Monomeric solution structure of the prototypical 'C' chemokine lymphotactin.
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.
Kuloglu
E S
ES
Department of Biochemistry, Biophysics Instrumentation Facility, and National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706, USA.
McCaslin
D R
DR
Kitabwalla
M
M
Pauza
C D
CD
Markley
J L
JL
Volkman
B F
BF
D000595:Amino Acid Sequence; D000818:Animals; D019744:Chemokines, C / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D002645:Chickens; D018360:Crystallography, X-Ray; D006801:Humans; D008222:Lymphokines / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D008253:Macaca mulatta; D051379:Mice; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D012795:Sialoglycoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D012996:Solutions; D013816:Thermodynamics
D019744:Chemokines, C; D008222:Lymphokines; D011993:Recombinant Fusion Proteins; D012795:Sialoglycoproteins; D012996:Solutions; C092390:XCL1 protein, human; C486249:Xcl1 protein, mouse; C018997:lymphotactin