Abstract
The apparent complexity of biology increases as more biomolecular interactions that mediate function become known. We have used NMR spectroscopy and molecular modeling to provide direct evidence that tetrameric platelet factor-4 (PF4) and dimeric interleukin-8 (IL8), two members of the CXC chemokine family, readily interact by exchanging subunits and forming heterodimers via extension of their antiparallel beta-sheet domains. We further demonstrate using functional assays that PF4/IL8 heterodimerization has a direct and significant consequence on the biological activity of both chemokines. Formation of heterodimers enhances the anti-proliferative effect of PF4 on endothelial cells in culture, as well as the IL8-induced migration of CXCR2 vector-transfected Baf3 cells. These results suggest that CXC chemokine biology, and perhaps cytokine biology in general, may be functionally modulated at the molecular level by formation of heterodimers. This concept, in turn, has implications for designing chemokine/cytokine variants with modified biological properties.
Highlights
Platelet factor-4 (PF4)1 and interleukin-8 (IL8) are members of the CXC chemokine family of small (8 –10 kDa) proteins, a subfamily of chemokines within the cytokine superfamily [1]
In its well known capacity to bind heparin, PF4 functions optimally as a tetramer in vitro [18, 19] and presumably as a tetramer in vivo due both to the presence of heparan sulfate normally found on the surface of cells and because it is released from ␣-granules of platelets in relatively large quantities upon tissue injury
Interactions between PF4 (PF4M2) and IL8 Defined Using NMR—To investigate molecular interactions between PF4 and IL8, nonisotopically enriched IL8 was titrated into a solution of uniformly 15N-enriched PF4, and chemical shift changes in 15N-PF4 were monitored by recording 1H-15N HSQC spectra upon addition of IL8
Summary
Proteins—Recombinant PF4, PF4M2, and IL8 were expressed in Escherichia coli and purified as described previously [22]. Three additional equations are required to calculate the concentration of monomers, homodimers, homotetramers, and heterodimers. We assume that dimers and tetramers are spherical, allowing us to express the diffusion coefficient using the Stokes-Einstein relationship, D ϭ kT/6R, where T is the temperature, R is the radius of the molecule, and is the viscosity of pure solvent as shown, ͱ ͱ DD ϭ 3 2 DM, DT ϭ 3 4 DM (Eq 7). Solving Equations 3, 5, and 6 yields the concentration of monomers, homodimers, homotetramers, and heterodimers. To construct PF4/IL8 and PF4M2/IL8 -sheet sandwiched heterodimers, two adjacent and overlapping monomers from each AB-type dimer in the PF4 tetramer were first removed, leaving the -sheet sandwiched homodimer. The same equation for PF4/IL8 heterodimer receptor binding was obtained by assuming that binding of the heterodimer to the receptor is described by the following equilibrium shown in Equation 13
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