Molecular interaction between kisspeptin decapeptide analogs and a lipid membrane Ju Yeon Lee a , Jung Sun Moon b , Young-Jae Eu a , Chul Won Lee a , Sung-Tae Yang a , Seung Kyu Lee a , Hyun Ho Jung a , Ha Hyung Kim c , Hyewhon Rhim d , Jae Young Seong b , Jae Il Kim a,e, * a Department of Life Science, Research Center for Bio-imaging, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea b Research Laboratory of G Protein Coupled Receptors, Korea University College of Medicine, Seoul 136-705, Republic of Korea c College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea d Life Sciences Division, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea e Department of Life Science, Gwangju Institute of Science and Technology, AnyGen Co. Ltd., 1 Oryong-dong, Puk-gu Gwangju 500-712, Republic of Korea article info Article history: Received 2 January 2009 and in revised form 1 March 2009 Available online 9 March 2009 Keywords: Kisspeptin GPR54 Peptide–membrane interaction NMR DPC abstract Kisspeptin-10 is the C-terminal decapeptide amide of kisspeptin, an endogenous ligand for GPR54, and exhibits the same binding and agonist activity as the parent molecule. Although GPR54 is a mem- brane-embedded protein, details of the molecular interaction between kisspeptin-10 and lipid mem- branes remain unclear. Here, we performed a series of structural analyses using alanine-scanning analogs of kisspeptin-10 in membrane-mimetic medium. We found that there is a close correlation between lipid membrane binding and agonist activity. For instance, the F10A and non-amidated (NH 2 ? OH) analogs showed little or no GPR54-agonist activity and elicited no blue shift in tryptophan fluorescence. NMR analysis of kisspeptin-10 analog in DPC micelles revealed it to contain several tight turn structures, encompassing residues Trp3 to Phe10, but no helical conformation like that seen previ- ously with SDS micelles. Together, our results suggest that kisspeptin-10 may activate GPR54 via a ligand transportation pathway incorporating a lipid membrane. Ó 2009 Elsevier Inc. All rights reserved. 1. Introduction G protein-coupled receptors (GPCRs) are the largest family of cell surface membrane receptors and regulate a variety of physio- logical responses by mediating transmission of extracellular sig- nals into cells [1]. GPR54 is a GPCR family member that is widely expressed in the central nervous system and shares about 45% identity with the galanine receptor [2]. Galanine, however, does not activate or even bind to GPR54. Instead, kisspeptin (also known as metastin), a 54-amino acid peptide fragment of the protein en- coded by the KiSS-1 metastasis suppressor gene, appears to be the cognate ligand for GPR54 [3]. It was previously reported that the 10 C-terminal residues of kisspeptin (kisspeptin-10; Tyr45–Phe54) are critical for specific binding to GPR54 and exhibit agonist activ- ity comparable to that of wild-type kisspeptin [3,4]. Moreover, when the structure–activity relationship of kisspeptin-10 was examined through amino acid substitutions and NMR structure analysis, it was found that the C-terminal region of kisspeptin-10 forms a helical structure in the presence of SDS micelles and plays an especially important role in its binding to and activation of GPR54 [5–7]. On the other hand, although it is well known that GPR54 is a membrane-embedded protein, details of the molecular interaction between kisspeptin-10 and membrane lipids remain unclear. There are two alternative mechanisms by which a ligand may interact with a membrane-embedded receptor [8,9]. The first is a ligand transportation model [10], in which initial binding of a ligand to the membrane is followed by lateral diffusion to the receptor. The second is a direct transfer of the ligand from the aqueous solu- tion to the receptor. In this study, we tried to assess whether the interaction between kisspeptin-10 and GPR54 is consistent with the ‘‘membrane compartment theory” [11,12] and the kisspeptin- 10 structure in DPC micelles is similar to that in SDS micelles. Interestingly, it was revealed that there is a close correlation be- tween the membrane binding of kisspeptin-10 analogs and their agonist activity. 2. Materials and methods 2.1. Peptide synthesis All kisspeptin-10 analogs were synthesized through solid phase peptide synthesis performed manually using 9-fluorenylmethoxy- 0003-9861/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2009.03.002 * Corresponding author. Address: Department of Life Science, Gwangju Institute of Science and Technology, AnyGen Co. Ltd., 1 Oryong-dong, Puk-gu Gwangju 500- 712, Republic of Korea. Fax: +82 62 970 2484. E-mail addresses: jikim@gist.ac.kr, ljy@gist.ac.kr (J.I. Kim). Archives of Biochemistry and Biophysics 485 (2009) 109–114 Contents lists available at ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi