title: Electron microscopy analysis of a disaccharide analog complex reveals receptor interactions of adeno-associated virus.
abstract: Mechanistic studies of macromolecular complexes often feature X-ray structures of complexes with bound ligands. The attachment of adeno-associated virus (AAV) to cell surface glycosaminoglycans (GAGs) is an example that has not proven amenable to crystallography, because the binding of GAG analogs disrupts lattice contacts. The interactions of AAV with GAGs are of interest in mediating the cell specificity of AAV-based gene therapy vectors. Previous electron microscopy led to differing conclusions on the exact binding site and the existence of large ligand-induced conformational changes in the virus. Conformational changes are expected during cell entry, but it has remained unclear whether the electron microscopy provided evidence of their induction by GAG-binding. Taking advantage of automated data collection, careful processing and new methods of structure refinement, the structure of AAV-DJ complexed with sucrose octasulfate is determined by electron microscopy difference map analysis to 4.8Å resolution. At this higher resolution, individual sulfate groups are discernible, providing a stereochemical validation of map interpretation, and highlighting interactions with two surface arginines that have been implicated in genetic studies. Conformational changes induced by the SOS are modest and limited to the loop most directly interacting with the ligand. While the resolution attainable will depend on sample order and other factors, there are an increasing number of macromolecular complexes that can be studied by cryo-electron microscopy at resolutions beyond 5Å, for which the approaches used here could be used to characterize the binding of inhibitors and other small molecule effectors when crystallography is not tractable.
authors.affiliation: Department of Biochemistry & Molecular Biology, School of Medicine, Oregon Health &v Science University, Portland, OR 97239-3098, USA.
authors.forename: Qing
authors.initials: Q
authors.lastname: Xie
authors.forename: Michael
authors.initials: M
authors.lastname: Spilman
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Meyer
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Lerch
authors.forename: Scott M
authors.initials: SM
authors.lastname: Stagg
authors.forename: Michael S
authors.initials: MS
authors.lastname: Chapman
mesh_terms: D001665:Binding Sites; D036022:Capsid Proteins / Q000737:chemistry / Q000648:ultrastructure; D002478:Cells, Cultured; D020285:Cryoelectron Microscopy; D000229:Dependovirus / Q000648:ultrastructure*; D004187:Disaccharides / Q000737:chemistry; D008958:Models, Molecular; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011991:Receptors, Virus / Q000737:chemistry / Q000648:ultrastructure; D014771:Virion / Q000737:chemistry / Q000648:ultrastructure
chemical_list: D036022:Capsid Proteins; D004187:Disaccharides; D011991:Receptors, Virus
keywords: AAV; Attachment; CTF; Difference map; EM; FSC; Fourier shell correlation; HBD; HSPG; Heparin; Parvovirus; Receptor; Refinement; VLP; adeno-associated virus; contrast transfer function; electron microscopy; heparan sulfate proteoglycan; heparin-binding domain; virus-like particle
references.citation: Curr Opin Mol Ther. 2010 Oct;12(5):553-60
references.pmid: 20886387
references.citation: Elife. 2013 Mar 05;2:e00218
references.pmid: 23483797
references.citation: J Virol. 2011 Nov;85(22):11791-9
references.pmid: 21900159
references.citation: J Virol. 2006 Jul;80(14):7265-9
references.pmid: 16809332
references.citation: Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905-21
references.pmid: 9757107
references.citation: EMBO J. 1993 Jul;12(7):2589-99
references.pmid: 8334984
references.citation: J Virol. 2003 Oct;77(20):11072-81
references.pmid: 14512555
references.citation: Structure. 2008 Mar;16(3):441-8
references.pmid: 18334219
references.citation: J Virol. 2006 Sep;80(18):8961-9
references.pmid: 16940508
references.citation: Curr Gene Ther. 2005 Jun;5(3):299-310
references.pmid: 15975007
references.citation: J Struct Biol. 2008 Oct;164(1):170-5
references.pmid: 18614378
references.citation: J Virol. 2008 Jun;82(12):5887-911
references.pmid: 18400866
references.citation: EMBO J. 1999 Feb 1;18(3):543-54
references.pmid: 9927414
references.citation: J Struct Biol. 2009 Apr;166(1):95-102
references.pmid: 19263523
references.citation: J Struct Biol. 1999 Dec 1;128(1):82-97
references.pmid: 10600563
references.citation: J Virol. 2012 Jul;86(13):7326-33
references.pmid: 22514350
references.citation: Mol Ther. 2006 Apr;13(4):683-93
references.pmid: 16387552
references.citation: Nat Med. 1999 Jan;5(1):78-82
references.pmid: 9883843
references.citation: Nature. 1997 Mar 6;386(6620):88-91
references.pmid: 9052786
references.citation: Cell. 1997 Dec 12;91(6):721-30
references.pmid: 9413981
references.citation: Virology. 2009 Mar 15;385(2):434-43
references.pmid: 19144372
references.citation: Acta Crystallogr D Biol Crystallogr. 2009 Oct;65(Pt 10):1074-80
references.pmid: 19770504
references.citation: J Virol. 2012 Oct;86(19):10408-17
references.pmid: 22787229
references.citation: J Virol. 1998 Feb;72(2):1438-45
references.pmid: 9445046
references.citation: J Virol. 2005 Jan;79(1):609-14
references.pmid: 15596854
references.citation: Acta Crystallogr D Biol Crystallogr. 2000 Oct;56(Pt 10):1270-7
references.pmid: 10998623
references.citation: J Mol Biol. 1966 Jan;15(1):175-92
references.pmid: 5912039
references.citation: Nature. 2013 Feb 21;494(7437):385-9
references.pmid: 23395961
references.citation: J Virol. 2006 Oct;80(19):9831-6
references.pmid: 16973587
references.citation: J Struct Biol. 2007 Jan;157(1):117-25
references.pmid: 16828314
references.citation: Proc Natl Acad Sci U S A. 2002 Aug 6;99(16):10405-10
references.pmid: 12136130
references.citation: J Mol Biol. 2003 Oct 31;333(4):721-45
references.pmid: 14568533
references.citation: J Virol. 2006 Jan;80(2):1015-24
references.pmid: 16379002
references.citation: J Biol Chem. 2011 Apr 15;286(15):13532-40
references.pmid: 21330365
references.citation: Nat Med. 1999 Jan;5(1):71-7
references.pmid: 9883842
references.citation: J Mol Biol. 1964 Jan;8:96-104
references.pmid: 14149967
references.citation: J Virol. 2001 Aug;75(15):6884-93
references.pmid: 11435568
references.citation: J Struct Biol. 2009 Mar;165(3):146-56
references.pmid: 19121398
references.citation: Elife. 2013 Feb 19;2:e00461
references.pmid: 23427024
references.citation: Virology. 2012 Feb 5;423(1):6-13
references.pmid: 22169623
references.citation: Science. 1986 Sep 19;233(4770):1286-93
references.pmid: 3018924
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):486-501
references.pmid: 20383002
references.citation: Nat Biotechnol. 2010 Jan;28(1):79-82
references.pmid: 20037580
references.citation: AIDS Res Hum Retroviruses. 1993 Feb;9(2):167-74
references.pmid: 8096145
references.citation: J Virol. 2000 Mar;74(6):2777-85
references.pmid: 10684294
references.citation: J Virol. 2007 Nov;81(22):12260-71
references.pmid: 17728238
references.citation: J Struct Biol. 2007 Jan;157(1):201-10
references.pmid: 17029845
references.citation: J Virol. 2012 Jun;86(12):6947-58
references.pmid: 22496238
references.citation: Virology. 2012 Sep 15-30;431(1-2):40-9
references.pmid: 22682774
references.citation: Nat Biotechnol. 2006 Feb;24(2):198-204
references.pmid: 16429148
references.citation: N Engl J Med. 2011 Dec 22;365(25):2357-65
references.pmid: 22149959
references.citation: J Virol. 2006 Nov;80(22):11040-54
references.pmid: 16956943
references.citation: Mol Ther. 2012 Apr;20(4):699-708
references.pmid: 22273577
references.citation: FEBS J. 2013 Jan;280(1):28-45
references.pmid: 23181775
references.citation: J Struct Biol. 2005 Jul;151(1):41-60
references.pmid: 15890530
references.citation: J Virol. 2003 Jun;77(12):6995-7006
references.pmid: 12768018
references.citation: Structure. 2012 Aug 8;20(8):1310-20
references.pmid: 22727812
references.citation: J Struct Biol. 2013 Apr;182(1):10-21
references.pmid: 23376441
references.citation: Nature. 1965 May 22;206(4986):761-3
references.pmid: 5840126
references.citation: Adv Virus Res. 2007;70:183-232
references.pmid: 17765706
references.citation: J Virol. 2006 Dec;80(24):12017-24
references.pmid: 17020940
references.citation: J Virol. 2006 Nov;80(22):11393-7
references.pmid: 16943302
references.citation: Ultramicroscopy. 2005 Aug;104(1):8-29
references.pmid: 15935913
references.citation: Virology. 1999 Nov 25;264(2):436-40
references.pmid: 10562505
references.citation: Biochemistry. 2013 Sep 10;52(36):6275-85
references.pmid: 23952613