Determination of a high precision structure of a novel protein, Linum usitatissimum trypsin inhibitor (LUTI), using computer-aided assignment of NOESY cross-peaks
T. Cierpicki et J. Otlewski, Determination of a high precision structure of a novel protein, Linum usitatissimum trypsin inhibitor (LUTI), using computer-aided assignment of NOESY cross-peaks, J MOL BIOL, 302(5), 2000, pp. 1179-1192
The solution structure of a novel 69 residue proteinase inhibitor, Linum us
itatissimum trypsin inhibitor (LUTI), was determined using a method based o
n computer aided assignment of nuclear Overhauser enhancement spectroscopy
(NOESY) data. The approach applied uses the program NOAH/DYANA for automati
c assignment of NOESY cross-peaks. Calculations were carried out using two
unassigned NOESY peak lists and a set of determined dihedral angle restrain
ts. In addition, hydrogen bonds involving amide protons were identified dur
ing calculations using geometrical criteria and values of HN temperature co
efficients. Stereospecific assignment of beta-methylene protons was carried
out using a standard procedure based on nuclear Overhauser enhancement int
ensities and (3)J(alpha beta) coupling constants. Further stereospecific as
signment of methylene protons and diastereotopic methyl groups were establi
shed upon structure-based method available in the program GLOMSA and chemic
al shift calculations. The applied algorithm allowed us to assign 1968 out
of 2164 peaks (91%) derived from NOESY spectra recorded in H2O and (H2O)-H-
2. The final experimental data input consisted of 1609 interproton distance
restraints, 88 restraints for 44 hydrogen bonds, 63 torsion angle restrain
ts and 32 stereospecifically assigned methylene proton pairs and methyl gro
ups. The algorithm allowed the calculation of a high precision protein stru
cture without the laborious manual assignment of NOESY cross-peaks. For the
20 best conformers selected out of 40 refined ones in the program CNS, the
calculated average pairwise rmsd values for residues 3 to 69 were 0.38 Ang
strom (backbone atoms) and 1.02 Angstrom tall heavy atoms). The three-dimen
sional LUTI structure consists of a mixed parallel and antiparallel beta-sh
eet, a single alpha-helix and shows the fold of the potato 1 family of prot
einase inhibitors. Compared to known structures of the family LUTI contains
Arg and Trp residues at positions P-6' and P-8', respectively, instead of
two Arg residues, involved in the proteinase binding loop stabilization. A
consequence of the Arg --> Trp substitution at P-8' is a slightly more comp
act conformation of the loop relative to the protein core. (C) 2000 Academi
c Press.