Abstract In the context of drug design, C-H···O hydrogen bonds have received little attention so far, mostly because they are considered weak relative to other noncovalent interactions such as O-H···O hydrogen bonds, π/π interactions, and van der Waals interactions. Herein, we demonstrate the significance of hydrogen bonds between C-H groups adjacent to an ammonium cation and an oxygen atom (N + -C-H···O hydrogen bonds) in protein-ligand complexes. Quantum chemical calculations revealed details on the strength and geometrical requirements of these N + -C-H···O hydrogen bonds, and a subsequent survey of the Protein Data Bank (PDB) based on these criteria suggested that numerous protein-ligand complexes contain such N + -C-H···O hydrogen bonds. An ensuing experimental investigation into the G9a-like protein (GLP)-inhibitor complex demonstrated that N + -C-H···O hydrogen bonds affect the activity of the inhibitors against the target enzyme. These results should provide the basis for the use of N + -C-H···O hydrogen bonds in drug discovery.
A number of diverse cell-surface proteins are anchored to the cytoskeleton via scaffold proteins. Na+/H+ exchanger regulatory factor-1 (NHERF1), encoded by the Slc9a3r1 gene, functions as a scaffold protein, which is implicated in the regulation of membrane expression of various cell-surface proteins. Here, we demonstrate that the circadian clock component PERIOD2 (PER2) modulates transcription of the mouse Slc9a3r1 gene, generating diurnal accumulation of NHERF1 in the mouse liver. Basal expression of Slc9a3r1 was dependent on transcriptional activation by p65/p50. PER2 bound to p65 protein and prevented p65/p50-mediated transactivation of Slc9a3r1. The time-dependent interaction between PER2 and p65 underlay diurnal oscillation in the hepatic expression of Slc9a3r1/NHERF1. The results of immunoprecipitation experiments and liquid chromatography-mass spectrometry analysis of mouse liver revealed that NHERF1 time-dependently interacted with fatty acid transport protein-5 (FATP5). Temporary accumulation of NHERF1 protein stabilized plasmalemmal localization of FATP5, thereby enhancing hepatic uptake of fatty acids at certain times of the day. Our results suggest an unacknowledged role for PER2 in regulating the diurnal expression of NHERF1 in mouse liver. This machinery also contributed to diurnal changes in the ability of hepatic cells to uptake fatty acids.
Src homology 2 (SH2) domains play a critical role in cellular signal transduction. They bind to peptides containing phosphotyrosine (pY) with various specificities that depend on the flanking amino-acid residues. The SH2 domain of growth-factor receptor-bound protein 2 (Grb2) specifically recognizes pY-X-N-X, whereas the SH2 domains in phosphatidylinositol 3-kinase (PI3K) recognize pY-X-X-M. Binding of the pY site in CD28 (pY-M-N-M) by PI3K and Grb2 through their SH2 domains is a key step that triggers the CD28 signal transduction for T cell activation and differentiation. In this study, we determined the crystal structure of the Grb2 SH2 domain in complex with a pY-containing peptide derived from CD28 at 1.35 Å resolution. The peptide was found to adopt a twisted U-type conformation, similar to, but distinct from type-I β-turn. In all previously reported crystal structures, the peptide bound to the Grb2 SH2 domains adopts a type-I β-turn conformation, except those with a proline residue at the pY+3 position. Molecular modeling also suggests that the same peptide bound to PI3K might adopt a very different conformation.
We examined the changes in prolylhydroxylase (PH) and interstitial collagenase (CA) activities in the healing of acetic acid-induced gastric ulcers in rats. Gastric ulcers were induced by subserosal injection of acetic acid, and confirmed by endoscopy on the third day. At the acute stage of ulceration, PH activity was not high but CA activity was increased. At the healing stage, PH activity was increased but CA activity gradually decreased to a normal level. Cimetidine did not effect PH activity but tended to increase CA activity in the healing. On the contrary, elcatonin increased PH activity but had little effect on CA activity. The above enzymes seems to be indispensable in the healing of gastric ulcers and effects of antiulcer agents on these enzymes must be considered.
