Secondary structure and topology of interleukin-1 receptor antagonist protein determined by heteronuclear three-dimensional NMR spectroscopy

Abstract

Interleukin-1 (IL-1) proteins, such as IL-1 beta, play a key role in immune and inflammatory responses. Interaction of these cytokines with the IL-1 receptor induces a variety of biological changes in neurologic, metabolic, hematologic, and endocrinologic systems. Interleukin-1 receptor antagonist protein (IRAP) is a naturally occurring inhibitor of the interleukin-1 receptor. The 153-residue protein binds to the receptor with an affinity similar to that of IL-1 beta but does not elicit any physiological responses. As a first step toward understanding IRAP's mode of action, we have used multidimensional, heteronuclear NMR spectroscopy to determine the antagonist's solution secondary structure and global fold. Using a combination of 3D 1H-15N NOESY-HMQC and TOCSY-HMQC and 3D 1H-15N-13C HNCA and HN(CO)CA experiments on uniformly 15N- or doubly 13C/15N-enriched IRAP, we have made resonance assignments for more than 90% of the main-chain atoms. Analysis of short- and long-range NOE's indicates that IRAP is predominantly beta-sheet, with the same overall topology as IL-1 beta but with different regions of the primary sequence comprising the beta-strands. Two short helical segments also were identified. The 14% sequence identity between IL-1 beta and IRAP increases to 25% when differences in the locations of secondary structure elements in the primary sequences are taken into account. Still, numerous differences in side chains, which ultimately play a major role in receptor interaction, exist.(ABSTRACT TRUNCATED AT 250 WORDS)

Initial crystallographic analysis of a recombinant human interleukin-1 receptor antagonist protein

“…structural class. Secondary structure assignments extracted from our model agree closely with those observed by NMR for IRAP in solution (Stockman et al, 1992). 12 fl-strands characteristic of molecules that share this architecture are found as arranged in IL-lfl, with six strands distributed around one end of a six-stranded fl-barrel.…”

The 13C Chemical-Shift Index: A simple method for the identification of protein secondary structure using 13C chemical-shift data

“…Conse- (Grzesiek et al, 1992) 92 88 -89 94 Calmodulin* (Ikura et al, 1990(Ikura et al, , 1991 91 76 98 82 94 Ribonuclease H* (Yamazaki et al, 1991(Yamazaki et al, , 1993 86 75 94 85 94 Interleukin 4* (Powers et al, 1992) 90 93 99 90 93 hn RNP c* (Wittekind et al, 1992) 88 89 -84 93 SH2 Domain-pl T (J. Forman-Kay) b 88 -83 83 93 FKB 506 (Xu et al, 1993) 88 74 88 89 93 Digoxin antibody (Constantine et al, 1993) 88 -87 87 92 Tendamistat* (Kessler et al, 1990) 85 -78 85 91 Glucose Perm. IIA* (Fairbrother et al, 1992) 85 --84 -IRAP* (Stockman et al, 1992) 82 --81 -BPTI* (Wagner and Bruhwiler, 1986) 78 --78 -* Indicates that the chemical shifts from these proteins were used in compiling " Chemical-shift data unavailable or not published. b Personal communication.…”

Solution structure of human interleukin‐1 receptor antagonist protein

“…lB, which shows an overlay of the backbone atoms of the two proteins. The alignment is based on homologous B-strands as defined previously [16,17]. For this subset of 100 residues, the RMS deviation between the two proteins for the backbone N, C" and C' atoms is 2.27 A.…”

“…Uniformly 15N-and "C/"N-enriched IRAP, a prerequisite for solution structure determination, was prepared using a bacterial expression system as described previously [16]. Samples for NMR spectroscopy typically were 2 mM IRAP, 50 mM 2H,-ethanolamine and 300 mM NaCl at pH 6.4.…”

“…Although several hundred intra-residue NOES were also assigned, they were not incorporated into the structure calculations since they provided little meaningful information. The 419 NOE-derived distance constraints were supplemented with 96 distance constraints assigned to hydrogen bonds based on slowly exchanging amide protons [16]. All 96 distance restraints represented hydrogen bonds between adjacent /?-sheet strands.…”