SOLUTION STRUCTURE AND BACKBONE DYNAMICS OF THE HUMAN ALPHA-3-CHAIN TYPE-VI COLLAGEN C-TERMINAL KUNITZ DOMAIN

The solution structure and backbone dynamics of the 58-residue C-termi nal Kunitz domain fragment [alpha 3(VI)] of human alpha 3-chain type V I collagen has been studied by two-dimensional H-1-H-1 and H-1-N-15 nu clear magnetic resonance spectroscopy at 303 K. The solution structure is represented by an ensemble of 20 structures calculated with X-PLOR using 612 distance and 47 dihedral angle restraints. The distance res traints were obtained by a complete relaxation matrix analysis using M ARDIGRAS. The root mean squared (rms) deviation is 0.91 Angstrom for t he backbone atoms of the residues Thr2(8)-Gly12(18), Arg15(21)-Tyr35(4 1), and Gly40(46)-Pro57(63). The central beta-sheet [residues Ile18(24 )-Tyr35(41)] and the C-terminal alpha-helix [residues Gln48(54)-Cys55( 61)] are better defined with a backbone rms deviation of 0.46 Angstrom . The solution structure of alpha 3(VI) is virtually identical to the crystal structure of alpha 3(VI) and to the solution structure of bovi ne pancreatic trypsin inhibitor (BPTI). The N-15 spin-lattice and spin -spin relaxation rates and the H-1-N-15 heteronuclear nuclear Overhaus er enhancement (NOE) were analyzed using both the ''model-free'' forma lism [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559 and 4559-4570] and the reduced spectral density mapping procedure [Far row, N. A., Szabo, A., Torchia, D. A., & Kay, L. E. (1995) J. Biomol. NMR 6, 153-162]. The results obtained from the ''model-free'' analysis include an overall correlation time tau(c), of 3.00 ns and backbone o rder parameters S-2 in the range from 0.28 to 0.93. The necessity of i ncluding an exchange term in the analysis of the relaxation data from 14 residues indicated that these residues are involved in motions on t he micro-to millisecond time scale. The majority of the 14 residues ar e located in the vicinity of the Cys14(20)-Cys38(44) disulfide bond, s uggesting the presence of a disulfide bond isomerization similar to th e one observed in BPTI [Otting, G., Liepinsh, E., & Wuthrich, K. (1993 ) Biochemistry 32, 3571-3582]. It is suggested that this disulfide bon d isomerization is the main reason for the surprisingly small effect o n trypsin inhibition observed when Thr13(19) of alpha 3(VI) is substit uted with Pro.