Structure/function studies of the human chemokine lymphotactin
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NMR structure of human lymphotactin.
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Chemokines mediate the immune response by attracting and, in some cases, activating specific populations of leukocytes. These small (8-10 kDa) proteins bind specifically to G protein-coupled receptors of the seven-transmembrane (7-TM) type. Nearly all contain two disulfide bridges and are highly basic proteins with the capacity for binding cell-surface glycosaminoglycans (GAGs). Most of the human chemokines have probably now been identified through genome sequencing efforts and currently number about 50. Four chemokine families are distinguished and named by the spacing of cysteine residues near the N-terminus. Members of the CXC, CC and CX3C subclasses contain two disulfide bridges, whereas the single chemokine of the C class, lymphotactin, has only one. Lymphotactin is also unusual for its ~25 residue C-terminal extension, a feature that is not found in other chemokine sequences. The three-dimensional structures of a number of chemokines have been determined, revealing a conserved tertiary structure stabilized by two disulfide bonds. We have completed the NMR structure of lymphotactin at 10C in solution conditions containing 200 mM NaCl (Kuloglu et al, 2001). Many other chemokines are known to form dimers at micromolar concentrations, and, using NMR pulsed-field gradient self-diffusion and sedimentation measurements, we found that Ltn forms dimers with a Kd of ~1 mM. The weakness of this self-association has so far prevented us from identifying the dimerization interface.
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Comparison of the backbone 30's loop conformations of hLtn (green), IL-8 (yellow), RANTES (red) and Fractalkine (blue).
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Analysis of the NMR structure showed that Ltn adopts the conserved chemokine fold, which is characterized by a three-stranded beta-sheet and a C-terminal alpha-helix. Structural comparisons with representative CXC, CC and CX3C chemokines showed that, with backbone atomic root mean square deviations of ~1 Angstrom to the others, the unique sequence features of Ltn do not translate into significant conformational divergence from the chemokine superfamily. In all other chemokines, two disulfide bonds tether the N-terminus to beta-strand 3 and to the loop connecting strand 1 and strand 2 (the 30's loop). Compared to the others, Ltn has a shorter 30's loop with no disulfide to position it relative to the N-terminus. Because this loop has been implicated in other chemokine-receptor interactions, we speculate that these differences in structure and dynamics may play a role in the specificity of Ltn for the XCR1 receptor. In addition, since the other major unique feature of Ltn, the extended C-terminal region, is completely unstructured in solution, it seems likely that its functional requirement must derive from specific Ltn-XCR1 interactions.
We have also found that Ltn undergoes a novel conformational rearrangement that depends on temperature and ionic strength (Kuloglu et al, 2002). Our ongoing studies of this novel signaling molecule are directed at understanding the specific elements of its receptor and glycosaminoglycan binding sites, as well as the functional implications of each of the two Ltn structures present at physiological solution conditions.
References
Tikhonov, I., Kitabwalla, M., Wallace, M., Malkovsky, M., Volkman, B. F., and Pauza, C. D., Staphylococcal superantigens induce lymphotactin production by human CD4+ and CD8+ T cells, (2001) Cytokine, 16, 73-8.
Kuloglu, E. S., McCaslin, D. R., Kitabwalla, M., Pauza, C. D., Markley, J. L. and Volkman, B. F., Monomeric Solution Structure of the Prototypical 'C' Chemokine Lymphotactin, (2001) Biochemistry, 40, 12486-12496.
Kuloglu, E.S., McCaslin, D.R., Markley, J.L. and Volkman, B.F. , Structural rearrangement of human lymphotactin, a C chemokine, under physiological solution conditions, (2002) J. Biol. Chem., 277, 17863-70.
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