Conformational Plasticity of an Enzyme during Catalysis: Intricate Coupling between Cyclophilin A Dynamics and Substrate Turnover

Enzyme catalysis is central to almost all biochemical processes, speeding up rates of reactions to biological relevant timescales. Enzymes make use of a large ensemble of conformations in recognizing their substrates and stabilizing the transition states, due to the inherent dynamical nature of biomolecules. The exact role of these diverse enzyme conformations and the interplay between enzyme conformational dynamics and catalysis is, according to the literature, not well understood. Here, we use molecular dynamics simulations to study human cyclophilin A (CypA), in order to understand the role of enzyme motions in the catalytic mechanism and recognition. Cyclophilin A is a tractable model system to study using classical simulation methods, because catalysis does not involve bond formation or breakage. We show that the conformational dynamics of active site residues of substrate-bound CypA is inherent in the substrate-free enzyme. CypA interacts with its substrate via conformational selection as the configurations of the substrate changes during catalysis. We also show that, in addition to tight intermolecular hydrophobic interactions between CypA and the substrate, an intricate enzyme-substrate intermolecular hydrogen-bonding network is extremely sensitive to the configuration of the substrate. These enzyme-substrate intermolecular interactions are loosely formed when the substrate is in the reactant and product states and become well formed and reluctant to break when the substrate is in the transition state. Our results clearly suggest coupling among enzyme-substrate intermolecular interactions, the dynamics of the enzyme, and the chemical step. This study provides further insights into the mechanism of peptidyl-prolyl cis/trans isomerases and the general interplay between enzyme conformational dynamics and catalysis

Replica-Exchange Accelerated Molecular Dynamics (REXAMD) Applied to Thermodynamic Integration

Accelerated molecular dynamics (AMD) is an efficient strategy for accelerating the sampling of molecular dynamics simulations, and observable quantities such as free energies derived on the biased AMD potential can be reweighted to yield results consistent with the original, unmodified potential. In conventional AMD the reweighting procedure has an inherent statistical problem in systems with large acceleration, where the points with the largest biases will dominate the reweighted result and reduce the effective number of data points. We propose a replica exchange of various degrees of acceleration (REXAMD) to retain good statistics while achieving enhanced sampling. The REXAMD method is validated and benchmarked on two simple gas-phase model systems, and two different strategies for computing reweighted averages over a simulation are compared

Coupling Accelerated Molecular Dynamics Methods with Thermodynamic Integration Simulations

In this work we propose a straightforward and efficient approach to improve accuracy and convergence of free energy simulations in condensed-phase systems. We also introduce a new accelerated Molecular Dynamics (MD) approach in which molecular conformational transitions are accelerated by lowering the energy barriers while the potential surfaces near the minima are left unchanged. All free energy calculations were performed on the propane-to-propane model system. The accuracy of free energy simulations was significantly improved when sampling of internal degrees of freedom of solute was enhanced. However, accurate and converged results were only achieved when the solvent interactions were taken into account in the accelerated MD approaches. The analysis of the distribution of boost potential along the free energy simulations showed that the new accelerated MD approach samples efficiently both low- and high-energy regions of the potential surface. Since this approach also maintains substantial populations in regions near the minima, the statistics are not compromised in the thermodynamic integration calculations, and, as a result, the ensemble average can be recovered

Identification of an L-Phenylalanine Binding Site Enhancing The Cooperative Responses of The Calcium Sensing Receptor to Calcium

Functional positive cooperative activation of the extracellular calcium ([Ca2+]o)-sensing receptor (CaSR), a member of the family C G protein-coupled receptors (GPCRs), by [Ca2+]o or amino acids elicits intracellular Ca2+ ([Ca2+]i) oscillations. Here, we report the central role of predicted Ca2+-binding Site 1 within the hinge region of the extracellular domain (ECD) of CaSR and its interaction with other Ca2+-binding sites within the ECD in tuning functional positive homotropic cooperativity caused by changes in [Ca2+]o. Next, we identify an adjacent L-Phe-binding pocket that is responsible for positive heterotropic cooperativity between [Ca2+]o and L- Phe in eliciting CaSR-mediated [Ca2+]i oscillations. The hetero-communication between Ca2+ and an amino acid globally enhances functional positive homotropic cooperative activation of CaSR in response to [Ca2+]o signaling by positively impacting multiple [Ca2+]o-binding sites within the ECD. Elucidation of the underlying mechanism provides important insights into the longstanding question of how the receptor transduces signals initiated by [Ca2+]o and amino acids into intracellular signaling events

Role of Ca2+ and L-Phe in Regulating Functional Cooperativity of Disease- Associated ‘‘Toggle’’ Calcium-Sensing Receptor Mutations

The Ca2+-sensing receptor (CaSR) regulates Ca2+ homeostasis in the body by monitoring extracellular levels of Ca2+ ([Ca2+]o) and amino acids. Mutations at the hinge region of the N-terminal Venus flytrap domain (VFTD) produce either receptor inactivation (L173P, P221Q) or activation (L173F, P221L) related to hypercalcemic or hypocalcemic disorders. In this paper, we report that both L173P and P221Q markedly impair the functional positive cooperativity of the CaSR as reflected by [Ca2+]o–induced [Ca2+]i oscillations, inositol-1-phosphate (IP1) accumulation and extracellular signal-regulated kinases (ERK1/2) activity. In contrast, L173F and P221L show enhanced responsiveness of these three functional readouts to [Ca2+]o. Further analysis of the dynamics of the VFTD mutants using computational simulation studies supports disruption in the correlated motions in the loss-offunction CaSR mutants, while these motions are enhanced in the gain-of-function mutants. Wild type (WT) CaSR was modulated by L-Phe in a heterotropic positive cooperative way, achieving an EC50 similar to those of the two activating mutations. The response of the inactivating P221Q mutant to [Ca2+]o was partially rescued by L-Phe, illustrating the capacity of the L-Phe binding site to enhance the positive homotropic cooperativity of CaSR. L-Phe had no effect on the other inactivating mutant. Moreover, our results carried out both in silico and in intact cells indicate that residue Leu173, which is close to residues that are part of the L-Phe-binding pocket, exhibited impaired heterotropic cooperativity in the presence of L-Phe. Thus, Pro221 and Leu173 are important for the positive homo- and heterotropic cooperative regulation elicited by agonist binding

Extracellular Calcium Modulates Actions of Orthosteric and Allosteric Ligands on Metabotropic Glutamate Receptor 1alpha

SUMMARY: Metabotropic glutamate receptor 1α (mGluR1α), a member of the family C G protein-coupled receptors (GPCRs), is emerging as a potential drug target for various disorders including chronic neuronal degenerative diseases. In addition to being activated by glutamate, mGluR1α is also modulated by extracellular Ca2+. However, the underlying mechanism is unknown. Moreover, it has long been challenging to develop receptor-specific agonists due to homologies within the mGluR family, and the Ca2+-binding site(s) on mGluR1α may provide an opportunity for receptor-selective targeting by therapeutics. In the present study, we show that our previously predicted Ca2+-binding site in the hinge region of mGluR1α is adjacent to the site where orthosteric agonists and antagonists bind on the extracellular domain of the receptor. Moreover, we have found that extracellular Ca2+ enhances mGluR1α-mediated intracellular Ca2+ responses evoked by the orthosteric agonist, L-quisqualate. Conversely, extracellular Ca2+ diminishes the inhibitory effect of the mGluR1α orthosteric antagonist, (s)-MCPG. In addition, selective positive (Ro 67-4853) and negative (CPCCOEt) allosteric modulators of mGluR1α potentiate and inhibit responses to extracellular Ca2+, respectively, in a manner similar to their effects on the response of mGluR1α to glutamate. Mutations at residues predicted to be involved in Ca2+-binding, including E325I, have significant effects on the modulation of responses to the orthosteric agonist, L-quisqualate, and the allosteric modulator Ro 67-4853 by extracellular Ca2+. These studies reveal that binding of extracellular Ca2+ to the predicted Ca2+-binding site in the ECD of mGluR1α modulates not only glutamate-evoked signaling but also the actions of both orthosteric ligands and allosteric modulators on mGluR1α

Cyclodextrin Complexes of Reduced Bromonoscapine in Guar Gum Microspheres Enhance Colonic Drug Delivery

Here, we report improved solubility and enhanced colonic delivery of reduced bromonoscapine (Red-Br-Nos), a cyclic ether brominated analogue of noscapine, upon encapsulation of its cyclodextrin (CD) complexes in bioresponsive guar gum microspheres (GGM). Phase−solubility analysis suggested that Red-Br-Nos complexed with β-CD and methyl-β-CD in a 1:1 stoichiometry, with a stability constant (Kc) of 2.29 × 103 M−1 and 4.27 × 103 M−1. Fourier transforms infrared spectroscopy indicated entrance of an O−CH2 or OCH3−C6H4−OCH3 moiety of Red-Br-Nos in the β-CD or methyl-β- CD cavity. Furthermore, the cage complex of Red-Br-Nos with β-CD and methyl-β-CD was validated by several spectral techniques. Rotating frame Overhauser enhancement spectroscopy revealed that the Ha proton of the OCH3−C6H4−OCH3 moiety was closer to the H5 proton of β-CD and the H3 proton of the methyl-β-CD cavity. The solubility of Red-Br-Nos in phosphate buffer saline (PBS, pH ∼ 7.4) was improved by ∼10.7-fold and ∼21.2-fold when mixed with β-CD and methyl-β-CD, respectively. This increase in solubility led to a favorable decline in the IC50 by ∼2-fold and ∼3-fold for Red-Br-Nos−β-CD-GGM and Red-Br-Nos−methyl-β-CD-GGM formulations respectively, compared to free Red-Br-Nos−β-CD and Red-Br-Nos−methyl-β-CD in human colon HT-29 cells. GGM-bearing drug complex formulations were found to be highly cytotoxic to the HT-29 cell line and further effective with simultaneous continuous release of Red-Br-Nos from microspheres. This is the first study to showing the preparation of drug-complex loaded GGMS for colon delivery of Red-Br-Nos that warrants preclinical assessment for the effective management of colon cancer

Deciphering the role of glucosamine-6-phosphate in the riboswitch action of glmS ribozyme

The GlmS ribozyme is believed to exploit a general acid-base catalytic mechanism in the presence of glucosamine-6-phosphate (GlcN6P) to accelerate self-cleavage by approximately six orders of magnitude. The general acid and general base are not known, and the role of the GlcN6P cofactor is even less well understood. The amine group of GlcN6P has the ability to either accept or donate a proton and could therefore potentially act as an acid or a base. In order to decipher the role of GlcN6P in the self-cleavage of glmS, we have determined the preferred protonation state of the amine group in the wild-type and an inactive G40A mutant using molecular dynamics simulations and free energy calculations. Here we show that, upon binding of GlcN6P to wild-type glmS, the pKa of the amine moiety is altered by the active site environment, decreasing by about 2.2 from a solution pKa of about 8.2. On the other hand, we show that the pKa of the amine group slightly increases to about 8.4 upon binding to the G40A inactive mutant of glmS. These results suggest that GlcN6P acts as a general acid in the self-cleavage of glmS. Upon binding to glmS, GlcN6P can easily release a proton to the 5′-oxygen of G1 during self-cleavage of the backbone phosphodiester bond. However, in the G40A inactive mutant of glmS, the results suggest that the ability of GlcN6P to easily release its proton is diminished, in addition to the possible lack of G40 as an effective base