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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Binding of Hemopressin Peptide with Cannabinoid CB1Receptor: A Structural StudyMario Scrima 1 , Sara Di Marino 1 , Manuela Grimaldi 1 ,Ettore Novellino 2 , Maurizio Bifulco 1 , and Anna Maria D’Ursi 1*1 Department of Pharmaceutical Sciences, University of Salerno, I-84084, Fisciano, Italy;2 Department of Pharmaceutical and Toxicological Chemistry, University of NaplesFederico II, I-80131, Naples, ItalyIntroductionHemopressin (PVNFKFLSH), a bioactive nonapeptide derived from the 1-chain ofhemoglobin, was recently shown to possess selective antagonist activity at cannabinoidCB 1 receptor [1]. CB 1 receptor antagonists have been extensively studied for their possibletherapeutic use in the treatment of obesity, drug abuse and heroin addiction.Using circular dichroism and nuclear magnetic resonance spectroscopy, the presentwork reports the conformational analysis of hemopressin and its truncated, biologicallyactive fragment hemopressin(1-6). The binding modes of both hemopressin andhemopressin(1-6) are investigated by molecular docking calculations.Results and DiscussionCD Spectroscopy. The quantitative evaluation of the CD curves, indicated that hemopressinand hemopressin(1-6) in a water solution at pH 5.4 assume random coil conformations withminimal amounts of turn and strand structures. In mixed DPC/SDS micelles, hemopressinand hemopressin(1-6) prevalently assume -helical and -turn (negative ellipticity value at218 nm), with small amounts of random coil conformations.NMR Spectroscopy. An entire set of 1D and 2D proton spectra (COSY, TOCSY andNOESY) of hemopressin and hemopressin(1-6) (peptide concentration range of 0.5-15mM) were recorded in water and in DPC/SDS (90/10 M:M) mixed micelles. Analysis ofhemopressin structure bundles according to the PROMOTIF procedure points to thepresence of regular type I -turn structures on residues Phe4-Leu7, Lys5-Ser8, and Phe6-His9. H-bonds between C=O (Phe4) and HN (Leu7) as well as C=O (Phe6) and HN (His9)stabilize the -turn structures. Analysis of hemopressin(1-6) structure bundles following theprocedure reported for hemopressin indicates that this shorter fragment assumes -turnstructures centered on residues Phe4-Phe6.Homology modeling. CB1 receptor shares 21%identity with visual rhodopsin (code PDB: 1GZM)[2]. Based on previously performed alignmentstudies [3,4], we built a molecular model of theCB1 receptor using MODELLER software.(Figure 1). The stereochemical quality of the CB1model was assessed using PROCHECK software.The final results showed 94.8% of residues in themost favored regions, 4% in additional allowedregions, 0.4% in generously allowed regions and0.8% in disallowed regions; the residues indisallowed regions were not located in the bindingpocket.Molecular docking. Molecular docking calculationsof hemopressin at the CB1 binding site wereconducted starting from the low-energyhemopressin NMR structure using Autodock 4.0software. The binding of hemopressin andhemopressin (1-6) at the CB1 binding site areshown in Figure 2 and 3, respectively.Fig. 1. Molecular model of CB1receptor in the inactive state aspredicted by HomologyModeling. On the right residuescrucial to stabilize the receptorthree-dimensional structure.342