Solution Structure of a Chemosensory Protein from the Desert Locust Schistocerca gregaria ²,‡ Simona Tomaselli, § Orlando Crescenzi, § Domenico Sanfelice, § Eiso AB, |,⊥ Rainer Wechselberger, | Sergio Angeli, # Andrea Scaloni, 4 Rolf Boelens, | Teodorico Tancredi, ) Paolo Pelosi, # and Delia Picone* ,§ Department of Chemistry, UniVersity of Naples Federico II, 80126 Napoli, Italy, Department of NMR Spectroscopy, BijVoet Center for Biomolecular Research, Utrecht UniVersity, 3584 CH Utrecht, The Netherlands, Department of Agricultural Chemistry and Biotechnology, UniVersity of Pisa, 56124 Pisa, Italy, Proteomics and Mass Spectrometry Laboratory, ISPAAM, National Research Council, 80147 Napoli, Italy, and Institute of Biomolecular Chemistry, National Research Council, Napoli, Italy ReceiVed May 19, 2006; ReVised Manuscript ReceiVed July 7, 2006 ABSTRACT: Chemical stimuli, generally constituted by small volatile organic molecules, are extremely important for the survival of different insect species. In the course of evolution, insects have developed very sophisticated biochemical systems for the binding and the delivery of specific semiochemicals to their cognate membrane-bound receptors. Chemosensory proteins (CSPs) are a class of small soluble proteins present at high concentration in insect chemosensory organs; they are supposed to be involved in carrying the chemical messages from the environment to the chemosensory receptors. In this paper, we report on the solution structure of CSPsg4, a chemosensory protein from the desert locust Schistocerca gregaria, which is expressed in the antennae and other chemosensory organs. The 3D NMR structure revealed an overall fold consisting of six R-helices, spanning residues 13-18, 20-31, 40-54, 62-78, 80-90, and 97-103, connected by loops which in some cases show dihedral angles typical of -turns. As in the only other chemosensory protein whose structure has been solved so far, namely, CSP from the moth Mamestra brassicae, four helices are arranged to form a V-shaped motif; another helix runs across the two V’s, and the last one is packed against the external face. Analysis of the tertiary structure evidenced multiple hydrophobic cavities which could be involved in ligand binding. In fact, incubation of the protein with a natural ligand, namely, oleamide, produced substantial changes to the NMR spectra, suggesting extensive conformational transitions upon ligand binding. Chemical signals regulate most aspects of insect life. Insects have developed very sensitive and sophisticated systems to detect and correctly recognize different molecules present in the environment. Receptors are distributed on the surface of sensory organs, such as antennae, tarsi, and mouth parts, housing dendrites of chemosensory neurons. G-protein- coupled receptors, spanning the neuronal membrane with seven R-helical segments, have been generally recognized as the biochemical elements responsible for recognition of olfactory and taste stimuli (1, 2). Before interacting with their cognate membrane-bound receptors, chemical stimuli, mainly consisting of small hydrophobic molecules, have to cross an aqueous barrier. According to a current view, this process is assisted by small soluble proteins capable of reversibly binding a great variety of these organic molecules (3-10). These proteins are classified in two main groups, based mainly on sequence analysis, namely, odorant/pheromone binding proteins (OBPs/ PBPs) 1 and chemosensory proteins (CSPs). Their specific roles in chemoreception have not been entirely elucidated, although it has been demonstrated that olfactory receptors (e.g., those expressed in heterologous systems) can correctly recognize odorant molecules even in the absence of OBPs or CSPs (11, 12). Other experiments seem to indicate that LUSH, a member of Drosophila OBPs, is required for both behavioral and electrophysiological response to the phero- mone vaccenyl acetate (13). Whatever their functions are, the presence of PBPs and CSPs seems crucial for these important physiological pro- cesses; their biosynthesis in exceptionally large amounts requires a significant investment of energy by the insect and suggests an important role, both for the survival of the individual and for the conservation of the species (10). Moreover, the extremely high concentrations of OBPs and CSPs in the sensillar lymph (around 10 mM) (14) do not ² Financial support was from the Italian MIUR (FIRB RBNE03B8KK). ‡ The coordinates have been deposited into the PDB together with the NMR data. The accession codes are 2GVS and 7184, respectively. * Address correspondence to this author. Fax: + 39 081 674409. Tel: + 39 081 674406. E-mail: delia.picone@unina.it. § University of Naples Federico II. | Utrecht University. ⊥ Current address: Gorlaeus Laboratories, Einsteinweg 55, 2333 CC Leiden, The Netherlands. # University of Pisa. 4 Proteomics and Mass Spectrometry Laboratory, ISPAAM, National Research Council. ) Institute of Biomolecular Chemistry, National Research Council. 1 Abbreviations: CSP, chemosensory protein; OBP, odorant binding protein; PBP, pheromone binding protein; CSPsg4, CSP4 from Schis- tocerca gregaria; CSPMbra, CSP from Mamestra brassicae; HSQC, heteronuclear single-quantum coherence; TOCSY, total correlated spectroscopy; NOESY, nuclear Overhauser enhancement spectroscopy; NOE, nuclear Overhauser effect. 10606 Biochemistry 2006, 45, 10606-10613 10.1021/bi060998w CCC: $33.50 © 2006 American Chemical Society Published on Web 08/09/2006