1H and 15N resonance assignment and secondary structure of capsicein, an alpha-elicitin, determined by three-dimensional heteronuclear NMR.
The backbone 1H and 15N resonance assignments and solution secondary structure determination of capsicein, a protein of 98 residues with a molecular mass of 10161 Da, are presented. Capsicein belongs to the elicitin family, elicitor molecules having toxic and signaling properties that are secreted by Phytophthora fungi, responsible for the incompatible hypersensitive reaction of diverse plant species leading to resistance against fungal or bacterial plant pathogens. The protein was uniformly labeled with 15N to overcome spectral overlap of the proton resonances. A combination of 3D HOHAHA-HMQC and 3D NOESY-HMOC experiments allowed the identification of spin systems with through-bond correlations, which were in turn correlated by through-space connections. The sequential assignment was obtained for main- and side-chain resonances and led to the identification of all secondary structures. A 3D HMQC-NOESY-HMQC experiment was performed which characterized the NH(i)-NH(i+1) connections specific to alpha-helical structures. This proved particularly useful for the assignment of degenerate amide protons of successive residues in alpha-helical structures. The data show five alpha-helical regions comprising residues 5-18, 26-33, 44-58, 59-67, and 86-98 and a two-stranded antiparallel beta-sheet involving residues 70-75 and 80-85, packed around a hydrophobic core grouping all of the aromatic residues. The C-terminal secondary structure motifs of capsicein evoke phospholipase structural features, which suggests that elicitins might interact with the lipidic molecules of the plasma membrane.
Laboratoire de Résonance Magnétique Nucléaire, Institut de Chimie des Substances naturelles du CNRS, Gif-sur-Yvette, France.
S
S
Bouaziz
C
C
van Heijenoort
J C
JC
Huet
J C
JC
Pernollet
E
E
Guittet
D000595:Amino Acid Sequence; D004220:Disulfides / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010838:Phytophthora / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012996:Solutions
D004220:Disulfides; D005656:Fungal Proteins; D012996:Solutions; C061033:Alpha-elicitin capsicein protein, Phytophthora capsici