Chemokine Fold Research Articles

Investigation of CC and CXC chemokine quaternary state mutants

The chemokine family forms two different types of homodimer despite members sharing nearly identical folds. To study the formation of quaternary structure in this family, rational mutagenesis was employed on a representative member of each subfamily (MIP-1β and IL-8). The variants were studied by analytical ultracentrifugation and NMR, and it was determined that formation of a folded monomer from a natural chemokine dimer is reasonably facile, while conversion between dimer types is not. Monomeric variants of MIP-1β and IL-8 were randomly mutated and a λ phage-based selection system was employed in a novel way to screen for dimerization. A total of 6,000,000 random mutants were screened, but no dimers were formed, suggesting again that the chemokine fold is robust and amenable to sequence variation, while the chemokine dimer is much more difficult to attain. This work represents a biophysical analysis of an array of chemokine quaternary state variants.

Mapping the Binding of the N-terminal Extracellular Tail of the CXCR4 Receptor to Stromal Cell-derived Factor-1α

The solution structure of monomeric stromal cell-derived factor-1alpha (SDF-1alpha), the natural ligand for the CXCR4 G-coupled receptor, has been solved by multidimensional heteronuclear NMR spectroscopy. The structure has a characteristic chemokine fold and is in excellent agreement with the individual subunits observed in the crystal structures of dimeric SDF-1alpha. Using various peptides derived from the N-terminal extracellular tail of the CXCR4 receptor, we show that the principal determinants of binding reside in the N-terminal 17 residues of CXCR4, with a major contribution from the first six residues. From 15N/1HN chemical shift pertubation studies we show that the interaction surface on SDF-1alpha is formed by the undersurface of the three-stranded antiparallel beta-sheet bounded by the N-terminal loop on one side and the C-terminal helix on the other. This surface overlaps with but is not identical to that mapped on several other chemokines for the binding of equivalent peptides derived from their respective receptors.

NMR structure of CXCR3 binding chemokine CXCL11 (ITAC).

CXCL11 (ITAC) is one of three chemokines known to bind the receptor CXCR3, the two others being CXCL9 (Mig) and CXCL10 (IP-10). CXCL11 differs from the other CXCR3 ligands in both the strength and the particularities of its receptor interactions: It has a higher affinity, is a stronger agonist, and behaves differently when critical N-terminal residues are deleted. The structure of CXCL11 was determined using solution NMR to allow comparison with that of CXCL10 and help elucidate the source of the differences. CXCL11 takes on the canonical chemokine fold but exhibits greater conformational flexibility than has been observed for related chemokines under the same sample conditions. Unlike related chemokines such as IP-10 and IL-8, ITAC does not appear to form dimers at millimolar concentrations. The origin for this behavior can be found in the solution structure, which indicates a beta-bulge in beta-strand 1 that distorts the dimerization interface used by other CXC chemokines.

Solution Structure and Dynamics of Myeloid Progenitor Inhibitory Factor-1 (MPIF-1), A Novel Monomeric CC Chemokine

MPIF-1, a CC chemokine, is a specific inhibitor of myeloid progenitor cells and is the most potent activator of monocytes. The solution structure of myeloid progenitor inhibitor factor-1 (MPIF-1) has been determined by NMR spectroscopy. The structure reveals that MPIF-1 is a monomer with a well defined core except for termini residues and adopts the chemokine fold of three beta-strands and an overlying alpha-helix. In addition to the four cysteines that characterize most chemokines, MPIF-1 has two additional cysteines that form a disulfide bond. The backbone dynamics indicate that the disulfide bonds and the adjacent residues that include the functionally important N-terminal and N-terminal loop residues show significant dynamics. MPIF-1 is a highly basic protein (pI >9), and the structure reveals distinct positively charged pockets that could be correlated to proteoglycan binding. MPIF-1 is processed from a longer proprotein at the N terminus and the latter is also functional though with reduced potency, and both proteins exist as monomers under a variety of solution conditions. MPIF-1 is therefore unique because longer proproteins of all other chemokines oligomerize in solution. The MPIF-1 structure should serve as a template for future functional studies that could lead to therapeutics for preventing chemotherapy-associated myelotoxicity.