STRUCTURE OF THE BACILLUS-SUBTILIS PHAGE SPO1-ENCODED TYPE-II DNA-BINDING PROTEIN TF1 IN SOLUTION

The solution structure of a type II DNA-binding protein, the bacteriop hage SPO1-encoded transcription factor 1 (TF1), was determined using N MR spectroscopy. Selective H-2-labeling, C-13-labeling and isotopic he terodimers were used to distinguish contacts between and within monome rs of the dimeric protein. A total of 1914 distance and dihedral angle constraints derived from NMR experiments were used in structure calcu lations using restrained molecular dynamics and simulated annealing pr otocols. The ensemble of 30 calculated structures has a root-mean-squa re deviation (r.m.s.d.) of 0.9 Angstrom, about the average structure f or the backbone atoms, and 1.2 Angstrom for all heavy-atoms of the dim eric core (helices 1 and 2) and the beta-sheets. A severe helix distor tion at residues 92-93 in the middle of helix 3 is associated with r.m .s.d, of similar to 1.5 Angstrom for the helix 3 backbone. Deviations of similar to 5 Angstrom or larger are noted for the very flexible bet a-ribbon arms that constitute part of a proposed DNA-binding region. A structural model of TF1 has been calculated based on the previously r eported crystal structure of the homologous HU protein and this model was used as the starting structure for calculations. A comparison betw een the calculated average solution structure of TF1 and a solution st ructure of HU indicates a similarity in the dimeric core (excluding th e nine amino acid residue tail) with pairwise deviations of 2 to 3 Ang strom. The largest deviations between the average structure and the HU solution structure were found in the beta-ribbon arms, as expected. A 4 Angstrom deviation is found at residue 15 of TF1 which is in a loop connecting two helical segments; it has been reported that substituti on of Glu15 by Gly increases the thermostability of TF1. The homology between TF1 and other proteins of this family leads us to anticipate s imilar tertiary structures. (C) 1996 Academic Press Limited