NMR SOLUTION STRUCTURE OF THE C-TERMINAL FRAGMENT-255-316 OF THERMOLYSIN - A DIMER FORMED BY SUBUNITS HAVING THE NATIVE STRUCTURE

The solution structure of the C-terminal fragment 255-316 of thermolys in has been determined by two-dimensional proton NMR methods. For this disulfide-free fragment there was a previous proposal according to wh ich it would fold into a stable helical structure forming a dimer at c oncentrations above 0.06 mM. A complete assignment of the proton NMR r esonances of the backbone and amino acid side chains of the fragment w as first performed using standard sequential assignment methods. On th e basis of 729 distance constraints derived from unambiguously assigne d nuclear Overhauser effect (NOE) proton connectivities, the three-dim ensional structure of a monomeric unit was then determined by using di stance geometry and restrained molecular dynamic methods. The globular structure of fragment 255-316 of thermolysin in solution, composed of three helices, is largely coincident with that of the corresponding r egion in the crystallographic structure of intact thermolysin [Holmes, M. A., and Matthews, B. W. (1982) J. Mel. Biol. 160, 623-639]. This f act allowed identification as intersubunit of up to 52 NOE cross corre lations, which were used to dock the two subunits into a symmetric dim er structure. The obtained dimeric structure served as the starting st ructure in a final restrained molecular dynamic calculation subjected to a total of 1562 distance constraints. In the resulting dimeric stru cture, the interface between the two subunits, of a marked hydrophobic character, coincides topologically with the one between the 255-316 f ragment and the rest of the protein in the intact enzyme. The present work decisively shows that the thermolysin fragment 255-316 can attain a stable and nativelike structure independently of the rest of the po lypeptide chain. Considering that the thermolysin molecule is constitu ted of two structural domains of equal size (residues 1-157 and 158-31 6), the results of this study show that autonomously folding units can be substantially smaller than entire domains.