Ritonavir and xk263 Binding-Unbinding with HIV-1 Protease: Pathways, Energy and Comparison

Understanding non-covalent biomolecular recognition, which includes drug–protein bound states and their binding/unbinding processes, is of fundamental importance in chemistry, biology, and medicine. Fully revealing the factors that govern the binding/unbinding processes can further assist in designing drugs with desired binding kinetics. HIV protease (HIVp) plays an integral role in the HIV life cycle, so it is a prime target for drug therapy. HIVp has flexible flaps, and the binding pocket can be accessible by a ligand via various pathways. Comparing ligand association and dissociation pathways can help elucidate the ligand–protein interactions such as key residues directly involved in the interaction or specific protein conformations that determine the binding of a ligand under certain pathway(s). Here, we investigated the ligand unbinding process for a slow binder, ritonavir, and a fast binder, xk263, by using unbiased all-atom accelerated molecular dynamics (aMD) simulation with a re-seeding approach and an explicit solvent model. Using ritonavir-HIVp and xk263-HIVp ligand–protein systems as cases, we sampled multiple unbinding pathways for each ligand and observed that the two ligands preferred the same unbinding route. However, ritonavir required a greater HIVp motion to dissociate as compared with xk263, which can leave the binding pocket with little conformational change of HIVp. We also observed that ritonavir unbinding pathways involved residues which are associated with drug resistance and are distal from catalytic site. Analyzing HIVp conformations sampled during both ligand–protein binding and unbinding processes revealed significantly more overlapping HIVp conformations for ritonavir-HIVp rather than xk263-HIVp. However, many HIVp conformations are unique in xk263-HIVp unbinding processes. The findings are consistent with previous findings that xk263 prefers an induced-fit model for binding and unbinding, whereas ritonavir favors a conformation selection model. This study deepens our understanding of the dynamic process of ligand unbinding and provides insights into ligand–protein recognition mechanisms and drug discovery.

Explanation of Structure and Function of kv1.3 Potent Blocker From Mesobuthus eupeus Venom Gland: A New Promise in Drug Development

Background: Scorpions and other venomous animals are sought with great concern because venom is a source of novel peptides with exciting features. Some toxins of scorpion venom are effectors of potassium channels. Previous studies strongly support the importance of potassium channel toxins for use as pharmacological tools or potential drugs. Objectives: Here, a three-dimensional (3-D) structure and function of a potent acidic blocker of the human voltage-gated potassium ion channel, Kv1.3, previously identified in the scorpion Mesobuthus eupeus venom gland, were interpreted. Methods: The 3-D structure of meuK2-2 was generated using homology modeling. The interaction of meuK2-2 with the Kv1.3 channel was evaluated using a computational protocol employing peptide-protein docking experiments, pose clustering, and 100 ns molecular dynamic simulations to make the 3-D models of the meuK2-2/Kv1.3 complex trustworthy. Results: A CSα/β (cysteine-stabilized α-helical and β-sheet) fold was found for the 3-D structure of meuK2-2. In a different mechanism from what was identified so far, meuK2-2 binds to both turret and pore loop of Kv1.3 through two key residues (Ala28 and Ser11) and H-bonds. The binding of meuK2-2 induces some conformational changes to Kv1.3. Eventually, the side chain of a positively charged amino acid (His9) occupies the channel's pore. All together blocks the ion permeation pathway. Conclusions: MeuK2-2 could block Kv1.3 by a new mechanism. So, it could be a unique target for further investigations to develop a pharmacological tool and potential drug.

A New L-Proline Amide Hydrolase with Potential Application within the Amidase Process

L-proline amide hydrolase (PAH, EC 3.5.1.101) is a barely described enzyme belonging to the peptidase S33 family, and is highly similar to prolyl aminopeptidases (PAP, EC. 3.4.11.5). Besides being an S-stereoselective character towards piperidine-based carboxamides, this enzyme also hydrolyses different L-amino acid amides, turning it into a potential biocatalyst within the Amidase Process. In this work, we report the characterization of L-proline amide hydrolase from Pseudomonas syringae (PsyPAH) together with the first X-ray structure for this class of L-amino acid amidases. Recombinant PsyPAH showed optimal conditions at pH 7.0 and 35 °C, with an apparent thermal melting temperature of 46 °C. The enzyme behaved as a monomer at the optimal pH. The L-enantioselective hydrolytic activity towards different canonical and non-canonical amino-acid amides was confirmed. Structural analysis suggests key residues in the enzymatic activity.

Database of glutamate gated chloride (GluCl) subunits across 125 nematode species: patterns of gene accretion and sequence diversification

Glutamate Gated Chloride (GluCl) channels belong to the Cys-loop receptor superfamily. GluCl channels are activated by glutamate (Glu) and form substrates for the anti-parasitic drugs from the avermectin family. GluCl channels are pentameric, and each subunit contains an N-terminal extracellular domain that binds Glu and four helical transmembrane domains (TMs), which contain binding sites for avermectin drugs. In order to provide more insight into phylum-wide patterns of GluCl subunit gene expansion and sequence diversity across nematodes, we have developed a database of predicted GluCl subunit genes from 125 nematode species. Our analysis into this dataset described assorted patterns of species-specific GluCl gene counts across different nematodes as well as sequence diversity in key residues thought to be involved in avermectin binding.

Molecular Dissection of the Primase and Polymerase Activities of Deep-Sea Phage NrS-1 Primase-Polymerase

PrimPols are a class of primases that belong to the archaeo-eukaryotic primase (AEP) superfamily but have both primase and DNA polymerase activities. Replicative polymerase from NrS-1 phage (NrSPol) is a representative of the PrimPols. In this study, we identified key residues for the catalytic activity of NrSPol and found that a loop in NrSPol functionally replaces the zinc finger motif that is commonly found in other AEP family proteins. A helix bundle domain (HBD), conserved in the AEP superfamily, was recently reported to bind to the primase recognition site and to be crucial for initiation of primer synthesis. We found that NrSPol can recognize different primase recognition sites, and that the initiation site for primer synthesis is not stringent, suggesting that the HBD conformation is flexible. More importantly, we found that although the HBD-inactivating mutation impairs the primase activity of NrSPol, it significantly enhances the DNA polymerase activity, indicating that the HBD hinders the DNA polymerase activity. The conflict between the primase activity and the DNA polymerase activity in a single protein with the same catalytic domain may be one reason for why DNA polymerases are generally unable to synthesize DNA de novo.

From Crystal Structures of RgIA4 in Complex with Ac-AChBP to Molecular Determinants of Its High Potency of α9α10 nAChR

α9-containing nicotinic acetylcholine receptors (nAChRs) have been shown to play critical roles in neuropathic pain. The α-conotoxin (α-CTx) RgIA and its analog RgIA4 were identified as the most selective inhibitor of α9α10 nAChR. However, the mechanism of their selectivity toward α9α10 nAChR remains elusive. Here, we reported the co-crystal structure of RgIA and RgIA4 in complex with Aplysia californica acetylcholine binding protein (Ac-AChBP) at resolution of 2.6 Å, respectively. Based on the structure of the complexes, together with molecular dynamic simulation (MD-simulation), we suggested the key residues of α9α10 nAChR in determining its high affinity for RgIA/RgIA4. This is the first time the complex between pain-related conotoxins and Ac-AChBP was reported and the complementary side of α9 subunit in binding of the antagonists shown. These results provide realistic template for the design of new therapeutic in neuropathic pain.

Symmetric activation and modulation of the human calcium-sensing receptor

The human extracellular calcium-sensing (CaS) receptor controls plasma Ca2+ levels and contributes to nutrient-dependent maintenance and metabolism of diverse organs. Allosteric modulation of the CaS receptor corrects disorders of calcium homeostasis. Here, we report the cryogenic-electron microscopy reconstructions of a near–full-length CaS receptor in the absence and presence of allosteric modulators. Activation of the homodimeric CaS receptor requires a break in the transmembrane 6 (TM6) helix of each subunit, which facilitates the formation of a TM6-mediated homodimer interface and expansion of homodimer interactions. This transformation in TM6 occurs without a positive allosteric modulator. Two modulators with opposite functional roles bind to overlapping sites within the transmembrane domain through common interactions, acting to stabilize distinct rotamer conformations of key residues on the TM6 helix. The positive modulator reinforces TM6 distortion and maximizes subunit contact to enhance receptor activity, while the negative modulator strengthens an intact TM6 to dampen receptor function. In both active and inactive states, the receptor displays symmetrical transmembrane conformations that are consistent with its homodimeric assembly.

Dissociation pathways of the p53 DNA binding domain from DNA and critical roles of key residues elucidated by dPaCS-MD/MSM

The dissociation process of the DNA binding domain of p53 (p53-DBD) from a DNA duplex that contains the consensus sequence, which is the specific target of p53-DBD, was investigated by a combination of dissociation parallel cascade selection molecular dynamics (dPaCS-MD) and the Markov state model (MSM). Based on an all-atom model including explicit solvent, we first simulated the p53-DBD dissociation processes by 75 trials of dPaCS-MD, which required an average simulation time of 11.2 ± 2.2 ns per trial. By setting the axis of the DNA duplex as the Z-axis and the binding side of p53-DBD on DNA as the + side of the X-axis, we found that dissociations took place along the +X and −Y directions (−Y directions) in 93% of the cases, while 7% of the cases moved along +X and +Y directions (+Y directions). Toward the −Y directions, p53-DBD dissociated first from the major groove and then detached from the minor groove, while unbinding from the minor groove occurred first in dissociations along the +Y directions. Analysis of the free energy landscape by MSM showed that loss of the minor groove interaction with p53-DBD toward the +Y directions incurred a relatively high energy cost (1.1 kcal/mol) upon a critical transition, whereas major groove detachment more frequently occurred with lower free energy costs. The standard binding free energy calculated from the free energy landscape was −10.9 ± 0.4 kcal/mol, which agrees with an experimental value of –11.1 kcal/mol. These results indicate that the dPaCS-MD/MSM combination can be a powerful tool to investigate dissociation mechanisms of two large molecules. Minor groove binding is mainly stabilized by R248, identified as the most important residue that tightly binds deep inside the minor groove. Analysis of the p53 key residues for DNA binding indicates high correlations with cancer-related mutations, confirming that impairment of the interactions between p53-DBD and DNA can be frequently related to cancer.