Ferredoxin-dependent glutamate synthase (Fd-GltS) is reported as an enzymatic bioelectrocatalyst for the first time. By configuring mediated electrochemical interfaces with mediators of different redox potentials, we realize bioelectrosynthesis or bioelectrooxidation of glutamate with recombinant Fd-GltS from cyanobacteria. Particularly, bioelectrocatalytic oxidation of glutamate by Fd-GltS is demonstrated to be oxygen independent. This study reinforces a new catalytic option for developing enzymatic bioelectronic devices for powering, sensing or synthesis.
Aminoacyl-tRNA synthetases (aaRSs), a family of ubiquitous and essential enzymes, can bind target tRNAs and catalyze the aminoacylation reaction in genetic code translation. In this work, we explore the dynamic properties and allosteric communication of human mitochondrial phenylalanyl-tRNA synthetase (hmPheRS) in free and bound states to understand the mechanisms of its tRNA Phe recognition and allostery using molecular dynamics simulations combined with the torsional mutual information-based network model. Our results reveal that hmPheRS's residue mobility and inter-residue motional coupling are significantly enhanced by tRNA Phe binding, and there occurs a strong allosteric communication which is critical for the aminoacylation reaction, suggesting the vital role of tRNA Phe binding in the enzyme's function. The identified signaling pathways mainly make the connections between the anticodon binding domain (ABD) and catalytic domain (CAD), as well as within the CAD composed of many functional fragments and active sites, revealing the co-regulation role of them to act coordinately and achieve hmPheRS's aminoacylation function. Besides, several key residues along the communication pathways are identified to be involved in mediating the coordinated coupling between anticodon recognition at the ABD and activation process at the CAD, showing their pivotal role in the allosteric network, which are well consistent with the experimental observation. This study sheds light on the allosteric communication mechanism in hmPheRS and can provide important information for the structure-based drug design targeting aaRSs.
Dynamical properties of proteins play an essential role in their function exertion. The elastic network model (ENM) is an effective and efficient tool in characterizing the intrinsic dynamical properties encoded in biomacromolecule structures. The Gaussian network model (GNM) and anisotropic network model (ANM) are the two often-used ENM models. Here, we introduce an equally weighted multiscale ENM (equally weighted mENM) based on the original mENM (denoted as mENM), in which fitting weights of Kirchhoff/Hessian matrixes in mENM are removed since they neglect the details of pairwise interactions. Then, we perform its comparison with the mENM, traditional ENM, and parameter-free ENM (pfENM) in reproducing dynamical properties for the six representative proteins whose molecular dynamics (MD) trajectories are available in http://mmb.pcb.ub.es/MoDEL/. In the results, for B-factor prediction, mENM performs best, while the equally weighted mENM performs also well, better than the traditional ENM and pfENM models. As to the dynamical cross-correlation map calculation, mENM performs worst, while the results produced from the equally weighted mENM and pfENM models are close to those from MD trajectories with the latter a little better than the former. Furthermore, encouragingly, the equally weighted mANM displays the best performance in capturing the functional motional modes, followed by pfANM and traditional ANM models, while the mANM fails in all the cases. This work is helpful for strengthening the understanding of the elastic network model and provides a valuable guide for researchers to utilize the model to explore protein dynamics.
The binding interactions of small nuclear RNAs (snRNA) and the associated protein factors are critical to the function of spliceosomes in alternatively splicing primary RNA transcripts. Although molecular dynamics simulations are a powerful tool to interpret the mechanism of biological processes, the atomic-level simulations are, however, too expensive and with limited accuracy for the large-size systems, such as snRNA-protein complexes. We extend the coarse-grained Gaussian network model, which models the RNA-protein complexes as a harmonic chain of Ca, P, and O4 0 atoms, to investigating the impact of the snRNA-binding interaction on the dynamic stability of the human U1A protein, which is a major component of the spliceosomal U1 small nuclear ribonucleoprotein particle. The results reveal that the first and third loops and the C-terminal helix regions of the U1A domain undergo a significant loss of flexibility upon the RNA binding due to the forming of mostly electrostatic and hydrogen bond interactions with RNA 5 0 stem and loop. By examining the residues whose mutations significantly change the binding free energy between U1A and snRNA, the Gaussian network model-based calculations show that not only the residues at the binding sites that are traditionally considered to play a major role in U1A-RNA association but also those residues that are far away from the RNA-binding interface can participate in the long-range allosteric signal transmission; these calculations are quantitatively consistent with the data observed in the recent snRNA binding experiments. The study demonstrates a useful avenue to utilize the simplified elastic network model to investigate the dynamics characteristics of the biologically important macromolecular interactions.
Human multidrug resistance protein 1 (hMRP1) is an important member of the ATPbinding cassette (ABC) transporter superfamily. It can extrude a variety of anticancer drugs and physiological organic anions across the plasma membrane, which is activated by substrate binding, and is accompanied by large-scale cooperative movements between different domains. Currently, it remains unclear completely about how the specific interactions between hMRP1 and its substrate are and which critical residues are responsible for allosteric signal transduction. To the end, we first construct an inward-facing state of hMRP1 using homology modeling method, and then dock substrate proinflammatory agent leukotriene C4 (LTC4) to hMRP1 pocket. The result manifests LTC4 interacts with two parts of hMRP1 pocket, namely the positively charged pocket (P pocket) and hydrophobic pocket (H pocket), similar to its binding mode with bMRP1 (bovine MRP1). Additionally, we use the Gaussian network model (GNM)-based thermodynamic method proposed by us to identify the key residues whose perturbations markedly alter their binding free energy. Here the conventional GNM is improved with covalent/non-covalent interactions and secondary structure information considered (denoted as sscGNM). In the result, sscGNM improves the flexibility prediction, especially for the nucleotide binding domains with rich kinds of secondary structures. The 46 key residue clusters located in different subdomains are identified which are highly consistent with experimental observations. Furtherly, we explore the long-range cooperation within the transporter. This study is helpful for strengthening the understanding of the work mechanism in ABC exporters and can provide important information to scientists in drug design studies.binding mode, Gaussian network model, hMRP1, key residues, thermodynamic cycle | INTRODUCTIONMultidrug resistance (MDR) to chemotherapy is a major obstacle in the treatment of cancer patients. Human multidrug resistance protein 1 (hMRP1), a member of the ATP-binding cassette (ABC) transporter superfamily, can export a wide variety of anticancer drugs such as vincristine, etoposide, anthracyclines, methotrexate, and physiological organic anions antioxidants GSH and proinflammatory agent leukotriene C4 (LTC4) across the cell membrane utilizing the energy from ATP binding and hydrolysis. 1 Studies have shown that the process of substrate transport is accompanied by large-scale cooperative motions between spatially separated subdomains. 2 Thus, there must exist a network of key residues mediating long-range signal transmission and allosteric coupling. 2,3 Identification of key functional residues is very important not only for an "in-depth" understanding of the mechanism of signal transmission but also for the structure-based drug design.
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