Crystal Structure of HIV-1 gp41 Including Both Fusion Peptide and Membrane Proximal External Regions


The HIV-1 envelope glycoprotein (Env) composed of the receptor binding domain gp120 and the fusion protein subunit gp41 catalyzes virus entry and is a major target for therapeutic intervention and for neutralizing antibodies. Env interactions with cellular receptors trigger refolding of gp41, which induces close apposition of viral and cellular membranes leading to membrane fusion. The energy released during refolding is used to overcome the kinetic barrier and drives the fusion reaction. Here, we report the crystal structure at 2 Å resolution of the complete extracellular domain of gp41 lacking the fusion peptide and the cystein-linked loop. Both the fusion peptide proximal region (FPPR) and the membrane proximal external region (MPER) form helical extensions from the gp41 six-helical bundle core structure. The lack of regular coiled-coil interactions within FPPR and MPER splay this end of the structure apart while positioning the fusion peptide towards the outside of the six-helical bundle and exposing conserved hydrophobic MPER residues. Unexpectedly, the section of the MPER, which is juxtaposed to the transmembrane region (TMR), bends in a 90°-angle sideward positioning three aromatic side chains per monomer for membrane insertion. We calculate that this structural motif might facilitate the generation of membrane curvature on the viral membrane. The presence of FPPR and MPER increases the melting temperature of gp41 significantly in comparison to the core structure of gp41. Thus, our data indicate that the ordered assembly of FPPR and MPER beyond the core contributes energy to the membrane fusion reaction. Furthermore, we provide the first structural evidence that part of MPER will be membrane inserted within trimeric gp41. We propose that this framework has important implications for membrane bending on the viral membrane, which is required for fusion and could provide a platform for epitope and lipid bilayer recognition for broadly neutralizing gp41 antibodies.


Vyšlo v časopise: Crystal Structure of HIV-1 gp41 Including Both Fusion Peptide and Membrane Proximal External Regions. PLoS Pathog 6(5): e32767. doi:10.1371/journal.ppat.1000880
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1000880

Souhrn

The HIV-1 envelope glycoprotein (Env) composed of the receptor binding domain gp120 and the fusion protein subunit gp41 catalyzes virus entry and is a major target for therapeutic intervention and for neutralizing antibodies. Env interactions with cellular receptors trigger refolding of gp41, which induces close apposition of viral and cellular membranes leading to membrane fusion. The energy released during refolding is used to overcome the kinetic barrier and drives the fusion reaction. Here, we report the crystal structure at 2 Å resolution of the complete extracellular domain of gp41 lacking the fusion peptide and the cystein-linked loop. Both the fusion peptide proximal region (FPPR) and the membrane proximal external region (MPER) form helical extensions from the gp41 six-helical bundle core structure. The lack of regular coiled-coil interactions within FPPR and MPER splay this end of the structure apart while positioning the fusion peptide towards the outside of the six-helical bundle and exposing conserved hydrophobic MPER residues. Unexpectedly, the section of the MPER, which is juxtaposed to the transmembrane region (TMR), bends in a 90°-angle sideward positioning three aromatic side chains per monomer for membrane insertion. We calculate that this structural motif might facilitate the generation of membrane curvature on the viral membrane. The presence of FPPR and MPER increases the melting temperature of gp41 significantly in comparison to the core structure of gp41. Thus, our data indicate that the ordered assembly of FPPR and MPER beyond the core contributes energy to the membrane fusion reaction. Furthermore, we provide the first structural evidence that part of MPER will be membrane inserted within trimeric gp41. We propose that this framework has important implications for membrane bending on the viral membrane, which is required for fusion and could provide a platform for epitope and lipid bilayer recognition for broadly neutralizing gp41 antibodies.


Zdroje

1. MooreJP

TrkolaA

DragicT

1997 Co-receptors for HIV-1 entry. Curr Opin Immunol 9 551 562

2. GalloSA

FinneganCM

ViardM

RavivY

DimitrovA

RawatSS

PuriA

DurellS

BR

2003 The HIV Env-mediated fusion reaction. Biochimica Biophysica Acta 1614 36 50

3. HarrisonSC

2005 Mechanism of membrane fusion by viral envelope proteins. Adv Virus Res 64 231 259

4. WeissenhornW

DessenA

HarrisonSC

SkehelJJ

WileyDC

1997 Atomic structure of the ectodomain from HIV-1 gp41. Nature 387 426 430

5. ChanDC

FassD

BergerJM

KimPS

1997 Core structure of gp41 from the HIV envelope glycoprotein. Cell 89 263 273

6. Munoz-BarrosoI

DurellS

SakaguchiK

AppellaE

BlumenthalR

1998 Dilation of the human immunodeficiency virus-1 envelope glycoprotein fusion pore revealed by the inhibitory action of a synthetic peptide from gp41. J Cell Biol 140 315 323

7. WildCT

ShugarsDC

GreenwellTK

McDanalCB

MatthewsTJ

1994 Peptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection. Proc Natl Acad Sci U S A 91 9770 9774

8. FurutaRA

WildCT

WengY

WeissCD

1998 Capture of an early fusion-active conformation of HIV-1 gp41. Nat Struct Biol 5 276 279

9. ChanDC

KimPS

1998 HIV entry and its inhibition. Cell 93 681 684

10. LuftigMA

MattuM

Di GiovineP

GeleziunasR

HrinR

BarbatoG

BianchiE

MillerMD

PessiA

CarfiA

2006 Structural basis for HIV-1 neutralization by a gp41 fusion intermediate-directed antibody. Nat Struct Mol Biol 13 740 747

11. CortiD

LangedijkJPM

HinzA

SeamanMS

VanzettaF

Fernandez-RodriguezBM

SilacciC

PinnaD

JarrossayD

Balla-JhagjhoorsinghS

WillemsB

ZekveldMJ

DrejaH

O'SullivanE

PadeC

OrkinC

JeffsSA

MontefioriDC

DavidD

WeissenhornW

McKnightA

HeeneyJL

SallustoF

SattentauQJ

WeissRA

LanzavecchiaA

2010 Analysis of Memory B Cell Responses and Isolation of Novel Monoclonal Antibodies with Neutralizing Breadth from HIV-1-Infected Individuals. PLoS ONE 5 e8805 doi:10.1371/journal.pone.0008805

12. DimitrovAS

JacobsA

FinneganCM

StieglerG

KatingerH

BlumenthalR

2007 Exposure of the membrane-proximal external region of HIV-1 gp41 in the course of HIV-1 envelope glycoprotein-mediated fusion. Biochemistry 46 1398 1401

13. FreyG

PengH

Rits-VollochS

MorelliM

ChengY

ChenB

2008 A fusion-intermediate state of HIV-1 gp41 targeted by broadly neutralizing antibodies. Proc Natl Acad Sci U S A 105 3739 3744

14. HaynesBF

FlemingJ

St ClairEW

KatingerH

StieglerG

KunertR

RobinsonJ

ScearceRM

PlonkK

StaatsHF

OrtelTL

LiaoHX

AlamSM

2005 Cardiolipin polyspecific autoreactivity in two broadly neutralizing HIV-1 antibodies. Science 308 1906 1908

15. OfekG

TangM

SamborA

KatingerH

MascolaJR

WyattR

KwongPD

2004 Structure and mechanistic analysis of the anti-human immunodeficiency virus type 1 antibody 2F5 in complex with its gp41 epitope. J Virol 78 10724 10737

16. CardosoRM

ZwickMB

StanfieldRL

KunertR

BinleyJM

KatingerH

BurtonDR

WilsonIA

2005 Broadly neutralizing anti-HIV antibody 4E10 recognizes a helical conformation of a highly conserved fusion-associated motif in gp41. Immunity 22 163 173

17. SunZY

OhKJ

KimM

YuJ

BrusicV

SongL

QiaoZ

WangJH

WagnerG

ReinherzEL

2008 HIV-1 broadly neutralizing antibody extracts its epitope from a kinked gp41 ectodomain region on the viral membrane. Immunity 28 52 63

18. AlamSM

MorelliM

DennisonSM

LiaoH-X

ZhangR

XiaS-M

Rits-VollochS

SunL

HarrisonSC

HaynesBF

ChenB

2009 Role of HIV membrane in neutralization by two broadly neutralizing antibodies. Proc Nat Acad Sci USA 106(48) 20234 9

19. MelikyanGB

MarkosyanRM

HemmatiH

DelmedicoMK

LambertDM

CohenFS

2000 Evidence that the transition of HIV-1 gp41 into a six-helix bundle, not the bundle configuration, induces membrane fusion. J Cell Biol 151 413 423

20. MarkosyanRM

CohenFS

MelikyanGB

2003 HIV-1 envelope proteins complete their folding into six-helix bundles immediately after fusion pore formation. Mol Biol Cell 14 926 938

21. KligerY

GalloSA

PeisajovichSG

Munoz-BarrosoI

AvkinS

BlumenthalR

ShaiY

2001 Mode of action of an antiviral peptide from HIV-1. Inhibition at a post-lipid mixing stage. J Biol Chem 276 1391 1397

22. ChernomordikLV

KozlovMM

2008 Mechanics of membrane fusion. Nat Struct Mol Biol 15 675 683

23. Munoz-BarrosoI

SalzwedelK

HunterE

BlumenthalR

1999 Role of the membrane-proximal domain in the initial stages of human immunodeficiency virus type 1 envelope glycoprotein-mediated membrane fusion. J Virol 73 6089 6092

24. Bellamy-McIntyreAK

LayC-S

BaarS

MaerzAL

TalboGH

DrummerHE

PoumbouriosP

2007 Functional Links between the Fusion Peptide-proximal Polar Segment and Membrane-proximal Region of Human Immunodeficiency Virus gp41 in Distinct Phases of Membrane Fusion. J Biol Chem 282 23104 23116

25. FrolovVA

ChoM-S

BronkP

ReeseTS

ZimmerbergJ

2000 Multiple Local Contact Sites are Induced by GPI-Linked Influenza Hemagglutinin During Hemifusion and Flickering Pore Formation. Traffic 1 622 630

26. HarrisonSC

2008 Viral membrane fusion. Nat Struct Mol Biol 15 690 698

27. ChernomordikLV

KozlovMM

2003 Protein-lipid interplay in fusion and fission of biological membranes. Annual Rev Biochem 72 175 207

28. LuM

BlacklowSC

KimPS

1995 A trimeric structural domain of the HIV-1 transmembrane glycoprotein. Nat Struct Biol 2 1075 1082

29. WeissenhornW

WhartonSA

CalderLJ

EarlPL

MossB

AliprandisE

SkehelJJ

WileyDC

1996 The ectodomain of HIV-1 env subunit gp41 forms a soluble, alpha-helical, rod-like oligomer in the absence of gp120 and the N-terminal fusion peptide. EMBO J 15 1507 1514

30. KielianM

ReyFA

2006 Virus membrane-fusion proteins: more than one way to make a hairpin. 4 67 76

31. LambRA

JardetzkyTS

2007 Structural basis of viral invasion: lessons from paramyxovirus F. Curr Opin Struct Biol 17(4) 427 436

32. RocheS

AlbertiniAA

LepaultJ

BressanelliS

GaudinY

2008 Structures of vesicular stomatitis virus glycoprotein: membrane fusion revisited. Cell Mol Life Sci 65 1716 1728

33. YangX

KurtevaS

RenX

LeeS

SodroskiJ

2006 Subunit stoichiometry of human immunodeficiency virus type 1 envelope glycoprotein trimers during virus entry into host cells. J Virol 80 4388 4395

34. MagnusC

RusertP

BonhoefferS

TrkolaA

RegoesRR

2009 Estimating the Stoichiometry of Human Immunodeficiency Virus Entry. J Virol 83 1523 1531

35. CampeloF

McMahonHT

KozlovMM

2008 The Hydrophobic Insertion Mechanism of Membrane Curvature Generation by Proteins. Biophys J 95 2325 2339

36. MartensS

KozlovMM

McMahonHT

2007 How Synaptotagmin Promotes Membrane Fusion. Science 316 1205 1208

37. SchibliDJ

MontelaroRC

VogelHJ

2001 The membrane-proximal tryptophan-rich region of the HIV glycoprotein, gp41, forms a well-defined helix in dodecylphosphocholine micelles. Biochemistry 40 9570 9578

38. PejchalR

GachJS

BrunelFM

CardosoRM

StanfieldRL

DawsonPE

BurtonDR

ZwickMB

WilsonIA

2009 A Conformational Switch in Human Immunodeficiency Virus gp41 Revealed by the Structures of Overlapping Epitopes Recognized by Neutralizing Antibodies. J Virol 83 8451 8462

39. LayCS

WilsonKA

KobeB

KempBE

DrummerHE

PoumbouriosP

2004 Expression and biochemical analysis of the entire HIV-2 gp41 ectodomain: determinants of stability map to N- and C-terminal sequences outside the 6-helix bundle core. FEBS Lett 567 183 188

40. NoahE

BironZ

NaiderF

ArshavaB

AnglisterJ

2008 The membrane proximal external region of the HIV-1 envelope glycoprotein gp41 contributes to the stabilization of the six-helix bundle formed with a matching N' peptide. Biochemistry 47 6782 6792

41. KozlovMM

ChernomordikLV

1998 A Mechanism of Protein-Mediated Fusion: Coupling between Refolding of the Influenza Hemagglutinin and Lipid Rearrangements. Biophys J 75 1384 1396

42. KuzminPI

ZimmerbergJ

ChizmadzhevYA

CohenFS

2001 A quantitative model for membrane fusion based on low-energy intermediates. Proc Nat Acad Sci USA 98 7235 7240

43. LiY

TammLK

2007 Structure and Plasticity of the Human Immunodeficiency Virus gp41 Fusion Domain in Lipid Micelles and Bilayers. Biophys J 93 876 885

44. SteinA

WeberG

WahlMC

JahnR

2009 Helical extension of the neuronal SNARE complex into the membrane. Nature 460 525 528

45. SalzwedelK

WestJT

HunterE

1999 A conserved tryptophan-rich motif in the membrane-proximal region of the human immunodeficiency virus type 1 gp41 ectodomain is important for Env-mediated fusion and virus infectivity. J Virol 73 2469 2480

46. ZwickMB

JensenR

ChurchS

WangM

StieglerG

KunertR

KatingerH

BurtonDR

2005 Anti-human immunodeficiency virus type 1 (HIV-1) antibodies 2F5 and 4E10 require surprisingly few crucial residues in the membrane-proximal external region of glycoprotein gp41 to neutralize HIV-1. J Virol 79 1252 1261

47. MarkosyanRM

LeungMY

CohenFS

2009 The six-helix bundle of human immunodeficiency virus Env controls pore formation and enlargement and is initiated at residues proximal to the hairpin turn. J Virol 83 10048 10057

48. LiuJ

DengY

DeyAK

MooreJP

LuM

2009 Structure of the HIV-1 gp41 Membrane-Proximal Ectodomain Region in a Putative Prefusion Conformation. Biochemistry 48 2915 2923

49. LeslieAGW

1992 Recent changes to the MOSFLM package for processing film and image plate data. Jnt CCP4/ESF-EACMB Newslett Protein Crystallogr 26

50. CCP4 1994 The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 50 157 163

51. EvansP

2006 Scaling and assessment of data quality. Acta Crystallogr D Biol Crystallogr 62 72 82

52. AdamsPD

Grosse-KunstleveRW

HungL-W

LoergerTR

McCoyAJ

MoriartyNW

ReadRJ

SacchettiniJC

K.SN

TerwilligerTC

2002 PHENIX: building new software for automated crystallographic structure determination. Acta Cryst D58 1948 1954

53. McCoyAJ

Grosse-KunstleveRW

AdamsPD

WinnMD

StoroniLC

ReadRJ

2007 Phaser crystallographic software. J Appl Crystallogr 40 658 674

54. EmsleyP

CowtanK

2004 Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60 2126 2132

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