Binding Mechanism of CD47 with SIRPα Variants and Its Antibody: Elucidated by Molecular Dynamics Simulations.

The intricate complex system of the differentiation 47 (CD47) and the signal-regulatory protein alpha (SIRPα) cluster is a crucial target for cancer immunotherapy. Although the conformational state of the CD47-SIRPα complex has been revealed through crystallographic studies, further characterization is needed to fully understand the binding mechanism and to identify the hot spot residues involved. In this study, molecular dynamics (MD) simulations were carried out for the complexes of CD47 with two SIRPα variants (SIRPαv1, SIRPαv2) and the commercially available anti-CD47 monoclonal antibody (B6H12.2). The calculated binding free energy of CD47-B6H12.2 is lower than that of CD47-SIRPαv1 and CD47-SIRPαv2 in all the three simulations, indicating that CD47-B6H12.2 has a higher binding affinity than the other two complexes. Moreover, the dynamical cross-correlation matrix reveals that the CD47 protein shows more correlated motions when it binds to B6H12.2. Significant effects were observed in the energy and structural analyses of the residues (Glu35, Tyr37, Leu101, Thr102, Arg103) in the C strand and FG region of CD47 when it binds to the SIRPα variants. The critical residues (Leu30, Val33, Gln52, Lys53, Thr67, Arg69, Arg95, and Lys96) were identified in SIRPαv1 and SIRPαv2, which surround the distinctive groove regions formed by the B2C, C'D, DE, and FG loops. Moreover, the crucial groove structures of the SIRPα variants shape into obvious druggable sites. The C'D loops on the binding interfaces undergo notable dynamical changes throughout the simulation. For B6H12.2, the residues Tyr32LC, His92LC, Arg96LC, Tyr32HC, Thr52HC, Ser53HC, Ala101HC, and Gly102HC in its initial half of the light and heavy chains exhibit obvious energetic and structural impacts upon binding with CD47. The elucidation of the binding mechanism of SIRPαv1, SIRPαv2, and B6H12.2 with CD47 could provide novel perspectives for the development of inhibitors targeting CD47-SIRPα.

MDBuilder: a PyMOL plugin for the preparation of molecular dynamics simulations.

The preprocessed initial files that feed the molecular dynamics (MD) simulation packages dramatically influence the outcome of the simulations. However, the popular MD simulation packages depend, to a great extent, on the user's experience in the preparation of MD simulation systems. In this work, we present an easy-to-use tool called MDBuilder, a PyMOL plugin that assists researchers in building the starting structures for multiple popular MD simulation packages. MDBuilder is not only designed to assist MD beginners to overcome the steep learning curve by providing a menu-oriented, point-and-click user graphic interface (GUI), but also to provide an alternative way to prepare the input files for some highly scalable CHARMM force field-based MD simulation packages. The platform-independent GUI is implemented as a PyMOL plugin using the Python language, and it has been tested on Windows and Linux platforms. The source code and documentation of MDBuilder can be downloaded freely from https://github.com/HuiLiuCode/MDBuilder under the GNU General Public License.

Mapping the Binding Hot Spots on Human Programmed Cell Death 1 and Its Ligand with Free-Energy Simulations.

The immune checkpoint pathway of human programmed cell death 1 (hPD-1) and human programmed cell death ligand 1 (hPD-L1) is a promising target for cancer treatment. The blockade of the interplay between hPD-1 and hPD-L1 has recently shown good therapeutic efficacy. Although crystallographic studies have provided static conformational snapshots of the interface between hPD-1 and hPD-L1, the hot spot residues on both proteins that play key roles in the association process still remain elusive. To this end, we performed a series of alchemical free-energy simulations to analyze the energetic contributions of the interfacial residues on both hPD-1 and hPD-L1 and investigated the distributional patterns of the residues that significantly contribute to the binding. The results suggest that the hot spots on hPD-1 comprise Tyr68, Gln75, Ile126, Leu128, Ile134, and Glu136, and the hot spots on hPD-L1 comprise LAsp26 (the L symbol refers to hPD-L1), LIle54, LTyr56, LMet115, LAsp122, LTyr123, and LLys124. Moreover, we found that the distribution of these hot spot residues is highly uneven with respect to either the energetic contribution or the side-chain polarity, with energetically important residues clustered within densely packed hydrophobic regions. The mechanism ruling the interaction of the two binding partners is also discussed in detail from the perspective of the O-ring theory. Our work provides clues for the future development of anticancer inhibitors targeting the hPD-1/hPD-L1 immune checkpoint pathway.

Modeling of the static batch desorption and dynamic column elution of geniposidic acid from a porous anion-exchanger.

For several decades, plenty of iridoid glycosides including geniposide (GS) and geniposidic acid (GSA) in the gardenia yellow pigment extraction waste water was not recovered effectively. This study is aimed to supply an efficient GSA recycling route. In this study, a model incorporating a superficial desorption rate constant was applied to the batch GSA desorption process, i.e., recycling, for verification. Then, the model was further developed to research the feasibility in dynamic column elutions simulation through porous uniform media. The simulation approach was done by coupling velocity field and mass transfer equations using COMSOL Multiphysics™ Finite element method, with appropriate mesh refinement was employed to solve the equation system. The HCl solutions ranging from 0.03 mol/L to 0.06 mol/L were used to desorb/elute the GSA from a presaturated polymeric porous anionic resin D08. Good results were accomplished in terms of ion exchange desorption rate and GSA recovery. The pore diffusion model (PDM) considering counter ion was established to describe the desorption/elution kinetics in the batch/column experiment. By the least square fitting method, the superficial desorption rate constant Kd of GSA/HCl reaction on the ion-exchange sites of porous resin was fitted to 0.116 L/(mol s). Subsequently, this value was sequentially applied in the simulation of the dynamic elution process. The individual pore diffusion coefficients for GSA and Cl- were estimated to be 5.07 × 10-10 and 1.77 × 10-9 m2/s, respectively. In order to validate the simulation feasibility of this pore diffusion model to a dynamic column elution process, the effects of HCl concentration, flow rate and column's height/diameter ratio on the column performance were investigated systematically. The results from this work should serve as motivation for further experimental and theoretical study in the scaling-up of GSA purification process. Finally, repeated adsorption-elution column cycles were simulated by the PDM model well.

Computational simulations of the mass transfer zone in GS adsorption column packed with Fe3+ type ion exchanger.

In this work, a mesoporous Fe3+ type ion exchanger (DOW-3) was used to adsorb the geniposide (GS) involved in gardenia yellow pigment waste water. To test its viability, the mass transfer zone (MTZ) in dynamic adsorption column that plays an important role in the future scale-up of the GS adsorption process was studied systematically. Simulation of the MTZ behavior under various operation conditions in a packed adsorption column enabled the predictions of breakthrough curves of GS for the mesoporous adsorbent. Several modes of mass transport and processes taking place simultaneously, such as permeation, convection, dispersion and adsorption equilibrium were incorporated in the proposed model. Modeling process firstly affected the behavior of MTZ when it formed and moved throughout the column, and then had a much greater impact on the shape of breakthrough curve in the simulation. The dynamics of MTZ were studied under different adsorption conditions such as various column heights, flow rates and inlet concentrations. Moreover, the post-processing technique and 3D visualization of MTZ simulation results were capable of demonstrating the expanding and progressing of the MTZ related to the adsorption isotherm. The basic findings of MTZ dynamics will be very useful for process scale up. Additionally, the effective molecule docking results obtained from this study, help us fully understand the contribution of various molecular forces in the adsorption mechanism.

Four residues of propeptide are essential for precursor folding of nattokinase.

Subtilisin propeptide functions as an intramolecular chaperone that guides precursor folding. Nattokinase, a member of subtilisin family, is synthesized as a precursor consisting of a signal peptide, a propeptide, and a subtilisin domain, and the mechanism of its folding remains to be understood. In this study, the essential residues of nattokinase propeptide which contribute to precursor folding were determined. Deletion analysis showed that the conserved regions in propeptide were important for precursor folding. Single-site and multi-site mutagenesis studies confirmed the role of Tyr10, Gly13, Gly34, and Gly35. During stage (i) and (ii) of precursor folding, Tyr10 and Gly13 would form the part of interface with subtilisin domain. While Gly34 and Gly35 connected with an α-helix that would stabilize the structure of propeptide. The quadruple Ala mutation, Y10A/G13A/G34A/G35A, resulted in a loss of the chaperone function for the propeptide. This work showed the essential residues of propeptide for precursor folding via secondary structure and kinetic parameter analyses.

Insights into the modulation of optimum pH by a single histidine residue in arginine deiminase from Pseudomonas aeruginosa.

Arginine deiminase (ADI) is a potential antitumor agent for the arginine deprivation treatment of L-arginine auxotrophic tumors. The optimum pH of ADI varies significantly, yet little is known about the origin of this variety. Here, Pseudomonas aeruginosa ADI (PaADI), an enzyme that functions only at acidic pH, was utilized as the model system. The results of UV-pH titration imply that the nucleophilic Cys406 thiol group is protonated in the resting state. The H405R single mutation resulted in an altered pH optimum (from pH 5.5 to 6.5), an increased k(cat) (from 9.8 s(-1) to 101.7 s(-1) at pH 6.5), and a shifted pH rate dependence (ascending limb pK(a) from 3.6 to 4.4). Other mutants were constructed to investigate the effects of hydrogen bonding, charge distribution, and hydrophobicity on the properties of the enzyme. The pH optima of His405 mutants were all shifted to a relatively neutral pH except for the H405E mutant. The results of kinetic characterizations and molecular dynamic simulations revealed that the active site hydrogen bonding network involving Asp280 and His405 plays an important role in controlling the dependence of PaADI activity on pH. Moreover, the H405R variant showed increased cytotoxicity towards arginine auxotrophic cancer cell lines.

NF-κB, JNK and p53 pathways are involved in tubeimoside-1-induced apoptosis in HepG2 cells with oxidative stress and G2/M cell cycle arrest.

Tubeimoside-1 is a triterpenoid saponin extracted from the traditional Chinese herb Bolbostemma paniculatum (Maxim.) Franquet (Cucurbitaceae). We investigated the cytotoxic effect and apoptosis mechanism of tubeimoside-1. Tubeimoside-1 was cytotoxic in seven human cancer cell lines, with HepG2 the most sensitive. Tubeimoside-1 induced apoptosis of HepG2 cells dose and time dependently. Both the extrinsic and intrinsic pathways were triggered by tubeimoside-1. Caspase-3, -8 and -9 were activated and the expression of Fas, Fas ligand, Bcl-2, Bak and Bax was regulated. Moreover, tubeimoside-1 induced accumulation of reactive oxygen species and arrested cell cycle at the G(2)/M phase, thus contributing to apoptosis, through signaling regulation by tumor necrosis factor α, nuclear factor κB (NF-κB), Jun N-terminal kinase (JNK) and p53. We provide further insight into the tubeimoside-1 cytotoxic effect for antitumor chemotherapeutic treatment.

Binding modes of flavones to human serum albumin: insights from experimental and computational studies.

Pharmaceutical interactions with human serum albumin (HSA) are of great interest, because HSA is a pharmacokinetic determinant and a good model for exploring the protein-ligand interactions. Due to their hydrophobic nature, naturally occurring flavones, which possess various pharmacological activities, bind to HSA in human plasma. Here, we have identified the binding modes of two representative flavones--baicalin (BLI) and its aglycon, baicalein (BLE)--to HSA using a combination of experimental and computational approaches. The association properties were measured by applying spectroscopic methods, and a higher affinity was found for BLE. As evidenced by displacement and chemical unfolding assays, both ligands bind at Sudlow site I. Furthermore, molecular docking was utilized to characterize the models of HSA-flavone complexes, and molecular dynamics (MD) simulations as well as free energy calculations were undertaken to examine the energy contributions and the roles of various amino acid residues of HSA in flavones binding; the mechanism whereby glycosylation affects the association was also discussed. The present work provides reasonable binding models for both flavones to HSA.