The origin of the FeIV=O intermediates in cytochrome aa3 oxidase

AbstractThe dioxygen reduction mechanism in cytochrome oxidases relies on proton control of the electron transfer events that drive the process. Proton delivery and proton channels in the protein that are relevant to substrate reduction and proton pumping are considered, and the current status of this area is summarized. We propose a mechanism in which the coupling of the oxygen reduction chemistry to proton translocation (P→F transition) is related to the properties of two groups of highly conserved residues, namely, His411/G386-T389 and the heme a3–propionateA–D399–H403 chain. This article is part of a Special Issue entitled: Respiratory Oxidases

Binding and Docking Interactions of NO, CO and O2 in Heme Proteins as Probed by Density Functional Theory

Dynamics and reactivity in heme proteins include direct and indirect interactions of the ligands/substrates like CO, NO and O2 with the environment. Direct electrostatic interactions result from amino acid side chains in the inner cavities and/or metal coordination in the active site, whereas indirect interactions result by ligands in the same coordination sphere. Interactions play a crucial role in stabilizing transition states in catalysis or altering ligation chemistry. We have probed, by Density Functional Theory (DFT), the perturbation degree in the stretching vibrational frequencies of CO, NO and O2 molecules in the presence of electrostatic interactions or hydrogen bonds, under conditions simulating the inner cavities. Moreover, we have studied the vibrational characteristics of the heme bound form of the CO and NO ligands by altering the chemistry of the proximal to the heme ligand. CO, NO and O2 molecules are highly polarizable exerting vibrational shifts up to 80, 200 and 120 cm−1, respectively, compared to the non-interacting ligand. The importance of Density Functional Theory (DFT) methodology in the investigation of the heme-ligand-protein interactions is also addressed

Corrigendum to "Recognition motifs for importin 4 [(L)PPRS(G/P)P] and importin 5 [KP(K/Y)LV] binding, identified by bio-informatic simulation and experimental in vitro validation" [Comput Struct Biotechnol J 20 (2022) 5952-5961]

Nuclear translocation of large proteins is mediated through karyopherins, carrier proteins recognizing specific motifs of cargo proteins, known as nuclear localization signals (NLS). However, only few NLS signals have been reported until now. In the present work, NLS signals for Importins 4 and 5 were identified through an unsupervised in silico approach, followed by experimental in vitro validation. The sequences LPPRS(G/P)P and KP(K/Y)LV were identified and are proposed as recognition motifs for Importins 4 and 5 binding, respectively. They are involved in the trafficking of important proteins into the nucleus. These sequences were validated in the breast cancer cell line T47D, which expresses both Importins 4 and 5. Elucidating the complex relationships of the nuclear transporters and their cargo proteins is very important in better understanding the mechanism of nuclear transport of proteins and laying the foundation for the development of novel therapeutics, targeting specific importins

p-cymene impairs SARS-CoV-2 and Influenza A (H1N1) viral replication: In silico predicted interaction with SARS-CoV-2 nucleocapsid protein and H1N1 nucleoprotein

Therapeutic regimens for the COVID-19 pandemics remain unmet. In this line, repurposing of existing drugs against known or predicted SARS-CoV-2 protein actions have been advanced, while natural products have also been tested. Here, we propose that p-cymene, a natural monoterpene, can act as a potential novel agent for the treatment of SARS-CoV-2-induced COVID-19 and other RNA-virus-induced diseases (influenza, rabies, Ebola). We show by extensive molecular simulations that SARS-CoV-2 C-terminal structured domain contains a nuclear localization signal (NLS), like SARS-CoV, on which p-cymene binds with low micromolar affinity, impairing nuclear translocation of this protein and inhibiting viral replication, as verified by preliminary in vitro experiments. A similar mechanism may occur in other RNA-viruses (influenza, rabies and Ebola), also verified in vitro for influenza, by interaction of p-cymene with viral nucleoproteins, and structural modification of their NLS site, weakening its interaction with importin A. This common mechanism of action renders therefore p-cymene as a possible antiviral, alone, or in combination with other agents, in a broad spectrum of RNA viruses, from SARS-CoV-2 to influenza A infections

Protein–protein interactions within photosystem II under photoprotection: the synergy between CP29 minor antenna, subunit S (PsbS) and zeaxanthin at all-atom resolution

The assembly and disassembly of protein complexes within cells are crucial life-sustaining processes. In photosystem II (PSII) of higher plants, there is a delicate yet obscure balance between light harvesting and photo-protection under fluctuating light conditions, that involves protein–protein complexes. Recent breakthroughs in molecular dynamics (MD) simulations are combined with new approaches herein to provide structural and energetic insight into such a complex between the CP29 minor antenna and the PSII subunit S (PsbS). The microscopic model involves extensive sampling of bound and dissociated states at atomic resolution in the presence of photo-protective zeaxanthin (Zea), and reveals well defined protein–protein cross-sections. The complex is placed within PSII, and macroscopic connections are emerging (PsbS–CP29–CP24–CP47) along the energy transfer pathways from the antenna to the PSII core. These connections explain macroscopic observations in the literature, while the previously obscured atomic scale details are now revealed. The implications of these findings are discussed in the context of the Non-Photochemical Quenching (NPQ) of chlorophyll fluorescence, the down-regulatory mechanism of photosynthesis, that enables the protection of PSII against excess excitation load. Zea is found at the PsbS–CP29 cross-section and a pH-dependent equilibrium between PsbS dimer/monomers and the PsbS–CP29 dissociation/association is identified as the target for engineering tolerant plants with increased crop and biomass yields. Finally, the new MD based approaches can be used to probe protein–protein interactions in general, and the PSII structure provided can initiate large scale molecular simulations of the photosynthetic apparatus, under NPQ conditions

Deciphering the QR Code of the CRISPR-Cas9 System: Synergy between Gln768 (Q) and Arg976 (R)

Markov state models (MSMs) and machine learning (ML) algorithms can extrapolate the long-time-scale behavior of large biomolecules from molecular dynamics (MD) trajectories. In this study, an MD-MSM-ML scheme has been applied to probe the large endonuclease (Cas9) in the bacterial adaptive immunity CRISPR-Cas9 system. CRISPR has become a programmable and state-of-the-art powerful genome editing tool that has already revolutionized life sciences. CRISPR-Cas9 is programmed to process specific DNA sequences in the genome. However, human/biomedical applications are compromised by off-target DNA damage. Characterization of Cas9 at the structural and biophysical levels is a prerequisite for the development of efficient and high-fidelity Cas9 variants. The Cas9 wild type and two variants (R63A-R66A-R70A, R69A-R71A-R74A-R78A) are studied herein. The configurational space of Cas9 is provided with a focus on the conformations of the side chains of two residues (Gln768 and Arg976). A model for the synergy between those two residues is proposed. The results are discussed within the context of experimental literature. The results and methodology can be exploited for the study of large biomolecules in general and for the engineering of more efficient and safer Cas9 variants for applications