Crystal structure of interleukin 8: symbiosis of NMR and crystallography.

Abstract

The crystal structure of a host defense system chemotactic factor, interleukin 8, has been solved by molecular replacement using as a model the solution structure derived from nuclear magnetic resonance experiments. The structure was refined with 2 A x-ray data to an R factor of 0.187 (0.217 at 1.6 A). A comparison indicates some potential differences between the structure in solution and in the crystalline state. Our analysis also predicts that residues 4 through 9 on the amino terminus and the (-bend, which includes His-33, may be important for receptor binding.Nuclear magnetic resonance (NMR) has been established in the last 5 years as the only viable alternative to x-ray crystallography for determining detailed three-dimensional structures of macromolecules up to 20 kDa molecular mass (1, 2). Although test calculations using crambin (3), tendamistat (4), and ubiquitin (5) have established the feasibility of utilizing NMR-derived models as starting points for solving x-ray structures by molecular replacement, no unknown crystal structures have been previously solved by that route. We present here the results of such an investigation. The highresolution x-ray structure ofinterleukin 8 (IL-8) was solved by a combination of NMR (6, 7) and crystallography, and the models obtained by these two techniques were compared.tt IL-8 is a small, chemotactic factor released by monocytes and a number of other cell types in response to inflammatory stimuli (8). The factor promotes the directed migration of neutrophils, basophils, and T cells (9) along a concentration gradient to the inflammatory focus. IL-8 has also been reported to initiate the oxidative burst response in neutrophils. The factor has been known as monocyte-derived neutrophil chemotactic factor (10) or neutrophil-activating protein (11,12) among others, but it is now generally designated IL-8 (9). IL-8 is a member of a family of similar immune system proteins including the monocyte chemoattractant factor (13), human melanoma growth-stimulating activity (14), murine macrophage inflammatory protein 2 (15), and bovine platelet factor 4 (BPF4) (16). Each of these protein sequences shows a conserved region containing four cysteines that form two disulfide bridges. Under the experimental conditions of the structural investigations IL-8 is a dimer (6, 7) and BPF4 is a tetramer (17). Materials and MethodsRecombinant human IL-8 that was used in the crystallographic studies was purified from Escherichia coli (18). All of the x-ray data used for this structure determination were collected on crystals that were grown using protein recycled from the NMR experiments of Clore and Gronenborn and coworkers (6, 7). Crystals belonging to the space group P3121, a = b = 40.3 A, c = 90.1 A, were grownfrom40%o ammonium sulfate (pH = 8.5) as described (19). Identical crystals have also been grown using chemically synthesized protein. Recently, workers at Sandoz Pharmaceutical have reported that crystals apparently identical to the ones described here could be grown from poly...

Hematopoietic cytokines: Similarities and differences in the structures, with implications for receptor binding

“…Their differences could be due to a combination of inaccuracies of each structure and genuine differences between the structures of proteins in crystals and in solution (Baldwin et al, 1991;Shaanan et al, 1992 (Powers et al, 1993) yield similar results. We may conclude that while some of ,the differences between the structures of the three cytokines might be due to the differences of crystal packing and to inaccuracies in structure determination, such influences are not major because they are on the same order as the differences found in the same structure studied by different means and in different environments.…”

Monomeric variants of IL-8: Effects of side chain substitutions and solution conditions upon dimer formation

“…The nature of the charge (Glu or Arg) was chosen to oppose the local charged residues seen in the dimer structure. For example, E24 of the first subunit forms an ion pair with R26' of the second subunit (Baldwin et al, 1991). Therefore, E24R was selected to disrupt the ion pair and provide repulsive interactions with R26' and I28'R.…”

“…IL-8 exists as a non-covalent homodimer of 8 kDa subunits both at high concentrations in solution and crystal structures (Clore et al, 1989(Clore et al, , 1990Baldwin et al, 1991;, raising the question of whether ligand dimerization is involved in signal transduction. The primary functional determinant for binding of IL-8 to its neutrophil receptors is a short peptide motif corresponding to the peptide sequence GluLeu-Arg ("ELR') near the amino terminus of IL-8 (Clark- Lewis et al, 1991Lewis et al, , 1993HCbert et al, 1991).…”

“…Additionally, a region of more hydrophobic character has been identified in the residues 12-2 1 region of IL-8 (the N-loop) that clearly contributes to highaffinity receptor binding (Clubb et al, 1994;Schraufstatter et al, 1995;Lowman et al, 1996;Williams et al, 1996). Both of these regions, however, are rather distal to the dimer interface, and well exposed to solvent in either dimeric (Baldwin et al, 1991;Clore & Gronenborn, 1991) or monomeric forms of the protein.…”

Interaction of Monomeric Interleukin-8 with CXCR1 Mapped by Proton-Detected Fast MAS Solid-State NMR

“…Its structure, determined by solution NMR spectroscopy (8) and x-ray crystallography (9), is characterized by having a flexible N-terminal segment containing a conserved Glu-Leu-Arg (ELR) motif just before the characteristic CXC arrangement of cysteine residues (X represents another type of amino acid) that participate in the two disulfide bonds that stabilize the tertiary fold of the three b-strands and single a-helical segment. Most CXC chemokines have between 70 and 130 residues with substantial variation of sequences.…”