CRYSTAL STRUCTURE  OF C-TERMINAL DOMAIN OF A MAJOR GRASS POLLEN ALLERGEN, phl p5b BY SIRAS METHOD

K.R. Rajashankar1, A. Bufe2, W. Weber1 and Ch. Betzel1

1 Institute fuer Physiologische Chemie, UKE, AG fuer Makromolekular Strukturanalyse, c/o DESY, Geb 22a, Notkestrasse 85, 22603, Hamburg
2 Forschungszentrum Borstel, Klinische Allergologie, Parkallee 22, 23845 Borstel
E-mail: raja@unisgi1.desy.de

Keywords: Allergy, Immunology, SIRAS, X-ray structure

Allergic diseases are increasingly becoming a major problem for  public health. Nearly 30% of bronchial asthma, allergic rhinitis and  atopic dermatitis are induced by grass pollen allergens. Development  of novel therapeutic methods for allergic diseases requires the molecular  structure of allergens and identification of their IgE-binding epitopes. We have determined the crystal structure of C-terminal domain of a major  pollen allergen, Phl p5b from timothy grass using SIRAS methods.

The parent recombinant Phl p5b was observed to undergo a  spontaneous conversion to various short forms, of molecular sizes between  10 to 29kDa. Interestingly, the C-terminal domain alone was selectively  crystallized from this heterogeneous mixture, indicating a stable and  compact domain structure(1). This domain is of high pharmacological  interest as it is found be preferentially recognized by IgE antibodies  of most pollen allergic patients.

The crystals are orthorhombic C2221 space group with  a=39.0, b=50.4, c=107.8 A with one molecule per asymmetric unit. Native  X-ray intensity data were collected to 2A using a flash frozen crystal and  employing synchrotron radiation on the beam line BW6(DESY). For obtaining  the phase information the crystals were soaked in Mercury Chloride  solution and X-ray data to 3A were collected using the home source.  Heavy atom positions were located using Patterson synthesis, and refined   using CCP4 suite of programs. Use of the partial anomalous data collected at  the beam line BM14 at ESRF augmented the structure determination. The model  was built to density modified SIRAS map using O program. Subsequent  refinement using X-PLOR and rebuilding resulted in present agreement factors  R=23.3% and R-Free=25.5% at 2A resolution.

As warranted by crystallization experiments, the molecule indeed adopts a compact structure, which is novel for allergen class of proteins. Details of the structure solution and structural description of the molecule will be presented.
 

1.  A. Bufe, Ch. Betzel, G. Shramm, A. Petersen, W.M. Becker, M. Schlaak, M. Perbandt, Z. Dauter & W. Weber, J.Biol.Chem. 271 (1996) 27193-27196