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J. Biol. Chem., Vol. 277, Issue 20, 17863-17870, May 17, 2002

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Structural Rearrangement of Human Lymphotactin, a C Chemokine, under Physiological Solution Conditions^*

E. Sonay Kulolu, Darrell R. McCaslin^§, John L. Markley^¶, and Brian F. Volkman^**

From the  Department of Biochemistry, the ^§ Biophysics Instrumentation Facility, and the ^¶ National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, Madison, Wisconsin 53706 and the  Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

NMR spectra of human lymphotactin (hLtn), obtained under various solution conditions, have revealed that the protein undergoes a major conformational rearrangement dependent on temperature and salt concentration. At high salt (200 mM NaCl) and low temperature (10 °C), hLtn adopts a chemokine-like fold, which consists of a three-stranded antiparallel -sheet and a C-terminal -helix (Kulolu, E. S., McCaslin, D. R., Kitabwalla, M., Pauza, C. D., Markley, J. L., and Volkman, B. F. (2001) Biochemistry 40, 12486-12496). We have used NMR spectroscopy, sedimentation equilibrium, and intrinsic fluorescence to monitor the reversible conformational change undergone by hLtn as a function of temperature and ionic strength. We have used two-, three- and four-dimensional NMR spectroscopy of isotopically enriched protein samples to determine structural properties of the conformational state stabilized at 45 °C and 0 mM NaCl. Patterns of NOEs and ¹H and ¹³C chemical shifts show that hLtn rearranges under these conditions to form a four-stranded, antiparallel -sheet with a pattern of hydrogen bonding that is completely different from that of the chemokine fold stabilized at 10 °C and 200 mM NaCl. The C-terminal -helix observed at 10 °C and 200 mM NaCl, which is conserved in other chemokines, is absent at 45 °C and no salt, and the last 38 residues of the protein are completely disordered, as indicated by heteronuclear ¹⁵N-¹H NOEs. Temperature dependence of the tryptophan fluorescence of hLtn in low and high salt confirmed that the chemokine conformation is stabilized by increased ionic strength. Sedimentation equilibrium analytical ultracentrifugation showed that hLtn at 40 °C in the presence of 100 mM NaCl exists mainly as a dimer. Under near physiological conditions of temperature, pH, and ionic strength, both the chemokine-like and non-chemokine-like conformations of hLtn are significantly populated. The functional relevance of this structural interconversion remains to be elucidated.

The NMR spectrosocopy data reported in this paper have been deposited in the BioMagResBank under BMRB accession number 5251.

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