A subdomain of the human leptin receptor encoding part of the extracellular domain (amino acids 428 to 635) was subcloned, expressed in a prokaryotic host, and purified to homogeneity, as evidenced by SDS-PAGE, with over 95% monomeric protein. The purified leptin-binding domain (LBD) exhibited the predicted β structure, was capable of binding human, ovine, and chicken leptins, and formed a stable 1:1 complex with all mammalian leptins. The binding kinetics, assayed by surface plasmon resonance methodology, showed respective kon andkoff values (mean ± S.E.) of 1.20 ± 0.23 × 10−5 mol−1 s−1 and 1.85 ± 0.30 × 10−3 s−1 and aKd value of 1.54 × 10−8m. Similar results were achieved with conventional binding experiments. LBD blocked leptin-induced, but not interleukin-3-induced, proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. The modeled LBD structure and the known three-dimensional structure of human leptin were used to construct a model of 1:1 LBD·human leptin complex. Two main residues, Phe-500, located in loop L3, and Tyr-441, located in L1, are suggested to contribute to leptin binding.
This work was supported by Israeli Science Foundation Research Grant 594/02 (to A. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
