Cross-saturation experiments allow the identification of the contact residues of large protein complexes (MW>50 K) more rigorously than conventional NMR approaches which involve chemical shift perturbations and hydrogen-deuterium exchange experiments [Takahashi et al. (2000) Nat. Struct. Biol., 7, 220-223]. In the amide proton-based cross-saturation experiment, the combined use of high deuteration levels for non-exchangeable protons of the ligand protein and a solvent with a low concentration of (1)H(2)O greatly enhanced the selectivity of the intermolecular cross-saturation phenomenon. Unfortunately, experimental limitations caused losses in sensitivity. Furthermore, since main chain amide protons are not generally exposed to solvent, the efficiency of the saturation transfer directed to the main chain amide protons is not very high. Here we propose an alternative cross-saturation experiment which utilizes the methyl protons of the side chains of the ligand protein. Owing to the fast internal rotation along the methyl axis, we theoretically and experimentally demonstrated the enhanced efficiency of this approach. The methyl-utilizing cross-saturation experiment has clear advantages in sensitivity and saturation transfer efficiency over the amide proton-based approach.

Glycoprotein VI (GPVI) is a major collagen receptor on the platelet surface that recognizes the glycine-proline-hydroxyproline (GPO) sequence in the collagen molecule and plays a crucial role in thrombus formation. Inhibitors that block the interaction of GPVI with collagen have potential for use as antithrombotic drugs. For low molecular weight drug design for GPVI, it is essential to obtain precise structural and interaction information about GPVI-binding ligands. However, experimentally obtained structural and interaction information of small ligands, such as peptides, in the GPVIbound state has not been reported. In this study, by screening a phage-displayed peptide library, we discovered a novel peptide ligand (pep-10L; YSDTDWLYFSTS) without any similarities to the sequence of collagen that inhibits GPVI-GPO binding. Systematic Ala scanning in surface plasmon resonance experiments and a saturation transfer difference NMR experiment revealed that Trp 6 , Leu 7 , Phe 9, and Ser 10 residues in the pep-10L peptide interacted with GPVI. Furthermore, the GPVIbound conformation of the pep-10L peptide was determined using transferred nuclear Overhauser effect analysis. The obtained structure has revealed that the central part of pep-10L (Asp 5 -Phe 9 ) has a helical conformation, the side chains of Trp 6 , Leu 7 , and Phe 9 form a hydrophobic side in the helix, and the Tyr 8 side chain faces the opposite direction from the hydrophobic side. Computational docking prediction has shown that the hydrophobic side of pep-10L sticks in the hydrophobic groove on the GPVI surface, which corresponds to the putative collagen-related peptide binding groove. These data could enable the structure-guided development of a small molecule GPVI antagonist. Glycoprotein VI (GPVI)3 is a major collagen receptor on the platelet surface that plays a crucial role in collagen-induced platelet activation, thrombus formation (1-3), and acute coronary syndrome (4). The platelets of GPVI null mice fail to aggregate in response to collagen and lack thrombus formation, but the mice do not show a severe bleeding tendency (5). In clinical studies, GPVI-deficient patients show only a mild bleeding tendency despite the fact that their platelets lack collagen-induced aggregation (6 -8). In addition, the Fab fragment of a monoclonal antibody, OM4, effectively inhibits thrombosis in rats without prolonging bleeding (9). These observations suggest that inhibitors that block the interaction of GPVI with collagen could be effective antithrombotic drugs without severe side effects. For low molecular weight drug design, it is essential to elucidate the ligand recognition mechanism and to obtain precise structural and interaction information for GPVI-binding ligands.GPVI recognizes the glycine-proline-hydroxyproline (GPO) sequence in the collagen molecule (10). In addition to collagen, synthetic collagen-related peptide (CRP), which contains 10 GPO repeats, is also a specific agonist for GPVI and is commonly used to study GPVI function (11). Recently, the min...

A wide variety of compound libraries are currently available to obtain active compounds for drug target proteins, but the affinities of initially screened compounds are usually too low and have to be improved by chemical modification of the compounds. In such cases, pharmacophore information of the compounds plays a key role for the next modification step. Structure determination of the protein-compound complex is too time-consuming to be applied for such situations. Therefore, many chemists would highly appreciate simple and accurate experimental procedures to obtain pharmacophore information.From this viewpoint, various ligand-observed NMR spectroscopy experiments have been proposed to characterize protein-ligand interactions.[1] Among them, experiments exploiting nuclear Overhauser effects (NOEs; transferred NOE, saturation transfer difference (STD), pumped NOE, water-ligand observed by gradient spectroscopy (water-LOGSY), etc.) are widely used and are also utilized as a ligand pharmacophore (or epitope) mapping technique. [2] However, it has recently been revealed that the difference of the longitudinal relaxation of each ligand proton severely interferes with the derived pharmacophore mapping result, [3] and it is crucial to evaluate intermolecular cross-relaxation terms for accurate pharmacophore mapping. With this in mind, pharmacophore mapping by diffusion NMR spectroscopy, [4] adiabatic fast passage NOESY (AFP-NOESY), [5] and group epitope mapping considering relaxation of the ligand (GEM-CRL) [6] quantitatively exploit the obtained intermolecular cross-relaxation effect.Herein, we propose a simple and rapid approach for pharmacophore mapping experiments, which utilizes the difference between the longitudinal relaxation rates of ligand protons with and without irradiation of the protons of the target protein.The longitudinal relaxation of ligand proton I is represented by the modified Bloch equation [Eq. (1)]where DI z = I z ÀI z 0 (I z represents the instantaneous longitudinal magnetization, I z 0 represents the thermal equilibrium values of I z ); DS z and DX z for protons S and X are defined correspondingly. R I represents the auto-relaxation rate constant of ligand proton I, s IS represents the intramolecular cross-relaxation rate constant between ligand proton I and another ligand proton S, and s IX represents the intermolecular cross-relaxation rate constant between ligand proton I and proton X of the target protein. In the case that longitudinal relaxation rates are measured by using an inversion-recovery method, and the initial 1808 inversion pulse is replaced with two consecutive 908 pulses with an appropriate phase cycling, [7] the thermal equilibrium term can be omitted from Equation (1) to give Equation (2).