Glycine Substitution Effects on the Supramolecular Morphology and Rigidity of Cell-Adhesive Amphiphilic Peptides.

Self-assembling peptides that are capable of adopting ß-sheet structures can generate nanofibers that lead to hydrogel formation. Herein, to tune the supramolecular morphologies, mechanical properties, and stimuli responses of the hydrogels, we investigated glycine substitution in a ß-sheet-forming amphiphilic peptide. Glycine substitution generally enhances conformational flexibility. Indeed, glycine substitution in an amphiphilic peptide weakened the hydrogels or even inhibited the gelation. However, unexpectedly, glycine substitution at the center of the peptide molecule significantly enhanced the hydrogel stiffness. The central glycine substitution affected the molecular packing and led to twisted ß-sheet structures and to nanofiber bundling, which likely led to the stiffened hydrogel. Importantly, the supramolecular structures were accurately predicted by molecular dynamics simulations, demonstrating the helpfulness of these techniques for the identification of self-assembling peptides. The hydrogel formed by the amphiphilic peptide with the central glycine substitution had cell adhesive function, and showed a reversible thermal gel-to-sol transition. Thus, glycine substitution is effective in modulating self-assembling structures, rheological properties, and dynamics of biofunctional self-assembling peptides.

A structural and mutagenic blueprint for molecular recognition of strychnine and d-tubocurarine by different cys-loop receptors.

Cys-loop receptors (CLR) are pentameric ligand-gated ion channels that mediate fast excitatory or inhibitory transmission in the nervous system. Strychnine and d-tubocurarine (d-TC) are neurotoxins that have been highly instrumental in decades of research on glycine receptors (GlyR) and nicotinic acetylcholine receptors (nAChR), respectively. In this study we addressed the question how the molecular recognition of strychnine and d-TC occurs with high affinity and yet low specificity towards diverse CLR family members. X-ray crystal structures of the complexes with AChBP, a well-described structural homolog of the extracellular domain of the nAChRs, revealed that strychnine and d-TC adopt multiple occupancies and different ligand orientations, stabilizing the homopentameric protein in an asymmetric state. This introduces a new level of structural diversity in CLRs. Unlike protein and peptide neurotoxins, strychnine and d-TC form a limited number of contacts in the binding pocket of AChBP, offering an explanation for their low selectivity. Based on the ligand interactions observed in strychnine- and d-TC-AChBP complexes we performed alanine-scanning mutagenesis in the binding pocket of the human α1 GlyR and α7 nAChR and showed the functional relevance of these residues in conferring high potency of strychnine and d-TC, respectively. Our results demonstrate that a limited number of ligand interactions in the binding pocket together with an energetic stabilization of the extracellular domain are key to the poor selective recognition of strychnine and d-TC by CLRs as diverse as the GlyR, nAChR, and 5-HT(3)R.

Protection of ATP-depleted cells by impermeant strychnine derivatives: implications for glycine cytoprotection.

Glycine and structurally related amino acids with activities at chloride channel receptors in the central nervous system also have robust protective effects against cell injury by ATP depletion. The glycine receptor antagonist strychnine shares this protective activity. An essential step toward identification of the molecular targets for these compounds is to determine whether they protect cells through interactions with intracellular targets or with molecules on the outer surface of plasma membranes. Here we report cytoprotection by a cell-impermeant derivative of strychnine. A strychnine-fluorescein conjugate (SF) was synthesized, and impermeability of plasma membranes to this compound was verified by fluorescence confocal microscopy. In an injury model of Madin-Darby canine kidney cells, ATP depletion led to lactate dehydrogenase release. SF prevented lactate dehydrogenase leakage without ameliorating ATP depletion. This was accompanied by preservation of cellular ultrastructure and exclusion of vital dyes. SF protection was also shown for ATP-depleted rat hepatocytes. On the other hand, when a key structural motif in the active site of strychnine was chemically blocked, the SF lost its protective effect, establishing strychnine-related specificity for SF protection. Cytoprotective effects of the cell-impermeant strychnine derivative provide compelling evidence suggesting that molecular targets on the outer surface of plasma membranes may mediate cytoprotection by strychnine and glycine.

Identifying agonistic and antagonistic mechanisms operative at the GABA receptor.

Based on our molecular modeling investigations of the glycinergic receptor, we expanded our studies to similarly investigate the GABAergic receptor. New data suggest there may exist a slightly different agonistic mechanism for the molecules described herein as compared to glycine. The origin of this is undoubtedly the fact that, while glycine has a positive and two negative binding sites, it is significantly shorter than GABA and the other GABA agonists. Clearly, discovery of more glycine agonists is needed to further clarify this point. Moreover, we find a remarkedly different antagonistic mechanism exists for this phylogenetically newer inhibitory system in the central nervous system (CNS) than recently reported for strychnine and eight weaker glycine antagonists. We used GABA and six agonists (muscimol, dihydromuscimol, THIP, isoguvacine, trans-3-aminocyclopentane-1-carboxylic acid, piperidine-4-sulfonic acid) and five antagonists (bicuculline-N15-methobromide, R5135, pitrazepin, iso-THAZ and securinine) to derive our conclusions. We found that each of the agonists have three clearly defined atoms that can serve as attachment points at the GABAA receptor site. One of the three attachment atoms includes a carbonyl or carboxylate oxygen. The role of the carbonyl or carboxylate atom is very important. First, we theorize that a rapid two-point attachment occurs (one from the positive end and one from one of the other two negative atoms on the ligand) at the recognition site in the receptor where GABA or a GABAergic agonist binds. The positive end of the agonist perhaps associates through hydrogen bonding to a beta-carboxyl group in one of the aspartate molecules in the polypeptide. The negative attachment points perhaps bind through hydrogen bonding to arginine molecules in this polypeptide. The second negative site in the agonist immediately triggers a conformational change by pulling together the aforementioned groups by electrostatic attraction, and hence opening the chloride channel. We propose the carbonyl oxygen is partly responsible for triggering the opening by formation of a double hydrogen bond to arginine. We postulate that this attraction is the first step inducing the conformational change. In the case of the GABA antagonists investigated, a fourth attachment site was not found. In fact only two sites have been identified similar to the group II glycine antagonists. Our data support a hypothesis for GABAergic antagonist activity which suggests that the antagonist simply binds to the recognition site and blocks the neurotransmitter, GABA, from entering this site thereby preventing the opening of the chloride channel; it just stays closed. This mechanism is different from the mechanism proposed for the large number of Group I glycine antagonists (Aprison et al.: J Neurosci Res 41: 259-269, 1995).