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Ivan L. Budyak Forschungszentrum J ü lich, Deutschland University of Pittsburgh, PA, USA

The investigations of structure and properties of membrane receptors: human EGFR and halobacterial HtrII. Ivan L. Budyak Forschungszentrum J ü lich, Deutschland University of Pittsburgh, PA, USA Московский физико-технический институт , Россия May 2006.

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Ivan L. Budyak Forschungszentrum J ü lich, Deutschland University of Pittsburgh, PA, USA

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  1. The investigations of structure and properties of membrane receptors: human EGFR and halobacterial HtrII Ivan L. Budyak Forschungszentrum Jülich, Deutschland University of Pittsburgh, PA, USA Московский физико-технический институт, Россия May 2006

  2. Part 1:halobacterial transducerII (HtrII)fromNatronobacterium pharaonis

  3. Retinal-containing proteins of N.pharaonis Engelhard M. et al. (2002), Archaeabacterial phototaxis, In Photoreceptors and Light Signaling (pp. 2-39), The Royal Society of Chemistry, UK

  4. Two-componentsignal transduction system in N.pharaonis ? • transducer sequences from archaea including N.pharaonis are homologous to those of eubacterial chemoreceptors • both signal through the classical two-component system Gordeliy V.I. et al., Nature, 419 (2002), pp. 484-487 Oprian D.D., TIBS, 28 (2003), pp. 167-169 Structures of the cytoplasmic domains of halobacterial transducers remain unknown

  5. Design, expression, purification and initial characterization of HtrII-cyt Tsr, T286 Tsr, A526 open the cells salting out HIC purification gel-filtration HtrII, D504 HtrII, M234 Le Moual H. and Koshland D.E., J.Mol.Biol., 261 (1996), pp. 568-585 1 , 2 m 1 2 3 5 4 3 4 5 The cytoplasmic fragment of HtrII can be expressed in E.coli and purified to homogeneity and is unstructured in solution

  6. Structural predictions for HtrII-cyt 76%α-helix and24%random coil Combet C. et al., TIBS, 291 (2000), pp. 147-150 Lupas A. et al., Science, 252 (1991), pp. 1162-1164 The cytoplasmic fragment of HtrII is predicted to be α-helical and to form coiled coil structure O. Lund et al., “CPHmodels 2.0: X3M a Computer Program to Extract 3D Models”, A102 abstract at the CASP5 conference, 2002

  7. Predictions of dynamic properties of HtrII Romero P. et al., Proteins: Struct. Funct. Gen., 42 (2001), pp. 38-48 The cytoplasmic domain of HtrII is predicted to be disordered Predictions of structural parameters are contradictory suggesting the possibility of structural transitions

  8. Conformational transitions KCl NaCl glycerol CONTIN algorithm: Van Stokkum I.H.M. et al., Anal.Biochem., 191 (1990), pp. 110-119 KCl, NaCl and glycerol induce conformational transitions from mainly random coil to a-helix

  9. Conformational transitions sucrose ammonium sulfate TFE CONTIN algorithm: Van Stokkum I.H.M. et al., Anal.Biochem., 191 (1990), pp. 110-119 Sucrose, ammonium sulfate and TFE also induce conformational transitions from mainly random coil to a-helix

  10. FTIR spectroscopy 10 mM Tris-HCl pH 9.0 in D2O dry film 1654 1644 adapted from Stuart B. (1997), Biological Applications of Infrared Spectroscopy, University of Greenwich, UK FTIR indicates random coil in solution and a-helix in dry film

  11. 10mM NaP pH 6.0 + 70% glycerol Red: 1H-15N HSQC Green: Trosy-HSQC 125 120 115 110 15N chemical shift, ppm 9.0 8.0 7.0 1H chemical shift, ppm • peaks shifted and broadened • strong Trosy effect NMR spectroscopy 10 mM NaP pH 6.0 10 9 8 7 6 5 4 3 2 1 0 125 120 115 110 15N chemical shift, ppm • minimal spectral dispersion • negative het-NOE signals (data not shown) 8.5 8.0 7.5 7.0 6.5 1H chemical shift, ppm NMR data support structural transitions in glycerol

  12. Analyticalgel-filtration chromatography (AGFC) ammonium sulfate KClandNaCl Abnormal retention volumesofthe cytoplasmic fragmentof HtrII evidence its non-globular shape; ammonium sulfate induces hydrophobic interactions with the column up to complete retention

  13. Analyticalgel-filtration chromatography (AGFC) and chemical cross-linking 1xPBS 1xPBS + 40% as 1xPBS + 10% as 1xPBS + 4 M KCl Cross-linking data evidence HtrII-cyt dimerization in 4 M KCl

  14. Small-angle neutron scattering and atomic force microscopy AFMwithHtrII-cyt in dry film SANS withHtrII-cyt (many thanks to Dirk Mayer, ISG-2) HtrII-cyt has characteristic size of~180-200 Å and elongatedshape in solution

  15. Conclusions, part 1 • The cytoplasmic domain of HtrII from N.pharaonis (HtrII-cyt) can be expressed in E.coli in soluble form and then successfully purified • HtrII-cyt is shown to be unstructured (disordered) in common aqueous solutions • Drying and certain additives render HtrII-cyt α-helical with different efficacy • HtrII-cyt exists in solution in monomeric form, 4 M NaCl and KCl induce oligomerization with dimers being the most abundant species • HtrII-cyt has a rod-like shape of ~200 Å long and ~14 Å in diameter for the monomeric form, and ~250 Å long and ~20 Å in diameter as a dimer

  16. Part 2:human Epidermal Growth Factor Receptor (hEGFR)

  17. Epidermal Growth Factor Receptor (EGFR):general information • found in a number of epithelial tissues in human • transmembrane, 1186 a.a. long, 170 kDa, 8 domains (precursor peptide – 1212 a.a. with 26 a.a. signal sequence) • has 3 homologous proteins in humans (ErbB2, ErbB3 and ErbB4) and one each from D.melanogaster and C.elegans • posttranslationally glycosylated (20% of protein mass) • binds EGF, TGF-α and neuregulins • exists both as monomers and dimers • implicated in a variety of human cancers (e.g. mammary carcinoma, glioblastomas etc.) Burgess A. et al., Mol.Cell, 12 (2003), pp. 541-552

  18. residues 1-619 + EGF Ogiso H. et al., Cell, 110 (2002), pp. 775-787 Stamos J. et al., J.Biol.Chem., 277 (2002), pp. 46265-46272 residues 672-998 + ATP / kinase inhibitor Epidermal Growth Factor Receptor (EGFR):extracellular and kinase domains Burgess A. et al., Mol.Cell, 12 (2003), pp. 541-552

  19. residues 621-654 Rigby A. et al., Biochim.Biophys.Acta, 1371 (1998), pp. 241-253 residues 645-697 Choowongkomon K. et al., J.Biol.Chem., 280 (2005), pp. 24043-52 Epidermal Growth Factor Receptor (EGFR):trans- and juxtamembrane domains Burgess A. et al., Mol.Cell, 12 (2003), pp. 541-552

  20. 151 151 312 312 481 481 621 621 687 687 955 955 1186 1186 L1 L1 CR1 CR1 L2 L2 CR2 CR2 Kinase Kinase CT CT JM JM 644 644 Extracellular portion Extracellular portion Intracellular portion Intracellular portion Important information about the tj-EGFR • 73 amino acid residues (615-686 a.a.) (without tags) • carries N-terminal 7His-tag (HHHHHHH) • carries C-terminal StrepII-tag (WSHPQFEK) • molecular weight is about 10,152 Da • pI is around 11.2 • contains no Cys residues

  21. 20 15 10 tj-EGFR: why two tags?Relation to the previous studies Expression and purification m 1 2 3 4 m His-blot SDS-PAGE MALDI-TOF 20 15 MHHHHHHHGPKIPSIATGMVGALLLLLVVAL GIGLFMRRRH IVRKR TLRR LLQERELVEPLTPSGEAPNQALLRILKETE The results with tj-EGFR carrying ONLY 7His-tag were unsatisfactory

  22. 20 15 10 Expression of tj-EGFR in pET 27b+ E.coli BL21(DE3) Codon Plus RP m b 4 16 24 m b 4 16 24 m b 4 16 24 m b 4 16 24 20 10 Strep-blot His-blot 4 – 4 hours after induction16 – 16 hours after induction24 – 24 hours after induction m – markerb – before inductionred– at +37°C / blue– at + 28°C The optimal expression conditions for tj-EGFR are: +28°C, 24 hours

  23. open cells chelating Cu2+ Strep-Tactin RPC Purification of tj-EGFR in OG on Chelating and Strep-Tactin Sepharose SDS-PAGE His-blot Strep-Blot 20 15 10 tj-EGFR can be purified to homogeneity

  24. MALDI-TOF analysis of tj-EGFR (many thanks to Axel Niebisch, IBT-1) Only full-length tj-EGFR is observed: no degradation products

  25. CD spectra of tj-EGFR and secondary structure predictions: water and TFE

  26. CD spectra of tj-EGFR and secondary structure predictions: detergents 50 mM NaP pH 6.0,100 mM detergent

  27. Sequence-basedsecondary structure predictions ~ 60% α-helix and ~ 40% random coil MHHHHHHHGPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVR KRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEWSHPQFEK There is a discrepancy between the experimental and predicted secondary structure structure content

  28. NMR spectra of tj-EGFR in SDS and DPC 10 mM NaP pH 6.0 + SDS 10 mM NaP pH 6.0 + DPC 2D HSQC NMR spectra look promising in terms of peak assignment

  29. Conclusions, part 2 • The transmembrane + juxtamembrane domainof EGFRfromH.sapiens (tj-EGFR) can be expressed in E.coli and then successfully purified • tj-EGFR is prone to oligomerization/aggregation • The secondary structure of tj-EGFR is almost independent of the type of detergent • The tertiary structure of tj-EGFR strongly depends on the type of detergent, e.g. the presence of charged heads

  30. Acknowledgements FIRST my BIG BOSSES: • Prof. Judith Klein-Seetharaman (University of Pittsburgh) • Prof. Georg Büldt (Forschungszentrum Jülich, IBI-2) • Dr. Ramona Schlesinger (Forschungszentrum Jülich, IBI-2) • Dr.Valentin Gordeliy (MIPT) ... and then my NICE COLLEAGUES: • Dr.Olga Mironova (HtrII-cyt, cloning & purification) • Vijayalaxmi Manoharan (HtrII-cyt, NMR) • Naveena Yanamala (tj-EGFR, NMR) • Prof. Joe Zaccai and Dr. Vitaliy Pipich (HtrII-cyt, SANS)

  31. What is yet to be done?– I’m not leaving you right now! • HtrII-cyt project: • finalize the papers; • mutagenesis (if necessary); • try to obtain diffracting crystals. • EGFR project: • tj-EGFR: final CD in lipids; • tj-EGFR: cross-linking in lipids and detergents; • write up the paper; • prepare 13C, 15N sample (if necessary); • N-EGFR: express in COS-1 and develop purification strategy.

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