AN H-1-NMR DETERMINATION OF THE 3-DIMENSIONAL STRUCTURES OF MIRROR-IMAGE FORMS OF A LEU-5 VARIANT OF THE TRYPSIN-INHIBITOR FROM ECBALLIUM-ELATERIUM (EETI-II)

The 3-dimensional structures of mirror-image forms of a Leu-5 variant of the trypsin inhibitor Ecballium elaterium (EETI-II) have been deter mined by H-1 NMR spectroscopy and simulated annealing calculations inc orporating NOE-derived distance constraints. Spectra were assigned usi ng 2-dimensional NMR methods at 400 MHz, and internuclear distances we re determined from NOESY experiments. Three-bond spin-spin couplings b etween C alpha H and amide protons, amide exchange rates, and the temp erature dependence of amide chemical shifts were also measured. The st ructure consists largely of loops and turns, with a short region of be ta-sheet. The Leu-5 substitution produces a substantial reduction in a ffinity for trypsin relative to native EETI-II, which contains an lie at this position. The global structure of the Leu-5 analogue studied h ere is similar to that reported for native EETI-II (Heitz A, Chiche L, Le-Nguyen D, Castro B, 1989, Biochemistry 28:2392-2398) and to X-ray and NMR structures of the related proteinase inhibitor CMTI-I (Bode W et al., 1989, FEBS Lett 242:285-292; Holak TA et al., 1989a, J Mol Bio l 210:649-654; Holak TA, Gondol D, Otlewski J, Wilusz T, 1989b, J Mol Biol 210:635-648; Holak TA, Habazettl J, Oschkinat H, Otlewski J, 1991 , J Am Chem Soc 113:3196-3198). The region near the scissile bond is t he most disordered part of the structure, based on geometric superimpo sition of 40 calculated structures. This disorder most likely reflects additional motion being present in this region relative to the rest o f the protein. This motional disorder is increased in the Leu-5 analog ue relative to the native form and may be responsible for its reduced trypsin binding. A second form of the protein synthesized with all (D) amino acids was also studied by NMR and found to have a spectrum iden tical with that of the (L) form. This is consistent with the (D) form being a mirror image of the (L) form and not distinguishable by NMR in an achiral solvent (i.e., H2O). The (D) form has no activity against trypsin, as would be expected for a mirror-image form.