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• W2013156594 abstract "Asparagine deamidation at the NGR sequence in the 5th type I repeat of fibronectin (FN-I5) generates isoDGR, an αvβ3 integrin-binding motif regulating endothelial cell adhesion and proliferation. By NMR and molecular dynamics studies, we analyzed the structure of CisoDGRC (isoDGR-2C), a cyclic β-peptide mimicking the FN-I5 site, and compared it with NGR, RGD, or DGR-containing cyclopeptides. Docking experiments show that isoDGR, exploiting an inverted orientation as compared with RGD, favorably interacts with the RGD-binding site of αvβ3, both recapitulating canonical RGD-αvβ3 contacts and establishing additional polar interactions. Conversely, NGR and DGR motifs lack the fundamental pharmacophoric requirements for high receptor affinity. Therefore, unlike NGR and DGR, isoDGR is a new natural recognition motif of the RGD-binding pocket of αvβ3. These findings contribute to explain the different functional properties of FN-I5 before and after deamidation, and provide support for the hypothesis that NGR → isoDGR transition can work as a molecular timer for activating latent integrin-binding sites in proteins, thus regulating protein function. Asparagine deamidation at the NGR sequence in the 5th type I repeat of fibronectin (FN-I5) generates isoDGR, an αvβ3 integrin-binding motif regulating endothelial cell adhesion and proliferation. By NMR and molecular dynamics studies, we analyzed the structure of CisoDGRC (isoDGR-2C), a cyclic β-peptide mimicking the FN-I5 site, and compared it with NGR, RGD, or DGR-containing cyclopeptides. Docking experiments show that isoDGR, exploiting an inverted orientation as compared with RGD, favorably interacts with the RGD-binding site of αvβ3, both recapitulating canonical RGD-αvβ3 contacts and establishing additional polar interactions. Conversely, NGR and DGR motifs lack the fundamental pharmacophoric requirements for high receptor affinity. Therefore, unlike NGR and DGR, isoDGR is a new natural recognition motif of the RGD-binding pocket of αvβ3. These findings contribute to explain the different functional properties of FN-I5 before and after deamidation, and provide support for the hypothesis that NGR → isoDGR transition can work as a molecular timer for activating latent integrin-binding sites in proteins, thus regulating protein function. A number of cellular interactions with the extracellular matrix (ECM) 6The abbreviations used are: APBS, adaptive Poisson-Boltzmann solver program; a.u., arbitrary units; ECM, extracellular matrix; FN, fibronectin; (hTNF)-α, human tumor necrosis factor; MD, molecular dynamics; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; OPLS, optimized parameters for liquid simulation; REMD, replica exchange molecular dynamics simulations; r.m.s.d., root mean square deviation; MIDAS, metal ion-dependent adhesion site. 6The abbreviations used are: APBS, adaptive Poisson-Boltzmann solver program; a.u., arbitrary units; ECM, extracellular matrix; FN, fibronectin; (hTNF)-α, human tumor necrosis factor; MD, molecular dynamics; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; OPLS, optimized parameters for liquid simulation; REMD, replica exchange molecular dynamics simulations; r.m.s.d., root mean square deviation; MIDAS, metal ion-dependent adhesion site. are mediated by fibronectins, which are large adhesive glycoproteins (∼450 kDa) involved in several key processes, including embryogenesis, angiogenesis, inflammation, hemostasis, thrombosis, and tissue repair (1Humphries M.J. Obara M. Olden K. Yamada K.M. Cancer Investig. 1989; 7: 373-393Crossref PubMed Scopus (97) Google Scholar, 2Pankov R. Yamada K.M. J. Cell Sci. 2002; 115: 3861-3863Crossref PubMed Scopus (1403) Google Scholar). Fibronectins are soluble elements of plasma and other body fluids, as well as constituents of the insoluble ECM (2Pankov R. Yamada K.M. J. Cell Sci. 2002; 115: 3861-3863Crossref PubMed Scopus (1403) Google Scholar, 3Mohri H. Peptides (N. Y.). 1997; 18: 899-907Crossref PubMed Scopus (27) Google Scholar). Human fibronectin (FN), which is typically composed of two almost identical subunits connected covalently by disulfide bonds at their C termini, is thus an abundant and ubiquitous ECM protein present in about 20 isoforms and consisting primarily of three types of repeating modules (FN-I, FN-II, and FN-III) (2Pankov R. Yamada K.M. J. Cell Sci. 2002; 115: 3861-3863Crossref PubMed Scopus (1403) Google Scholar, 3Mohri H. Peptides (N. Y.). 1997; 18: 899-907Crossref PubMed Scopus (27) Google Scholar, 4Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8941) Google Scholar, 5Kornblihtt A.R. Pesce C.G. Alonso C.R. Cramer P. Srebrow A. Werbajh S. Muro A.F. FASEB J. 1996; 10: 248-257Crossref PubMed Scopus (172) Google Scholar). Distinct FN modules contain varying binding sites for several different molecules, including sulfated glycosaminoglycans, syndecans, DNA, gelatin, heparin, and fibrin (2Pankov R. Yamada K.M. J. Cell Sci. 2002; 115: 3861-3863Crossref PubMed Scopus (1403) Google Scholar, 3Mohri H. Peptides (N. Y.). 1997; 18: 899-907Crossref PubMed Scopus (27) Google Scholar, 6Yamada K.M. Curr. Opin. Cell Biol. 1989; 1: 956-963Crossref PubMed Scopus (97) Google Scholar). In addition, fibronectins contain binding sites for about half of the known cell surface integrin receptors (7Johansson S. Svineng G. Wennerberg K. Armulik A. Lohikangas L. Front. Biosci. 1997; 2: 126-146Crossref PubMed Scopus (265) Google Scholar, 8Plow E.F. Haas T.A. Zhang L. Loftus J. Smith J.W. J. Biol. Chem. 2000; 275: 21785-21788Abstract Full Text Full Text PDF PubMed Scopus (1086) Google Scholar). Integrin receptors are present in many animal species, ranging from sponges to mammals (9Humphries M.J. Biochem. Soc. Trans. 2000; 28: 311-339Crossref PubMed Google Scholar), and play essential roles in cellular physiology (attachment, migration, proliferation, differentiation, and survival) and in disease (cancer, tumor metastasis, immune dysfunction, ischemia-reperfusion injury, viral infections, osteoporosis, and coagulopathies) (4Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8941) Google Scholar, 10Arnaout M.A. Immunol. Rev. 1990; 114: 145-180Crossref PubMed Scopus (250) Google Scholar). In this context, it is worth noting that FN-I5 (i.e. the 5th FN-I repeat) and FN-I7 modules contain a GNGRG loop that is conserved in human, bovine, murine, rat, bird, amphibian, and fish fibronectin, suggesting that this loop is functionally important (11Di Matteo P. Curnis F. Longhi R. Colombo G. Sacchi A. Crippa L. Protti M.P. Ponzoni M. Toma S. Corti A. Mol. Immunol. 2006; 43: 1509-1518Crossref PubMed Scopus (49) Google Scholar). In addition, we have recently shown that the deamidation of Asn263 at the Asn-Gly-Arg (NGR) sequence of FN-I5 and of peptides containing the NGR motif generates isoDGR (isoAsp-Gly-Arg), a novel cell adhesion motif binding to αvβ3 integrin (12Curnis F. Longhi R. Crippa L. Cattaneo A. Dondossola E. Bachi A. Corti A. J. Biol. Chem. 2006; 281: 36466-36476Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Consistently, cells from homozygous knock-in mice carrying the RGD → RGE mutation in fibronectin exhibited normal fibronectin fibril assembly, both in vitro and in vivo, exploiting the presence in the FN-I5 repeat of a NGR → isoDGR motif binding to αvβ3 (13Takahashi S. Leiss M. Moser M. Ohashi T. Kitao T. Heckmann D. Pfeifer A. Kessler H. Takagi J. Erickson H.P. Fassler R. J. Cell Biol. 2007; 178: 167-178Crossref PubMed Scopus (146) Google Scholar). Integrin αvβ3 is a relevant receptor in tumor angiogenesis and metastasis, viral infections, inflammation, and bone resorption (8Plow E.F. Haas T.A. Zhang L. Loftus J. Smith J.W. J. Biol. Chem. 2000; 275: 21785-21788Abstract Full Text Full Text PDF PubMed Scopus (1086) Google Scholar, 14Eliceiri B.P. Cheresh D.A. J. Clin. Invest. 1999; 103: 1227-1230Crossref PubMed Scopus (613) Google Scholar), and its ligands contain the Arg-Gly-Asp (RGD) sequence (15Xiong J.P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S.L. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1369) Google Scholar, 16Dechantsreiter M.A. Planker E. Matha B. Lohof E. Holzemann G. Jonczyk A. Goodman S.L. Kessler H. J. Med. Chem. 1999; 42: 3033-3040Crossref PubMed Scopus (744) Google Scholar). Importantly, a cyclic β-peptide containing the CisoDGRC motif (isoDGR-2C) is a competitive antagonist of RGD-containing ligands of αvβ3 and inhibits endothelial cell adhesion, proliferation, and tumor growth (12Curnis F. Longhi R. Crippa L. Cattaneo A. Dondossola E. Bachi A. Corti A. J. Biol. Chem. 2006; 281: 36466-36476Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Furthermore, analysis of competitive binding plots of RGD-2C and isoDGR-2C shows that both ligands have comparable binding affinity for αvβ3, whereas DGR-2C and NGR-2C cyclopeptides show a >2 order of magnitude lower affinity (12Curnis F. Longhi R. Crippa L. Cattaneo A. Dondossola E. Bachi A. Corti A. J. Biol. Chem. 2006; 281: 36466-36476Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), pointing to stereospecific isoDGR-αvβ3 interactions. The structural determinants dictating the interaction between isoDGR and αvβ3 are still unknown. To gain a structural insight into the different αvβ3 recognition mechanisms, we analyzed the conformation in solution of four ligands containing either the isoDGR, RGD, NGR, or DGR motifs (Fig. 1 and supplemental Fig. S1), and we created putative binding models of the four ligands with αvβ3 based on the crystallographic structure of αvβ3 in its “RGD-bound” conformation (15Xiong J.P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S.L. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1369) Google Scholar). Consistently with functional data, our docking studies show that the isoDGR motif can perfectly mimic the canonical RGD interactions with αvβ3, whereas NGR and DGR lack the stereochemical and electrostatic requirements for a correct recognition of the αvβ3-binding pocket. Cell Line and Reagents—EA.hy926 cells (human endothelial cell fused with human lung carcinoma A549) were cultured as described previously (17Curnis F. Gasparri A. Sacchi A. Cattaneo A. Magni F. Corti A. Cancer Res. 2005; 65: 2906-2913Crossref PubMed Scopus (85) Google Scholar). Human αvβ3 was from Immunological Science (Rome, Italy), and streptavidin peroxidase was from Società Prodotti Antibiotici (Milan, Italy). NGR-TNF and FN-I5 were prepared as described previously (12Curnis F. Longhi R. Crippa L. Cattaneo A. Dondossola E. Bachi A. Corti A. J. Biol. Chem. 2006; 281: 36466-36476Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Preparation and Characterization of Synthetic Peptides—CRGDCGVRY (RGD-2C), CDGRCGVRY (DGR-2C), CNGRCGVRY (NGR-2C), and CisoDGRCGVRY (isoDGR-2C) were prepared as described (12Curnis F. Longhi R. Crippa L. Cattaneo A. Dondossola E. Bachi A. Corti A. J. Biol. Chem. 2006; 281: 36466-36476Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The biotinylated peptides CNGRCGVRSSSRTPSDKYGK-bio and CARACGVRSSSRTPSDKYGK-bio (called bio-CNGRC-hTNF-(1-11) and bio-CARAC-hTNF-(1-11)) consist of CNGRCG or CARACG fused to the N-terminal sequence of human tumor necrosis factor (hTNF)-α followed by a Tyr, to enable detection, and a biotinylated Lys. These peptides were prepared and purified as described (11Di Matteo P. Curnis F. Longhi R. Colombo G. Sacchi A. Crippa L. Protti M.P. Ponzoni M. Toma S. Corti A. Mol. Immunol. 2006; 43: 1509-1518Crossref PubMed Scopus (49) Google Scholar). Deamidation of bio-CNGRC-hTNF-(1-11) was obtained by diluting bio-CNGRC-hTNF-(1-11) in 0.1 m ammonium bicarbonate buffer, pH 8.5, and incubating for 16 h at 37 °C. The resulting β-peptide was called “heat-treated” bio-CNGRC-hTNF-(1-11). All peptides were dissolved in sterile water and stored in aliquots at -20 °C. The molecular mass of each peptide was checked by matrix-assisted laser desorption ionization-time-of-flight analysis. Binding of Peptides to αvβ3 Integrin—The binding of biotinylated peptides (bio-CNGRC-hTNF-(1-11), heat-treated bio-CNGRC-hTNF-(1-11) at 37 °C, and bio-CARAC-hTNF-(1-11)) to αvβ3 integrin immobilized on microtiter plates was analyzed as described previously, using streptavidin peroxidase-peptide complexes (12Curnis F. Longhi R. Crippa L. Cattaneo A. Dondossola E. Bachi A. Corti A. J. Biol. Chem. 2006; 281: 36466-36476Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). These complexes were prepared by mixing various amounts of biotinylated peptides (4 to 0.062 μg) in phosphate-buffered saline with Ca2+ and Mg2+ (DPBS, Cambrex) containing 3% bovine serum albumin with 0.03 units of streptavidin peroxidase (binding capacity 1 μg of biotin/unit of streptavidin peroxidase, final volume of 15 μl). Complexes were diluted in 3% bovine serum albumin/DPBS (1:300), added to microtiter plates coated with purified human αvβ3 integrin (0.5 μg/ml), and incubated for 2 h at room temperature. After washing with DPBS, bound peroxidase was detected by chromogenic reaction with o-phenylenediamine. Cell Adhesion Assay—The effect of peptides on EA.hy926 cell adhesion was investigated by seeding EA.hy926 cells (7.5 × 103) in 96-well flat bottom plates in the presence of increasing concentration of ligands (NGR-2C, RGD-2C, isoDGR-2C, and DGR-2C). After 4 h of incubation at 37 °C, nonadherent cells were washed out. The amount of adherent cells was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay (18Traversari C. van der Bruggen P. van den Eynde B. Hainaut P. Lemoine C. Ohta N. Old L. Boon T. Immunogenetics. 1992; 35: 145-152Crossref PubMed Scopus (184) Google Scholar). NMR Experiments and Structure Calculations—For each ligand NMR spectra of an ∼5 mm sample (90% H2O, 10% D2O) at pH 3 were recorded at 280 K on a Bruker Avance-600 spectrometer (Bruker BioSpin) equipped with a triple-resonance TCI cryoprobe with an x, y, z shielded pulsed-field gradient coil. The experiments were performed at acidic pH to avoid both signal loss because of high exchange rates at neutral pH and deamidation of the asparagine in the NGR-2C peptide (12Curnis F. Longhi R. Crippa L. Cattaneo A. Dondossola E. Bachi A. Corti A. J. Biol. Chem. 2006; 281: 36466-36476Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Proton resonances were assigned by conventional two-dimensional experiments as follows: total correlation spectroscopy (tmix = 60 ms), nuclear Overhauser effect spectroscopy (NOESY), and rotational nuclear Overhauser effect spectroscopy (tmix = 100-400 ms) (19van Well R.M. Marinelli L. Altona C. Erkelens K. Siegal G. van Raaij M. Llamas-Saiz A.L. Kessler H. Novellino E. Lavecchia A. van Boom J.H. Overhand M. J. Am. Chem. Soc. 2003; 125: 10822-10829Crossref PubMed Scopus (63) Google Scholar). Cross-peaks intensities were measured from NOESY spectra at 200 ms. No differences were observed in the experiments at higher mixing times. Water proton signals were suppressed with excitation sculpting sequence (20Hwang T.L. Shaka A.J. J. Magn. Reson. Ser. A. 1995; 112: 275-279Crossref Scopus (1511) Google Scholar). All resonances of the four ligands have been assigned with the only exception of the amide proton of C1, which has a higher exchange rate with the solvent even at low pH (Biological Magnetic Resonance Data Bank accession codes: RGD, 1928339, isoDGR, 38926841; NGR, 32415285; and DGR, 52915018). The 3JHN-HA coupling constants were obtained directly from the resolved amide proton resonances of well digitized mono-dimensional spectrum. The temperature coefficients of the amide protons were obtained from linear fits of the chemical shift data from mono-dimensional spectra acquired in a temperature range from 280 to 300 K (5 K-increasing steps). Data were processed with NMRPipe (21Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11280) Google Scholar) and analyzed using the NMRView software (22Linge J.P. Nilges M. J. Biomol. NMR. 1999; 13: 51-59Crossref PubMed Scopus (239) Google Scholar). Structure Calculations—Structures were calculated using ARIA 1.2, ambiguous restraints for iterative assignment (23Linge J.P. O'Donoghue S.I. Nilges M. Methods Enzymol. 2001; 339: 71-90Crossref PubMed Scopus (329) Google Scholar), in combination with CNS 1.2, crystallography, and NMR systems (24Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16919) Google Scholar) using only manually assigned NOEs as experimental restraints. Coupling constants that were in the range between 6 and 8.5 Hz were not used in calculations because their values were suggestive of rapidly interconverting conformers coexisting in solution. To avoid bias in the calculations, hydrogen bonds inferred from temperature coefficients data were not included. These data were only used for structure validation. Calculations were carried out in the simplified all-hydrogen PARALLHDG5.3 force field with nonbonded interactions modeled by PROLSQ force field (22Linge J.P. Nilges M. J. Biomol. NMR. 1999; 13: 51-59Crossref PubMed Scopus (239) Google Scholar). Parameters for isoaspartic residue were derived from the aspartic residue applying appropriate dihedrals and improper angles. A total of eight iterations (200 structures per iteration) were performed. The ARIA default water refinement was performed on the 30 best structures of the final iteration. The stereochemical quality of the structures was assessed with PROCHECK-NMR program (25Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4285) Google Scholar). In the case of isoDGR-2C, residue 2 was excluded from torsion angle check. However, its torsion angle compared well with φ and ψ angles of isoaspartic residues measured in deposited x-ray structures (Protein Data Bank codes 1AT6, 1DY5, 1RTU, 2FI5, 2FTM, 1C9P, 1LSQ, 2FI4, and 2FTL). Molecular Dynamics Simulations—Simulations were performed on the lowest energy NMR structures of each ligand using the GROMACS 3.3.1 package (26Berendsen H.J.C. van der Spoel D. van Drunen R. Comp. Phys. Comm. 1995; 91: 43-56Crossref Scopus (6854) Google Scholar) with the optimized parameters for liquid simulation (OPLS) force field (27Jorgensen W.L. Maxwell D.S. Tirado-Rives J. J. Am. Chem. Soc. 1996; 118: 11225-11236Crossref Scopus (10145) Google Scholar). All trajectories were calculated in periodic cubic boxes (5 × 5 × 5 nm) of explicit SPC water molecules (28Hermans J. Berendsen H.J.C. van Gunsteren W.F. Postma J.P.M. Biopolymers. 1984; 23: 1513-1518Crossref Scopus (697) Google Scholar). The system was neutralized by 1-2 chloride ions according to the charge of the ligand. Bond lengths were constrained using the LINCS algorithm (29Hess B. Bekker H. Berendsen H.J.C. Fraaije J.G.E.M. J. Comput. Chem. 1997; 18: 1463-1472Crossref Scopus (10848) Google Scholar), and Lennard-Jones interactions were calculated with a 0.9-nm twin-range cutoff. Full electrostatic potentials were computed using the PME method (30Darden T. Perera L. Li L. Pedersen L. Structure (Camb.). 1999; 7: 55-60Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar) with a cutoff of 0.9 nm. The system was first minimized using steepest descent algorithm and then equilibrated at 300 K for 100 ps under NPT (number of molecules, volume, temperature) and periodic boundary conditions. REMD simulations were performed with 16 replicas run in parallel at the following temperatures from 293 to 353 K with 4 K-increasing steps. The temperatures were chosen so as to maintain an exchange rate of 5-10%. Transitions between adjacent temperatures were attempted every 500 MD steps (1 ps) using a Metropolis transition probability (31Sugita Y. Okamoto Y. Chem. Phys. Lett. 1999; 1-2: 141-151Crossref Scopus (3376) Google Scholar), which gives a probability of exchange between two replicas i and j: P(i,j) = exp(-(βi - βj)(Ej - Ei)), where β = 1/kBT; E is the potential energy of the system; T is the absolute temperature, and kB is Boltzmann's constant. Each replica was simulated for 2 ns, with an integration time step of 0.002 fs under NVT (number of molecules, volume, temperature) conditions, yielding a total sampling time of 32 ns. Configurations were saved prior to every attempted transition, leading to an ensemble at each temperature containing 2000 structures. The full trajectories were clustered over the backbone atoms of the macrocycle and the disulfide bridge using the GROMOS algorithm g_cluster (32Daura X. Antes I. van Gunsteren W.F. Thiel W. Mark A.E. Proteins. 1999; 36: 542-555Crossref PubMed Scopus (93) Google Scholar) as implemented in the GROMACS 3.3.1 package with a cutoff of 0.07 nm. Electrostatic Surface Potential Calculations—The electrostatic potential of the four ligands has been calculated using the adaptive Poisson-Boltzmann solver program (APBS 0.5) (33Baker N.A. Sept D. Joseph S. Holst M.J. McCammon J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10037-10041Crossref PubMed Scopus (5699) Google Scholar). The charges (Q) and radii (R) of atoms in the PQR file required by APBS were taken from the OPLS force field. Electrostatic potential was visualized using the PyMOL program (Delano Scientific LLC) with positive potential in blue and negative potential in red in a range between -5 and +5 kT/e. Molecular Docking Calculations—Docking calculations of the four ligands on the globular head of the extracellular part of αvβ3 in its ligand-bound conformation (Protein Data Bank code 1L5G), have been performed using the docking program HADDOCK2.0 (34de Vries S.J. van Dijk A.D. Krzeminski M. van Dijk M. Thureau A. Hsu V. Wassenaar T. Bonvin A.M. Proteins. 2007; 69: 726-733Crossref PubMed Scopus (476) Google Scholar, 35Dominguez C. Boelens R. Bonvin A.M. J. Am. Chem. Soc. 2003; 125: 1731-1737Crossref PubMed Scopus (2087) Google Scholar). For each ligand an ensemble of the best 30 NMR structures in terms of energy was docked onto αvβ3. The protocol follows a three-stage docking procedure, which includes the following: (a) randomization of orientations and rigid body minimization, (b) simulated annealing in torsion angle space, and (c) refinement in Cartesian space with explicit water. Ambiguous interaction restraints (αvβ3: Asp150, Asp218, Tyr122, Arg214, Asn215, and Arg216; ligand, residues 2-4) (35Dominguez C. Boelens R. Bonvin A.M. J. Am. Chem. Soc. 2003; 125: 1731-1737Crossref PubMed Scopus (2087) Google Scholar) were derived from the known interactions of the RGD motif of the cyclic pentapeptide in the Protein Data Bank structure 1L5G. OPLS force field was used. During the rigid body docking step 1000 structures were calculated, allowing the ligand to explore solutions rotated by 180°, thus increasing the sampling of the solutions. The best 200 solutions in terms of intermolecular energies were selected for a semiflexible simulated annealing in which the side-chains of αvβ3 and of the cyclic peptides located at the binding interface (αvβ3, residues 148-152, 216-220 on the αv domain and 118-120, 124-126 on β3 domain; ligand, residues 1-9) were allowed to move in a semi-rigid body docking protocol to search for conformational rearrangements. The models were then subjected to a water refinement step (TIP3P model). Backbone and side-chain of amino acids 6-9 were fully flexible in iteration 1 and in the water refinement step. Analogous calculations have been performed using for each ligand an ensemble of conformers composed by the centroids representing the highest populated clusters (80%) identified in the clusters of REMD trajectories (4, 23, 10, and 5 structures for RGD, isoDGR, NGR, and DGR, respectively). The analysis of the simulations was performed applying in-house Python and Tcl scripts. Root mean square deviation (r.m.s.d.) values were calculated using the ProFit program (available on line). The fitting of the protein was performed on the flexible residues (Table 3) using the McLachlan algorithm (36McLachlan A. Acta Crystallogr. Sect. A. 1982; 38: 871-873Crossref Scopus (330) Google Scholar). The r.m.s.d. values of the cyclopeptides were calculated on the backbone of residues 1-5 and on the sulfur and Cβ atoms of residues 1 and 5. The final r.m.s.d. matrix was then clustered using the algorithm described in Daura et al. (32Daura X. Antes I. van Gunsteren W.F. Thiel W. Mark A.E. Proteins. 1999; 36: 542-555Crossref PubMed Scopus (93) Google Scholar), where a cluster is defined as an ensemble of at least two conformations displaying a r.m.s.d. smaller than 1 Å. In the case of NGR-2C, the cut off was increased to 1.3 Å.TABLE 3Summary of the distances (Å) between the ligands and αvβ3 observed in the highest populated clusters (cluster 1)Ligandαvβ3RGD x-rayRGD x-ray dockRGD-2CisoDGR-2CNGR-2CDGR-2CCHNTermaHNTerm denotes N-terminal protons.Tyr122ObO denotes carbonyl oxygen.1.8 ± 0.1DOXcOX denotes carboxylate oxygen./IASOXTyr122HNdHN denotes amide proton.1.92.1 ± 0.12 ± 0.12.2 ± 0.1DOX/IASOXAsn215HN1.82.2 ± 0.12.2 ± 0.12.3 ± 0.2DOX/IASOXCa2+2.71.7 ± 0.11.7 ± 0.11.8 ± 0.1DOArg214HXeHX denotes guanidinium proton.3.02.0 ± 0.11.9 ± 0.12.3 ± 0.11.9 ± 0.2DHNAsp150OXDHNArg216O2.72.4 ± 0.12.2 ± 0.1isoDHNTyr122O2.0 ± 0.2NHXAsn215O2.1 ± 0.2NOArg214HX1.9 ± 0.1NOXAsn216HN2.2 ± 0.1GHNArg216O2.4 ± 0.1RHXAsp150OX2.92.2 ± 0.21.9 ± 0.21.9 ± 0.11.9 ± 0.2RHXAsp218OX1.91.7 ± 0.11.8 ± 0.22.0 ± 0.42.0 ± 0.3RHXGlu180OX2.71.9 ± 0.21.7 ± 0.12.0 ± 0.3RHXAsn215O1.8 ± 0.1RHXArg216O2.3 ± 0.1RHXTyr122O2.0 ± 0.2RHNAsp218OX2.4 ± 0.1a HNTerm denotes N-terminal protons.b O denotes carbonyl oxygen.c OX denotes carboxylate oxygen.d HN denotes amide proton.e HX denotes guanidinium proton. Open table in a new tab The final structures after water refinement were clustered and scored using a combination of energy terms defined as follows: 1.0 × EvdW + 1.0 × Eelec + 0.1 × EAIR + 1.0 × Edesolv, the subscripts are as follows: vdW, van der Waals energy; elec, the electrostatic energy; AIR, the ambiguous interaction restraint energy; and desolv, the desolvation energy calculated using the atomic desolvation parameters of Fernandez-Recio and colleagues (37Fernandez-Recio J. Totrov M. Abagyan R. J. Mol. Biol. 2004; 335: 843-865Crossref PubMed Scopus (227) Google Scholar). Unlike NGR-2C and DGR-2C, isoDGR-2C and RGD-2C Can Efficiently Bind αvβ3 Integrin—We have shown previously that peptides containing the NGR motif, such as CNGRC and FN-I5, can rapidly deamidate (half-life, 2-3 h) when incubated for 16 h at pH 8.5 and 37 °C. This reaction leads to the generation of species containing the DGR and isoDGR motifs (12Curnis F. Longhi R. Crippa L. Cattaneo A. Dondossola E. Bachi A. Corti A. J. Biol. Chem. 2006; 281: 36466-36476Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). To assess the effect of NGR deamidation on αvβ3 binding, we have analyzed the interaction of a biotinylated NGR peptide (bio-CNGRC-hTNF-(1-11)) to αvβ3 integrin before and after incubation at 37 °C. To this aim, we prepared complexes of biotinylated peptides and streptavidin-peroxidase, and we analyzed their binding to αvβ3 integrin adsorbed on microtiter plates. Although little or no binding was observed with bio-CNGRC-hTNF-(1-11) and bio-CARAC-hTNF-(1-11), a control peptide, a marked increase in binding occurred after heat treatment of bio-CNGRC-hTNF-(1-11) (Fig. 2A). Similar results were observed also with a FN-I5 peptide (data not shown). This suggests that peptide deamidation is very critical for αvβ3 binding. Given that NGR deamidation can lead to the formation of isoDGR and DGR, we performed additional assays to assess which of these molecular species was responsible for the increase in binding after heat treatment. To this aim we compared the capability of synthetic DGR-2C, isoDGR-2C, and NGR-2C to compete the binding of heat-treated bio-CNGRC-hTNF-(1-11) to αvβ3 integrin adsorbed on microtiter plates. The RGD-2C peptide was analyzed in parallel as a positive control. The results showed that isoDGR-2C and RGD-2C could inhibit the binding of heat-treated bio-CNGRC-hTNF-(1-11) to αvβ3 with similar potency, whereas >100-fold higher concentration of DGR and NGR were necessary to induce partial competition (Fig. 2B). These results confirm the hypothesis that isoDGR, unlike NGR and DGR, can efficiently mimic RGD in binding αvβ3. To assess whether these ligand-integrin interactions can also occur in living cells, we investigated the effect of RGD-2C, DGR-2C, isoDGR-2C, and NGR-2C on the adhesion of EA.hy926 cells. As expected both RGD-2C and isoDGR-2C, but not DGR-2C, efficiently inhibited cell adhesion (Fig. 2C). Furthermore, a 10-fold higher concentration of NGR-2C was necessary to compete to comparable levels. Considering that assay incubation was 3.5 h and that the half-life of NGR deamidation in cell culture medium is 2-3 h (12Curnis F. Longhi R. Crippa L. Cattaneo A. Dondossola E. Bachi A. Corti A. J. Biol. Chem. 2006; 281: 36466-36476Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), it is very likely that NGR competition was actually related to isoDGR formation during assay incubation. Taken together, these results suggest that isoDGR, unlike DGR and NGR, can functionally mimic RGD in the interaction with adhesion receptors, such as αvβ3 integrin. The modest activity observed with NGR could be due to deamidation occurring during assay incubation, conside" @default.
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• W2013156594 title "Structural Basis for the Interaction of isoDGR with the RGD-binding Site of αvβ3 Integrin" @default.
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• W2013156594 doi "https://doi.org/10.1074/jbc.m710273200" @default.
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