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

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

6. Structural predictions for HtrII-cyt Combet C. et al., TIBS, 291 (2000), pp. 147-150 76% α -helix and 24% random coil O. Lund et al., “CPHmodels 2.0: X3M a Computer Program to Extract a Computer Program to Extract 3D Models”, A102 abstract 3D Models”, A102 abstract at the CASP5 conference, 2002 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

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

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

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

10. FTIR spectroscopy wavenumber, cm -1 assignment 1621-1640, 1671-1679β-structure 1641-1647random coil 1651-1657α-helix 1658-1671, 1681-1690turns and bends 1644 1654 adapted from Stuart B. (1997), Biological Applications of Infrared Spectroscopy, University of Greenwich, UK FTIR indicates random coil in solution and  -helix in dry film 10 mM Tris-HCl pH 9.0 in D 2 O dry film

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

12. Analytical gel-filtration chromatography (AGFC) Abnormal retention volumes of the cytoplasmic fragment of HtrII evidence its non-globular shape; ammonium sulfate induces hydrophobic interactions with the column up to complete retention ammonium sulfate KCl and NaCl

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

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

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 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 HtrII-cyt is shown to be unstructured (disordered) in common aqueous solutions Drying and certain additives render HtrII-cyt α -helical with different efficacy 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 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 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 found in a number of epithelial tissues in human transmembrane, 1186 a.a. long, 170 kDa, 8 domains transmembrane, 1186 a.a. long, 170 kDa, 8 domains (precursor peptide – 1212 a.a. with 26 a.a. signal sequence) (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 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) posttranslationally glycosylated (20% of protein mass) binds EGF, TGF-α and neuregulins binds EGF, TGF-α and neuregulins exists both as monomers and dimers exists both as monomers and dimers implicated in a variety of human cancers (e.g. mammary carcinoma, glioblastomas etc.) 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. Epidermal Growth Factor Receptor (EGFR): extracellular and kinase domains Burgess A. et al., Mol.Cell, 12 (2003), pp. 541-552 Ogiso H. et al., Cell, 110 (2002), pp. 775-787 residues 1-619 + EGF Stamos J. et al., J.Biol.Chem., 277 (2002), pp. 46265-46272 residues 672-998 + ATP / kinase inhibitor

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

20. Important information about the tj-EGFR 73 amino acid residues (615-686 a.a.) (without tags) 73 amino acid residues (615-686 a.a.) (without tags) carries N-terminal 7His-tag (HHHHHHH) carries N-terminal 7His-tag (HHHHHHH) carries C-terminal StrepII-tag (WSHPQFEK) carries C-terminal StrepII-tag (WSHPQFEK) molecular weight is about 10,152 Da molecular weight is about 10,152 Da pI is around 11.2 pI is around 11.2 contains no Cys residues contains no Cys residues L1CR1L2CR2 JM KinaseCT 644 1513124816216879551186 Extracellular portionIntracellular portion L1CR1L2CR2 JM KinaseCT 644 1513124816216879551186 Extracellular portionIntracellular portion

21. MHHHHHHHGPKIPSIATGMVGALLLLLVVAL GIGLFMRRRH IVRKR TLRR LLQERELVEPLTPSGEAPNQALLRILKETE tj-EGFR: why two tags? Relation to the previous studies Expression and purification The results with tj-EGFR carrying ONLY 7His-tag were unsatisfactory MALDI-TOF 15 1020 m 1 2 3 4 15 20 m His-blot SDS-PAGE

22. Expression of tj-EGFR in pET 27b+ E.coli BL21(DE3) Codon Plus RP m – marker b – before induction red – at +37°C / blue – at + 28°C 4 – 4 hours after induction 16 – 16 hours after induction 24 – 24 hours after induction 15 102020 10 Strep-blotHis-blot m b 4 16 24 m b 4 16 24 The optimal expression conditions for tj-EGFR are: +28°C, 24 hours

23. Purification of tj-EGFR in OG on Chelating and Strep-Tactin Sepharose SDS-PAGE His-blot Strep-Blot open cells chelating Cu 2+ Strep-Tactin RPC 15 10 20 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 α-helixβ-sheetturnrandom water 18%29.5%22.5%30% TFE 40%13%18.5%28.5%

26. CD spectra of tj-EGFR and secondary structure predictions: detergents 50 mM NaP pH 6.0, 100 mM detergent α-helixβ-sheetturnrandom OG 21%28%23%28% SDS 21%29%20%30% DPC 32%21%20%27% DHPC 23%25%22%30% LPPG 25%26%19%30%

27. ~ 60% α-helix and ~ 40% random coil Sequence-based secondary structure predictions 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 2D HSQC NMR spectra look promising in terms of peak assignment 10 mM NaP pH 6.0 + SDS 10 mM NaP pH 6.0 + DPC

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

30. AcknowledgementsAcknowledgements FIRST my BIG BOSSES: Prof. Judith Klein-Seetharaman (University of Pittsburgh) Prof. Judith Klein-Seetharaman (University of Pittsburgh) Prof. Georg Büldt (Forschungszentrum Jülich, IBI-2) Prof. Georg Büldt (Forschungszentrum Jülich, IBI-2) Dr. Ramona Schlesinger (Forschungszentrum Jülich, IBI-2) Dr. Ramona Schlesinger (Forschungszentrum Jülich, IBI-2) Dr. Valentin Gordeliy (MIPT) Dr. Valentin Gordeliy (MIPT)... and then my NICE COLLEAGUES: Dr. Olga Mironova (HtrII-cyt, cloning & purification) Dr. Olga Mironova (HtrII-cyt, cloning & purification) Vijayalaxmi Manoharan (HtrII-cyt, NMR) Vijayalaxmi Manoharan (HtrII-cyt, NMR) Naveena Yanamala (tj-EGFR, NMR) Naveena Yanamala (tj-EGFR, NMR) Prof. Joe Zaccai and Dr. Vitaliy Pipich (HtrII-cyt, SANS) 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: HtrII-cyt project: - finalize the papers; - mutagenesis (if necessary); - try to obtain diffracting crystals. EGFR project: 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.