein, which provides an unambiguous mechanical fingerprint. We found that PimA exhibits weak mechanical stability albeit displaying β-sheet topology expected to unfold at much higher forces. Notably, PimA unfolds following heterogeneous multiple step mechanical unfolding pathways at low force akin to molten globule states. Interestingly, the ab initio low resolution envelopes obtained from small angle x-ray scattering of the unliganded PimA and the PimA·GDP complexed forms clearly demonstrate that not only the "open" and "closed" conformations of the GT-B enzyme are largely present in solution, but in addition, PimA experiences remarkable flexibility that undoubtedly corresponds to the N-terminal "Rossmann fold" domain, which has been proved to participate in protein-membrane interactions. Based on these results and on our previous experimental data, we propose a model wherein the conformational transitions are important for the mannosyltransferase to interact with the donor and acceptor substrates/membrane.
authors: From the Unidad de Biofísica, Consejo Superior de Investigaciones Científicas-Universidad del País Vasco/Euskal Herriko Unibertsitatea, Barrio Sarriena s/n, Leioa, Bizkaia 48940, Spain.
authors: David
authors: D
authors: Giganti
authors: Jorge
authors: J
authors: Alegre-Cebollada
authors: Saioa
authors: S
authors: Urresti
authors: David
authors: D
authors: Albesa-Jové
authors: Ane
authors: A
authors: Rodrigo-Unzueta
authors: Natalia
authors: N
authors: Comino
authors: Michael
authors: M
authors: Kachala
authors: Sonia
authors: S
authors: López-Fernández
authors: Dmitri I
authors: DI
authors: Svergun
authors: Julio M
authors: JM
authors: Fernández
authors: Marcelo E
authors: ME
authors: Guerin
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020043:Genes, Essential / Q000235:genetics; D006153:Guanosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D008364:Mannosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018625:Microscopy, Atomic Force / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000201:enzymology* / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D053838:Scattering, Small Angle; D013314:Stress, Mechanical; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D006153:Guanosine Diphosphate; D008364:Mannosyltransferases; C543796:phosphatidyl-myo-inositol mannosyltransferase PimA, Mycobacterium smegmatis
keywords: Glycobiology; Glycolipids; Glycosyltransferases; Membrane Proteins; Mycobacterium
references: J Biol Chem. 2010 Oct 29;285(44):33577-83
references: 20801880
references: Proc Natl Acad Sci U S A. 2009 Aug 18;106(33):13791-6
references: 19666489
references: J Appl Crystallogr. 2008 Aug 16;41(Pt 5):913-917
references: 25484841
references: Science. 1983 May 13;220(4598):671-80
references: 17813860
references: Proc Natl Acad Sci U S A. 2001 Sep 11;98(19):10682-6
references: 11526214
references: Nature. 2007 Nov 1;450(7166):124-7
references: 17972886
references: Mol Cell. 2011 Feb 4;41(3):249-62
references: 21292158
references: Annu Rev Biochem. 2008;77:521-55
references: 18518825
references: Glycobiology. 2006 Feb;16(2):29R-37R
references: 16037492
references: J Biol Chem. 2010 Apr 9;285(15):11235-42
references: 20139067
references: Proc Natl Acad Sci U S A. 2010 Oct 12;107(41):17563-8
references: 20876143
references: Biophys J. 2003 Jan;84(1):533-44
references: 12524305
references: J Biol Chem. 2000 May 12;275(19):14432-9
references: 10799526
references: Angew Chem Int Ed Engl. 2011 Nov 11;50(46):10897-901
references: 21953735
references: Nature. 1989 Aug 24;340(6235):609-16
references: 2770867
references: Nat Struct Biol. 1995 Feb;2(2):117-20
references: 7749914
references: Biophys J. 2005 Nov;89(5):3337-44
references: 16100277
references: J Biol Chem. 2002 Aug 30;277(35):31335-44
references: 12068013
references: J Mol Biol. 1999 Feb 19;286(2):553-61
references: 9973570
references: J Mol Biol. 2001 Nov 30;314(3):365-74
references: 11846551
references: Chem Biol. 2002 Dec;9(12):1287-96
references: 12498881
references: J Biol Chem. 2000 Dec 22;275(51):40568-75
references: 11001941
references: Proc Natl Acad Sci U S A. 2009 Dec 22;106(51):21555-60
references: 19926859
references: EMBO J. 1994 Aug 1;13(15):3413-22
references: 8062817
references: Proc Natl Acad Sci U S A. 2004 May 11;101(19):7299-304
references: 15123816
references: Glycobiology. 2011 May;21(5):547-52
references: 21098513
references: EMBO J. 2004 Aug 18;23(16):3196-205
references: 15272305
references: J Biol Chem. 2009 Sep 18;284(38):25687-96
references: 19638342
references: Biophys J. 1999 Jun;76(6):2879-86
references: 10354416
references: Nat Struct Biol. 2000 Dec;7(12):1117-20
references: 11101892
references: J Biol Chem. 2008 Jun 6;283(23):15834-44
references: 18390549
references: Cell. 2005 Apr 22;121(2):247-56
references: 15851031
references: Chem Biol. 2012 Jan 27;19(1):42-50
references: 22284353
references: Science. 1994 Sep 9;265(5178):1599-600
references: 8079175
references: Proc Natl Acad Sci U S A. 2009 Jun 30;106(26):10534-9
references: 19541635
references: Nat Chem Biol. 2011 Aug 07;7(9):631-8
references: 21822275
references: J Biol Chem. 2010 Jun 18;285(25):18961-6
references: 20382731
references: Curr Opin Struct Biol. 2012 Oct;22(5):540-9
references: 22819665
references: Annu Rev Biochem. 2009;78:515-40
references: 19489728
references: Biochim Biophys Acta. 2004 Nov 3;1666(1-2):40-50
references: 15519307
references: Proc Natl Acad Sci U S A. 2003 Feb 4;100(3):845-9
references: 12538870
references: J Biol Chem. 2007 Jul 13;282(28):20705-14
references: 17510062
references: J Biol Chem. 2003 Jun 13;278(24):21790-7
references: 12660251
references: J Biol Chem. 2009 Jun 26;284(26):17796-807
references: 19244233
references: J Biol Chem. 2012 Dec 28;287(53):44173-83
references: 23144457
references: Nature. 2002 Aug 29;418(6901):998-1002
references: 12198551
references: J Biol Chem. 2009 Aug 7;284(32):21613-25
references: 19520856
references: J Am Chem Soc. 2012 Mar 14;134(10):4743-52
references: 22352786
references: J Biol Chem. 2012 Sep 7;287(37):31494-502
references: 22782895
references: Biophys J. 2009 May 6;96(9):3810-21
references: 19413987
references: Biochim Biophys Acta. 2011 Jun;1810(6):630-41
references: 21477636
references: J Am Chem Soc. 2006 Dec 20;128(50):16030-1
references: 17165744
references: Biophys J. 2007 Jan 1;92(1):225-33
references: 17028145
references: Proc Natl Acad Sci U S A. 2012 Mar 6;109(10):3796-801
references: 22355138
references: Annu Rev Phys Chem. 2013;64:37-54
references: 23216378
references: J Appl Crystallogr. 2009 Apr 1;42(Pt 2):342-346
references: 27630371
references: Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):3694-9
references: 10097099
references: Acta Crystallogr D Biol Crystallogr. 2012 Apr;68(Pt 4):344-51
references: 22505255