Determination of the secondary structure and molecular topology of interleukin-1.beta. by use of two- and three-dimensional heteronuclear nitrogen-15-proton NMR spectroscopy

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Abstract
A study of the regular secondary structure elements of recombinant human interleukin-1.beta. has been carried out using NMR spectroscopy. Using a randomly 15N labeled sample, a number of heteronuclear three- and two-dimensional NMR experiments have been performed, which have enabled a complete analysis of short-, medium-, and long-range NOEs between protons of the polypeptide backbone, based on the sequence-specific resonance assignments that have been reported previously [Driscoll, P. C., Clore, G. M., Marion, D., Wingfield, P. T., and Gronenborn, A. M. (1990) Biochemistry 29, 3542-3556]. In addition, accurate measurements of a large number of 3JHN.alpha. coupling constants have been carried out by two-dimensional heteronuclear multiple-quantum-coherence-J spectroscopy. Amide NH solvent exchange rates have been measured by following the time dependence of the 15N-1H correlation spectrum of interleukin-1.beta. on dissolving the protein in D2O solution. Analysis of these data indicate that the structure of interleukin-1.beta. consists of 12 extended .beta.-strands aligned in a single extended network of antiparallel .beta.-sheet structure that in part folds into a skewed six-stranded .beta.-barrel. In the overall structure the .beta.-strands are connected by tight turns, short loops, and long loops in a manner that displays approximate pseudo-three-fold symmetry. The secondary structure analysis is discussed in the light of the unrefined X-ray structure of interleukin-1.beta. at 3-.ANG. resolution [Priestle, J. P., Schar, H.-P., and Grutter, M. G. (1988) EMBO J. 7, 339-343], as well as biological activity data. Discernible differences between the two studies are highlighted. Finally, we have discovered conformational heterogeneity in the structure of interleukin-1.beta., which is characterized by an exchange rate that is slow on the NMR chemical shift time scale.