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					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
              Part 1:

halobacterial transducer II (HtrII)
from Natronobacterium pharaonis
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
    Two-component signal 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
           Design, expression, purification and
           initial characterization of HtrII-cyt
                        Tsr, T286                                                                        Tsr, A526




          HtrII, M234                                                     HtrII, D504

                                                Le Moual H. and Koshland D.E., J.Mol.Biol., 261 (1996), pp. 568-585

  1,2                          m    1   2   3        4       5
  open the cells
     3
         salting out
 4
 HIC purification
     5
     gel-filtration



The cytoplasmic fragment of HtrII can be expressed in E.coli and
     purified to homogeneity and is unstructured in solution
            Structural predictions for HtrII-cyt


                                 76% α-helix and 24% random coil     Combet C. et al., TIBS, 291 (2000), pp. 147-150




                                                                   Lupas A. et al., Science, 252 (1991), pp. 1162-1164

O. Lund et al., “CPHmodels 2.0: X3M
   a Computer Program to Extract
                                      The cytoplasmic fragment of HtrII is predicted
      3D Models”, A102 abstract
   at the CASP5 conference, 2002
                                      to be α-helical and to form coiled coil structure
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
            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
          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
                   FTIR spectroscopy
  10 mM Tris-HCl pH 9.0 in D2O                    dry film

                                          1654
       1644




                                                             adapted from
               wavenumber, cm-1       assignment               Stuart B.
                                                                (1997),
              1621-1640, 1671-1679     β-structure             Biological
                  1641-1647           random coil            Applications of
                                                                Infrared
                  1651-1657             α-helix              Spectroscopy,
                                                              University of
              1658-1671, 1681-1690   turns and bends         Greenwich, UK


FTIR indicates random coil in solution and a-helix in dry film
                                                  NMR spectroscopy
         10 mM NaP pH 6.0                                                                            10mM NaP pH 6.0 + 70% glycerol




                                                                                                                                                     110
                                                                                                     Red:




                                                                                                                                                           chemical shift, ppm
                                                                                                     1H-15N HSQC

                                                                                                     Green:




                                                                                                                                                     115
                                                                                                     Trosy-HSQC
10   9     8    7         6   5     4   3     2   1     0




                                                                                                                                                     120
                                                                              chemical shift, ppm
                                                            125 120 115 110




                                                                                                                                                           15N
                                                                                                                                                     125
                                                                                                           9.0                 8.0             7.0
                                                                                                                    1H   chemical shift, ppm
                                                                              15N




     8.5
               1H
                    8.0       7.5       7.0       6.5                                                           peaks shifted and broadened
                    chemical shift, ppm
                                                                                                                strong Trosy effect
    minimal spectral dispersion
    negative het-NOE signals                                                                              NMR data support
     (data not shown)                                                                               structural transitions in glycerol
Analytical gel-filtration chromatography
                  (AGFC)
     ammonium sulfate                 KCl and NaCl




 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
Analytical gel-filtration chromatography
  (AGFC) and chemical cross-linking




                                      1xPBS + 4 M KCl 1xPBS + 40% as
1xPBS
1xPBS + 10% as




      Cross-linking data evidence HtrII-cyt dimerization in 4 M KCl
    Small-angle neutron scattering and
         atomic force microscopy
                                  AFM with HtrII-cyt in dry film
   SANS with HtrII-cyt




                                 (many thanks to Dirk Mayer, ISG-2)
HtrII-cyt has characteristic size of ~180-200 Å
       and elongated shape in solution
                    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
           Part 2:

human Epidermal Growth Factor
      Receptor (hEGFR)
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
Epidermal Growth Factor Receptor (EGFR):
     extracellular and kinase domains
                                 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



  Burgess A. et al., Mol.Cell,
   12 (2003), pp. 541-552
Epidermal Growth Factor Receptor (EGFR):
   trans- and juxtamembrane domains
                                  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

  Burgess A. et al., Mol.Cell,
   12 (2003), pp. 541-552
Important information about the tj-EGFR

     151     312    481      621   687         955       1186
 L1        CR1     L2     CR2      JM     Kinase       CT
                                644
     Extracellular portion            Intracellular portion

     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
               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
                                                                                                                    Voyager Spec #1=>BC=>NR(2.00)[BP = 5741.6, 10904]
                                                                                                                                               5742.33                                        1.1E+4
                                                                       100
                                                                                                                           4819.71

                                                                       90


                                                                       80


                                                                       70

                                                                                                                     4557.40
                                                                       60




                                                       % In ten sity
                                                                       50
                                                                                                                                               5757.75                         9109.28
                                                                       40
                                                                                 2111.82                             4564.95
                                                                                           2874.41
                                                                       30

                                20                                                                                                   5216.33
20                                                                     20                            3390.85         4533.96


                                                                       10
15
10                              15                                       0
                                                                        1957.0                             3810.8                          5664.6                     7518.4     9372.2
                                                                                                                                                                                              0
                                                                                                                                                                                          11226.0
                                                                                                                                                         Mass (m/z)




     MHHHHHHHGPKIPSIATGMVGALLLLLVVAL GIGLFMRRRH
     IVRKR TLRR LLQERELVEPLTPSGEAPNQALLRILKETE
           The results with tj-EGFR carrying ONLY 7His-tag
                          were unsatisfactory
       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
                                         20
                                         15
10                                       10


                Strep-blot                                  His-blot
     m – marker                               4 – 4 hours after induction
     b – before induction                     16 – 16 hours after induction
     red – at +37°C / blue – at + 28°C        24 – 24 hours after induction

        The optimal expression conditions for tj-EGFR are:
                        +28°C, 24 hours
  Purification of tj-EGFR in OG on
Chelating and Strep-Tactin Sepharose

                            SDS-PAGE His-blot Strep-Blot



   open cells

  chelating Cu2+

   Strep-Tactin
                       20



                       15
       RPC

                       10




       tj-EGFR can be purified to homogeneity
                           MALDI-TOF analysis of tj-EGFR
                                                     Voyager Spec #1=>RSM2000[BP = 10161.8, 7314]

                                                                                                         10161.32                  7313.8
                 100


                 90


                 80


                 70                                                                                      10217.60


                 60                                  5077.00
% In ten sity




                 50


                 40

                                                                                                          10274.98
                 30                                   5104.76

                                                                                                       10057.23
                 20                                   5135.56                                          10028.83
                                                    4954.85                                            10013.10
                 10                       4428.86


                  0                                                                                                                0
                  999.0          3799.4                           6599.8                      9400.2                 12200.6   15001.0
                                                                             Mass (m/z)


                (many thanks to Axel Niebisch, IBT-1)


                 Only full-length tj-EGFR is observed: no degradation products
CD spectra of tj-EGFR and secondary
structure predictions: water and TFE




            α-helix   β-sheet   turn    random

    water    18%      29.5%     22.5%    30%
    TFE      40%       13%      18.5%   28.5%
CD spectra of tj-EGFR and secondary
 structure predictions: detergents



                                50 mM NaP pH 6.0,
                                100 mM detergent




               α-helix   β-sheet      turn    random
        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%
              Sequence-based
       secondary structure predictions


MHHHHHHHGPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVR
KRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEWSHPQFEK




    ~ 60% α-helix and ~ 40% random coil

   There is a discrepancy between the experimental and
     predicted secondary structure structure content
   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
                   Conclusions, part 2


   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

   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
                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)
              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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