Elucidation of the origin of multiple conformations of the human alpha 3-chain type VI collagen C-terminal Kunitz domain: the reorientation of the Trp21 ring.

The human alpha 3-chain type VI collagen C-terminal Kunitz domain fragment (alpha 3(VI)) has been studied by two dimensional 1H-1H and 1H-13C NMR spectroscopy at 303 K. It is shown that the secondary structure of the protein is strikingly similar to that of BPTI, and a number of unusual H alpha chemical shifts, which are highly conserved in Kunitz-domain proteins, are also observed for a alpha 3(VI). Furthermore, a series of exchange cross peaks observed in 1H-1H spectra shows that a large number of protons in the central beta-sheet exist in two different chemical environments, corresponding to two unequally populated conformations that are slowly exchanging on the NMR time scale. Several protons, including Ser47(53) H alpha, Arg32(28) H(gamma 1) and H(gamma 2), and GLN48(54) H(beta 2), all located in the vicinity of the Trp21(27) ring in the crystal structure of alpha 3(VI) [Arnoux, B. et al. (1995) J. Mol. Biol., 246, 609-617], have very different chemical shifts in the two conformations, the most affected being Gln48(54) H(beta 2) (delta sigma = 3 ppm), which is placed directly above the Trp21(27) ring in the crystal structure of alpha 3(VI). It should be concluded that the origin of the multiple conformations of the central beta-sheet is a reorientation of the Trp21(27) ring. From the intensities of corresponding signals in the two conformations, the populations, the population of the minor conformation was found to be 6.4 +/- 0.2% of that of the major conformation, while a rate constant kM = 1.01 +/- 0.05 s-1 for the major to minor interconversion was obtained from a series of NOESY spectra with different mixing times. In addition, it is shown that Cys14(20)-Cys38(44) disulfide bond isomerization, previously observed in BPTI [Otting, G. et al. (1993) Biochemistry, 32, 3571-3582], is also likely to occur in alpha 3(VI).