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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, 2010Structure-Function Relationships of a Hexapeptide Fragment ofthe Carcinoembryonic AntigenSándor Lovas, Nicholas Y. Palermo, Peter Thomas, andRichard F. MurphyDepartment of Biomedical Sciences, Creighton University Medical Center,Omaha, NE, 68178, U.S.A.IntroductionCarcinoembryonic antigen (CEA), a 180,200 kD glycoprotein, binds to the heterogeneousribonucleoprotein M (hnRNP M) which acts as a cell surface receptor in Kupffer cells [1].The binding induces release of inflammatory cytokines and promotes colorectal cancermetastasis to the liver [2]. The amino acid sequence in CEA which binds the hnRNP Mreceptor is Tyr-Pro-Glu-Leu-Pro-Lys. In this study, the structure-function relationships ofAc-Tyr-Pro-Glu-Leu-Pro-Lys-NH 2 (YPELPK) was investigated by binding of the peptideand its Ala-scan analogs (Table 1) to hnRNP M using molecular docking calculations.Peptides were synthesized at the 0.1 mmol scale on a CEM Liberty microwave peptidesynthesizer using N α -Fmoc-protected amino acids. The structure of peptides weredetermined by 100 ns replica-exchange molecular dynamics (REMD) simulations [3] usingthe OPLS-AA/L force field [4] as implemented in the GROMACS package [5]. Peptideswere docked to the structure of hnRNP M (pbd id 2DGV) using the GLIDE program(Version 4.5, Schrödinger, LLC, New York, NY, 2007). The biological activity ofYPELPK and its analogs were studied using differentiated human THP-1 cells, whichexpress hnRNP M on their surface and secrete IL-6 when stimulated by CEA.Table 1. Relative free energy (ΔG rel ) of binding of YPELPK and its Ala-scan analogsPeptide Sequence ΔG rel / kcal mol -1YPELPK Ac-Tyr-Pro-Glu-Leu-Pro-Lys-NH 2 0.00 bA1 a Ac-Ala-Pro-Glu-Leu-Pro-Lys-NH 2 0.80A2 Ac-Tyr-Ala-Glu-Leu-Pro-Lys-NH 2 0.39A3 Ac-Tyr-Pro-Ala-Leu-Pro-Lys-NH 2 0.75A4 Ac-Tyr-Pro-Glu-Ala-Pro-Lys-NH 2 1.11A5 Ac-Tyr-Pro-Glu-Leu-Ala-Lys-NH 2 3.33A6 Ac-Tyr-Pro-Glu-Leu-Pro-Ala-NH 2 0.98a Residues of YPELPK replaced with Ala are indicated in boldface fonts. b Energies ofbinding are relative to that of YPELPK. A positive value indicates less binding affinity.Results and DiscussionThe REMD simulation of the structure of YPELPK folds the peptide into a stable PPIIhelix conformation, since the trajectory of the simulation has one dominant backboneconformation and 93.4% of the structures belong to a single cluster.In docking, analogs A1, A2, A3 and A6 have lower affinity for hnRNP M than doesYPELPK; the difference in energy is less than 1.0 kcal mol -1 (Table 1). A4 and A5 have theleast affinity for hnRNP M. The best ligand pose for YPELPK (Figure 1) is stabilized by amix of two hydrogen bonds, two salt bridges and four weakly polar interactions. Thebinding of YPELPK to the C-terminal region of hnRNP M which does not associate withthe cell membrane, indicates that no steric constraints would be imposed on binding. Thesmaller loss of binding energy, 0.80 kcal mol -1 in response to the replacement of the Tyr506