Determination of a high precision structure of a novel protein, Linum usitatissimum trypsin inhibitor (LUTI), using computer-aided assignment of NOESY cross-peaks

Citation
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
Citations number
54
Categorie Soggetti
Molecular Biology & Genetics
Journal title
JOURNAL OF MOLECULAR BIOLOGY
ISSN journal
00222836 → ACNP
Volume
302
Issue
5
Year of publication
2000
Pages
1179 - 1192
Database
ISI
SICI code
0022-2836(20001006)302:5<1179:DOAHPS>2.0.ZU;2-H
Abstract
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.