No abstract

Chemokines are small chemoattractant proteins that facilitate leukocyte migration and activation through binding to their seven-transmembrane helix G protein-coupled receptors and play a role in homeostasis, inflammation, and disease (1-3). Chemokine gradients formed through their interaction with cell surface GAGs, 1 together with chemokine oligomerization, are thought to be important in leukocyte recruitment (4). Chemokines are also involved in transendothelial migration of leukocytes, maturation of leukocytes, traffic and homing of lymphocytes, development of lymphoid tissues, and angiogenesis (5-10). Because of the broad range of their immunoregulatory roles, chemokines and their receptors are targets for drug development for control of allergic and autoimmune diseases as well as for HIV infection, because entry of the virus into cells is dependent on binding to a chemokine receptor.Chemokines are divided into four subclasses on the basis of the number of conserved cysteine residues and their spacing. Most of the ϳ50 known chemokines belong to either the CC or CXC subclass. The two other subclasses of chemokine each have a single known member: fractalkine for the CX 3 C class and lymphotactin for the C class (11, 12). Three-dimensional structures determined for a variety of chemokines have revealed a conserved, disulfide-stabilized chemokine fold that consists of a three-stranded antiparallel ␤-sheet and a C-terminal ␣-helix. Some CXC and CC chemokines form dimers, and while the tertiary structure of the subunits is invariant, substantial differences have been observed in quaternary structure (13-23). In CXC-class chemokines, the dimer interface primarily involves joining the ␤1-strand of each monomer to form a single six-stranded antiparallel ␤-sheet; by contrast, CC chemokines typically self-associate through an additional ␤-strand (␤0) in the N terminus of each monomer. Novel quaternary interactions have also been observed in x-ray crystal structures of fractalkine (24) and .Lymphotactin (Ltn) is unique among chemokines in that it (i) contains only one of the two disulfide bridges that are conserved in all other chemokines and (ii) possesses a unique C-terminal extension, which is required for biological activity (26,27). Originally identified as a T and NK cell-specific chemokine (12,28,29), Ltn has recently been found to chemoattract neutrophils and B cells through the XCR1 receptor (30 -32). A mediator of mucosal immunity, Ltn is thought to be a factor in acute allograft rejection (33) and inflammatory bowel diseases (34,35). We recently used NMR spectroscopy to determine the three-dimensional structure of human lymphotactin (hLtn) at 10°C in a solution containing 200 mM NaCl (23). Under these conditions, Ltn is predominantly monomeric and adopts the canonical chemokine fold; its C-terminal extension is disordered and highly mobile.NMR spectra of hLtn in the absence of NaCl acquired be-

CCL21 is a human chemokine that recruits normal immune cells and metastasizing tumor cells to lymph nodes through activation of the G protein-coupled receptor CCR7. The CCL21 structure solved by NMR contains a conserved chemokine domain followed by an extended, unstructured C-terminus that is not typical of most other chemokines. A sedimentation equilibrium study showed CCL21 to be monomeric. Chemical shift mapping indicates that the CCR7 N-terminus binds to the N-loop and third β-strand of CCL21’s chemokine domain. Details of CCL21-receptor recognition may enable structure-based drug discovery of novel anti-metastatic agents.

Bacterial surface motility works by retraction of surface-attached type IV pili. This retraction requires the PilT protein, a member of a large family of putative NTPases from type II and IV secretion systems. In this study, the PilT homologue from the thermophilic eubacterium Aquifex aeolicus was cloned, overexpressed, and purified. A. aeolicus PilT was shown to be a thermostable ATPase with a specific activity of 15.7 nmol of ATP hydrolyzed/min/mg of protein. This activity was abolished when a conserved lysine in the nucleotide-binding motif was altered. The substrate specificity was low; UTP, CTP, ATP, GTP, dATP, and dGTP served as substrates, UTP having the highest activity of these in vitro. Based on sedimentation equilibrium and size exclusion chromatography, PilT was identified as a Ϸ5-to 6-subunit oligomer. Potential implications of the NTPase activity of PilT in pilus retraction are discussed.

Lymphotactin, the sole identified member of the C class of chemokines, specifically attracts T lymphocytes and natural killer cells. This 93-residue protein lacks 2 of the 4 conserved cysteine residues characteristic of the other 3 classes of chemokines and possesses an extended carboxyl terminus, which is required for chemotactic activity. We have determined the three-dimensional solution structure of recombinant human lymphotactin by NMR spectroscopy. Under the conditions used for the structure determination, lymphotactin was predominantly monomeric; however, pulsed field gradient NMR self-diffusion measurements and analytical ultracentrifugation revealed evidence of dimer formation. Sequence-specific chemical shift assignments were determined through analysis of two- and three-dimensional NMR spectra of (15)N- and (13)C/(15)N-enriched protein samples. Input for the torsion angle dynamics calculations used in determining the structure included 1258 unique NOE-derived distance constraints and 60 dihedral angle constraints obtained from chemical-shift-based searching of a protein conformational database. The ensemble of 20 structures chosen to represent the structure had backbone and heavy atom rms deviations of 0.46 +/- 0.11 and 1.02 +/- 0.14 A, respectively. The results revealed that human lymphotactin adopts the conserved chemokine fold, which is characterized by a three-stranded antiparallel beta-sheet and a C-terminal alpha-helix. Two regions are dynamically disordered as evidenced by (1)H and (13)C chemical shifts and [(15)N]-(1)H NOEs: residues 1-9 of the amino terminus and residues 69-93 of the C-terminal extension. A functional role for the C-terminal extension, which is unique to lymphotactin, remains to be elucidated.