DETERMINATION OF THE SECONDARY STRUCTURE AND MOLECULAR TOPOLOGY OF INTERLEUKIN-1-BETA BY USE OF 2-DIMENSIONAL AND 3-DIMENSIONAL HETERONUCLEAR N-15-H-1 NMR-SPECTROSCOPY

A study of the regular secondary structure elements of recombinant human interleukin-1β 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., & Gronenborn, A. M. (1990) Biochemistry 29, 3542–3556]. In addition, accurate measurements of a large number of 3Jhnα 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-’H correlation spectrum of interleukin-1/3 on dissolving the protein in D20 solution. Analysis of these data indicate that the structure of interleukin-1/3 consists of 12 extended β-strands aligned in a single extended network of antiparallel β-sheet structure that in part folds into a skewed six-stranded β-barrel. In the overall structure the β-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-1β at 3-Å resolution [Priestie, J. P., Schär, H.-P., & Gnitter, 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 ß, which is characterized by an exchange rate that is slow on the NMR chemical shift time scale. © 1990, American Chemical Society. All rights reserved.