(1) Uniti de Biochimie & Structure des Protiines. INRA. 78352. Jouy-en-Josas.
France
(2) L.U.R.E (France) and MRC-LMB, Hills Road. Cambridge CB2 2QH (UK)
(3) IBS. 41, Av. des Martyrs. 38027 Grenoble. France
Elicitins are a family of more or less toxic elicitors (Mr=10kDa) secreted by Phytophthora fungi. They induce a hypersensitive reaction and serve as signals for the interaction between plants and pathogens. When applied to tobacco plant, they induce leaf necroses and, in addition elicit protection against a subsequent inoculation with the tobacco pathogen P. nicotianae which causes tobacco black shank. To date, 12 elicitins have been sequenced and regrouped in two groups according to pI: the a class includes acidic molecules whereas the b class regroups those having a basic pI. This classification also corresponds to their relative toxicity since, for a similar protection level, b elicitins are 100 fold more toxic than the a ones. Detailed sequence analysis (66% match between the whole set) led to the identification of a few residues correlated to the toxicity. This work deals with the structure determination of a basic elicitin, bcryptoge in, undertaken [1] to elucidate the molecular mechanisms involved in the signalling pathway.
The absence of sequence homology with structurally characterised proteins directed efforts towards a search for isomorphous heavy atom derivatives. Only a tetrachloroplatinate derivative of poor phasing power (absolute occupation factor = 0.35) was obtained. Nevertheless, this derivative was perfectly suitable to a MAD experiment. A 4 wavelengths data collection was carried out on the D2AM beam line at ESRF (Grenoble, France). The exceptional beam brilliance combined with the CCD detector speed allowed the accumulation of 4x135 frames during less then 6 hours on a cryocooled crystal. The 4 data sets were reduced using XDS (Rsym = 4.9%, completeness of 95% and 6.5 multiplicity over 8-2.2Å). This data enabled us to identify a single Pt site on both anomalous and dispersive Patterson maps.
Phasing efforts with MLPHARE (CCP4 suite) and HEAVY were unsuccessful. The maximum-likelihood heavy-atom parameters refinement performed by SHARP [2] and the addition of a native data set in the phases calculation led to the successful structure determination. The solvent flattening procedure carried out using SOLOMON clarified ambiguities due to enantiomorphicity of the space group (P4122) and solvent content (66%). The total lack of ambiguity combined with high quality electron density map continuity sped up the model building. The model refinement performed with X-PLOR has led to a final crystallographic R factor of 22.7% (Rfree=31.8%) between 8Å and 2.2Å. Rms deviations from bond lengths and bond angles stereochemical standards are 0.012Å and 1.500 respectively. The structure of cryptogein consists of 6 helices and a b sheet and seems to correspond to a new folding type.
[1] Guilloteau et al. J. Mol. Biol. (1993), 229, 564-565
[2] Bricogne, G. (1993). Acta Cryst. D49, 37-60;
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