Solution structure of a lipid transfer protein extracted from rice seeds -Comparison with homologous proteins

Nuclear magnetic resonance (NMR) spectroscopy was used to determine the thr ee dimensional structure of rice nonspecific lipid transfer protein (ns-LTP ), a 91 amino acid residue protein belonging to the broad family of plant n s-LTP. Sequence specific assignment was obtained for all but three HN backb one H-1 resonances and for more than 95% of the H-1 side-chain resonances u sing a combination of H-1 2D NOESY; TOCSY and COSY experiments at 293 K. Th e structure was calculated on the basis of four disulfide bridge restraints , 1259 distance constraints derived from H-1-H-1 Overhauser effects, 72 phi angle restraints and 32 hydrogen-bond restraints. The final solution struc ture involves four helices (H1.: Cys3-Arg18, H2: Ala25-Ala37, H3: Thr41-Ala 54 and H4: Ala66-Cys73) followed by a long C-terminal tail (T) with no obse rvable regular structure. N-capping residues (Thr2, Ser24, Thr-40), whose s idechain oxygen atoms are involved in hydrogen bonds with i + 3 amide proto n additionally stabilize the N termini of the first three helices. The four th helix involving Pro residues display a mixture of alpha and 3(10) confor mation. The rms deviation of 14 final structures with respect to the averag e structure is 1.14 +/- 0.16 Angstrom for all heavy atoms (C, N, O and S) a nd 0.72 +/- 0.01 Angstrom for the backbone atoms. The global fold of rice n s-LTP is close to the previously published structures of wheat, barley and maize ns-LTPs exhibiting nearly identical pattern of the numerous sequence specific interactions. As reported previously for different four-helix topo logy proteins, hydrophobic, hydrogen bonding and electrostatic mechanisms o f fold stabilization were found for the lice ns-LTP. The sequential alignme nt of 36 ns-LTP primary structures strongly suggests that there is a unifor m pattern of specific long-range interactions tin terms of sequence), which stabilize the fold of all plant ns-LTPs.