The¹H,¹³C,¹⁵N resonance assignment, solution structure, and residue level stability of eosinophil cationic protein/RNase 3 determined by NMR spectroscopy

Abstract

Eosinophil cationic protein (ECP)/human RNase 3, a member of the RNase A family, is a remarkably cytotoxic protein implicated in asthma and allergies. These activities are probably due to ECP's ability to interact with and disrupt membranes and depend on two Trp, 19 Arg, and possibly an extremely high conformational stability. Here, we have used NMR spectroscopy to assign essentially all (1)H, (15)N, and backbone (13)C resonances, to solve the 3D structure in aqueous solution and to quantify its residue-level stability. The NMR solution structure was determined on the basis of 2316 distance constraints and is well-defined (backbone RMSD = 0.81 A). The N-terminus and the loop composed of residues 114-123 are relatively well-ordered; in contrast, conformational diversity is observed for the loop segments 17-22, 65-68, and 92-95 and most exposed sidechains. The side chain NH groups of the two Trp and 19 Arg showed no significant protection against hydrogen/deuterium exchange. The most protected NH groups belong to the first and last two beta-strands, and curiously, the first alpha-helix. Analysis of their exchange rates reveals a strikingly high global stability of 11.8 kcal/mol. This value and other stability measurements are used to better quantify ECP's unfolding thermodynamics.

Antimicrobial Action and Cell Agglutination by the Eosinophil Cationic Protein Are Modulated by the Cell Wall Lipopolysaccharide Structure

“…Only the D21 strain contains the phosphate residues associated with the heptose residues. (C) Structure scheme for ECP and derived peptides showing [6][7][8][9][10][11][12][13][14][15][16][17]-Ahx- [23][24][25][26][27][28][29][30][31][32][33][34][35][36] structure based on nuclear magnetic resonance (NMR) information (16,21,35).…”

The sulfate-binding site structure of the human eosinophil cationic protein as revealed by a new crystal form

“…Interestingly, nitration of Tyr 33, which has been described to occur in vivo (Ulrich et al, 2008), would provide a negative charge that may enhance the interaction with the e amino group of Lys 38. Holding Lys 38 in an inactive conformation was suggested (Laurents et al, 2009) to be one of the reasons for ECP reduced catalytic activity.…”

“…The NMR structure, 2KB5 (Laurents et al, 2009), further defined the most mobile regions of the protein in solution. Conformational diversity was observed for loop segments 17-22, 65-68 and 92-95, where segment 92-95 (L7) showed the largest RMSD between NMR models.…”

“…1DYT (Mallorqui-Fernandez et al, 2000), and with the threedimensional NMR structure (2KB5 (Laurents et al, 2009)). The first crystal structure reported was solved at 2.4 Å (Boix et al, 1999a).…”

“…We also compare the new monoclinic crystal form to the previously reported hexagonal and tetragonal crystal forms (Boix et al, 1999a;Mallorqui-Fernandez et al, 2000) and to the NMR solution structure (Laurents et al, 2009). …”