1H and 15N Resonance Assignment and Secondary Structure of Capsicein, an .alpha.-Elicitin, Determined by Three-Dimensional Heteronuclear NMR

Abstract

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.

Three‐dimensional solution structure of β cryptogein, a β elicitin secreted by a phytopathogenic fungus phytophthora cryptogea

“…Three disulfide bridges (Cys3-Cys71, Cys27-Cys56, and Cys5I-Cys95) were established by NMR in / 3 cqptogein and are similar to those previously identified in capsicein (Bouaziz et al, 1994a(Bouaziz et al, , 1994b. The first one, linking a1 to the p-sheet, was also assessed by chemical means.…”

“…In vivo "N-labeling of p cryptogein was performed according to that of capsicein (Bouaziz et al, 1994b), except that I5N glycine was used as nitrogen source instead of nitrate. Samples were prepared by dissolving lyophilized unlabeled or ISN-labeled cryptogein in 0.3 mL at a 3mM concentration in H20/D20 (9:l) or and analyzed using FELIX (95.0).…”

Distant homology recognition using structural classification of proteins

“…Literature searches revealed that the secondary structure, disulfide bonds and some other long-range contacts, were determined for a close homologue of the target sequence. 13 No fold in SCOP was compatible with these data, so we were very confident in our ''negative'' prediction. In the other three new fold targets, T0005, T0010, and T0016, we could not rule out the possibility of distant relationship to known structures; therefore, with a different degree of confidence, we assigned a single SCOP superfamily for each of these targets.…”

Fold recognition using predicted secondary structure sequences and hidden Markov models of protein folds

“…A BLAST search 14 returned an alignment of the last 87 (out of 98), residues of T0032 to the alpha-elicitin capsicein protein (Swissprot ID: P15571) with 86% sequence identity. The secondary structure of this alpha-elicitin capsicein protein had already been determined by NMR spectroscopy by Bouaziz et al 15 As the sequence identity was quite high over an extended region, the secondary structure of T0032 was simply predicted to be identical to the NMR assignment of alpha-elicitin, and consisting of five alpha helices and two strands. This predicted sequence achieved the prediction accuracy Q3 ϭ 79.6%, with 97.8% of secondary structure elements correctly predicted.…”