Proteins often have multiple functional states, which might not always be accommodated by a single fold. Lymphotactin (Ltn) adopts two distinct structures in equilibrium, one corresponding to the canonical chemokine fold consisting of a monomeric threestranded ␤-sheet and carboxyl-terminal helix. The second Ltn structure solved by NMR reveals a dimeric all-␤-sheet arrangement with no similarity to other known proteins. In physiological solution conditions, both structures are significantly populated and interconvert rapidly. Interconversion replaces long-range interactions that stabilize the chemokine fold with an entirely new set of tertiary and quaternary contacts. The chemokine-like Ltn conformation is a functional XCR1 agonist, but fails to bind heparin. In contrast, the alternative structure binds glycosaminoglycans with high affinity but fails to activate XCR1. Because each structural species displays only one of the two functional properties essential for activity in vivo, the conformational equilibrium is likely to be essential for the biological activity of lymphotactin. These results demonstrate that the functional repertoire and regulation of a single naturally occurring amino acid sequence can be expanded by access to a set of highly dissimilar native-state structures.chemokine ͉ conformational change ͉ NMR spectroscopy

Chemokines adopt a conserved tertiary structure stabilized by two disulfide bridges and direct the migration of leukocytes. Lymphotactin (Ltn) is a unique chemokine in that it contains only one disulfide and exhibits large-scale structural heterogeneity. Under physiological solution conditions (37°C, 150 mM NaCl) Ltn is in equilibrium between the canonical chemokine fold (Ltn10) and a distinct 4-stranded β-sheet (Ltn40). Consequently, it has not been possible to address the biological significance of each structural species independently. To stabilize the Ltn10 structure independent of specific solution conditions, Ltn variants containing a second disulfide bridge were designed. Placement of the new cysteines was based on a sequence alignment of Ltn with either the first (Ltn-CC1) or third disulfide (Ltn-CC3) in the CC chemokine, HCC-2. NMR data demonstrate that both CC1 and CC3 retain the Ltn10 chemokine structure and no longer exhibit structural rearrangement. The ability of each mutant to activate the Ltn receptor, XCR1, has been tested using an intracellular Ca 2+ flux assay. These data support the conclusion that the chemokine fold of Ltn10 is responsible for receptor activation. We also examined the role of amino-and carboxyl-terminal residues in Ltnmediated receptor activation. In contrast to previous reports, we find that the 25 residues comprising the novel C-terminal extension do not participate in receptor activation, while the native N-terminus is absolutely required for Ltn function.Chemokines are small, secreted proteins that signal leukocytes to migrate during an immune response. Binding of chemokines to G-protein coupled receptors (GPCRs) 1 stimulates cell chemotaxis, while diffusion and glycosaminoglycan (GAG) binding are believed to establish the chemokine gradient that migrating cells follow. Approximately 50 chemokines have been identified and are classified into four subfamilies (CXC, CC, CX 3 C, and C) based on the spacing of two cysteine residues near the N-terminus, each of which contributes to a pair of conserved disulfide bonds (1).Lymphotactin (Ltn), the only example of a C-type chemokine, lacks the first and third cysteine residues found in all other chemokines and contains a unique extended C-terminal sequence that is conserved across species (2). We have previously shown that under physiological conditions (37 °C, 150 mM NaCl), Ltn exhibits reversible conformational heterogeneity, converting between two distinct structural species. The relative population of the two conformations is highly dependent on the temperature and ionic strength of the solution (3).*Address correspondence to: bvolkman@mcw.edu, . ‖ Present Address: The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399.1 The abbreviations used are: GCPR, G-coupled protein receptor; GAG, glycosaminoglycan; Ltn, lymphotactin; MALDI-MS, matrixassisted laser desorption ionization mass spectrometry; CNBr, cyanogen bromide; RANTES, regulated on activation normal T cell expressed and secreted; HCC-2, human che...

Chemokine-mediated recruitment of leukocytes in vivo depends on interactions with cell surface glycosaminoglycans. Lymphotactin, the unique member of the "C" chemokine subclass, is a highly basic protein that binds heparin, a glycosaminoglycan, with high affinity (ϳ10 nM). We detected lymphotactin-heparin binding by NMR and mapped this interaction to a narrow surface that wraps around the protein. Substitutions in and around this binding site and surface plasmon resonance analysis of heparin binding affinity identified two arginine residues of lymphotactin as critical for glycosaminoglycan binding. Both arginine mutant proteins and the combined double mutant had dramatically diminished in vivo activity in a leukocyte recruitment assay, suggesting that the lymphotactin-glycosaminoglycan interactions detected in vitro are important for the function of this chemokine. Our results demonstrate that like other chemokines, lymphotactin utilizes highly specific glycosaminoglycan-binding sites that represent potential targets for drug development.A family of small secreted proteins, chemokines recruit leukocytes from the circulatory system to mediate inflammatory responses in host defense and wound healing (1), control angiogenesis, and regulate lymphoid development (2, 3). Chemokine sequences are divided into four subfamilies, based on the number and spacing of conserved cysteine residues. Many chemokine structures are known, and members of all four subfamilies adopt a canonical tertiary fold, typically stabilized by a pair of disulfide bonds (4); some also form homodimeric structures (5). Chemokines stimulate leukocyte migration by activating specific G protein-coupled receptors expressed in their target cell populations (3, 6). This signaling network of roughly 50 chemokines and 20 receptors is both selective and redundant: specific leukocyte types express a particular receptor and are recruited only in response to their cognate chemokines, however, many receptors can be activated by more than one chemokine ligand (7).Human lymphotactin (hLtn/XCL1) 1 recruits T lymphocytes and natural killer cells through its specific receptor XCR1 in normal immune function and chronic inflammatory conditions (8, 9). For example, an inappropriate or uncontrolled T cell infiltration driven by hLtn expression is a factor in both Crohn's disease (10) and rheumatoid arthritis (11). Interestingly, the unique ability of hLtn to specifically chemoattract lymphocytes has been exploited therapeutically in combination with the T cell-activating cytokine interleukin-2 (IL-2). T cells recruited by hLtn and activated by IL-2 generated potent antitumor immunity in human clinical trials for treatment of neuroblastoma, where studies with either hLtn or IL-2 alone failed to generate a significant response (12). Hence, hLtn and its analogs with activity toward XCR1 will be extremely valuable as agents for T cell recruitment in cancer vaccines (6), whereas specific antagonists of hLtn activity may be useful for treating certain autoimmune or inflammator...

No abstract