A mutational analysis and molecular dynamics simulation of quinolone resistance proteins QnrA1 and QnrC from Proteus mirabilis

<p>Abstract</p> <p>Background</p> <p>The first report on the transferable, plasmid-mediated quinolone-resistance determinant <it>qnrA1 </it>was in 1998. Since then, <it>qnr </it>alleles have been discovered worldwide in clinical strains of Gram-negative bacilli. Qnr proteins confer quinolone resistance, and belong to the pentapeptide repeat protein (PRP) family. Several PRP crystal structures have been solved, but little is known about the functional significance of their structural arrangement.</p> <p>Results</p> <p>We conducted random and site-directed mutagenesis on <it>qnrA1 </it>and on <it>qnrC</it>, a newly identified quinolone-resistance gene from <it>Proteus mirabilis</it>. Many of the Qnr mutants lost their quinolone resistance function. The highly conserved hydrophobic Leu or Phe residues at the center of the pentapeptide repeats are known as <it>i </it>sites, and loss-of-function mutations included replacement of the <it>i </it>site hydrophobic residues with charged residues, replacing the <it>i</it>^-2site, N-terminal to the <it>i </it>residues, with bulky side-chain residues, introducing Pro into the β-helix coil, deletion of the N- and C-termini, and excision of a central coil. Molecular dynamics simulations and homology modeling demonstrated that QnrC overall adopts a stable β-helix fold and shares more similarities with MfpA than with other PRP structures. Based on homology modeling and molecular dynamics simulation, the dysfunctional point mutations introduced structural deformations into the quadrilateral β-helix structure of PRPs. Of the pentapeptides of QnrC, two-thirds adopted a type II β-turn, while the rest adopted type IV turns. A gap exists between coil 2 and coil 3 in the QnrC model structure, introducing a structural flexibility that is similar to that seen in MfpA.</p> <p>Conclusion</p> <p>The hydrophobic core and the β-helix backbone conformation are important for maintaining the quinolone resistance property of Qnr proteins. QnrC may share structural similarity with MfpA.</p

Detailed Regulatory Mechanism of the Interaction between ZO-1 PDZ2 and Connexin43 Revealed by MD Simulations

The gap junction protein connexin43 (Cx43) binds to the second PDZ domain of Zonula occludens-1 (ZO-1) through its C-terminal tail, mediating the regulation of gap junction plaque size and dynamics. Biochemical study demonstrated that the very C-terminal 12 residues of Cx43 are necessary and sufficient for ZO-1 PDZ2 binding and phosphorylation at residues Ser (-9) and Ser (-10) of the peptide can disrupt the association. However, only a crystal structure of ZO-1 PDZ2 in complex with a shorter 9 aa peptide of connexin43 was solved experimentally. Here, the interactions between ZO-1 PDZ2 and the short, long and phosphorylated Cx43 peptides were studied using molecular dynamics (MD) simulations and free energy calculation. The short peptide bound to PDZ2 exhibits large structural variations, while the extension of three upstream residues stabilizes the peptide conformation and enhanced the interaction. Phosphorylation at Ser(-9) significantly weakens the binding and results in conformational flexibility of the peptide. Glu210 of ZO-1 PDZ2 was found to be a key regulatory point in Cx43 binding and phosphorylation induced dissociation

The Conformational Transition Pathways of ATP-Binding Cassette Transporter BtuCD Revealed by Targeted Molecular Dynamics Simulation

BtuCD is a member of the ATP-binding cassette transporters in Escherichia coli that imports vitamin B12 into the cell by utilizing the energy of ATP hydrolysis. Crystal structures of BtuCD and its homologous protein HI1470/1 in various conformational states support the ‘‘alternating access’ ’ mechanism which proposes the conformational transitions of the substrate translocation pathway at transmembrane domain (TMD) between the outward-facing and inward-facing states. The conformational transition at TMD is assumed to couple with the movement of the cytoplasmic nucleotide-binding domains (NBDs) driven by ATP hydrolysis/binding. In this study, we performed targeted molecular dynamics (MD) simulations to explore the atomic details of the conformational transitions of BtuCD importer. The outward-facing to inward-facing (ORI) transition was found to be initiated by the conformational movement of NBDs. The subsequent reorientation of the substrate translocation pathway at TMD began with the closing of the periplasmic gate, followed by the opening of the cytoplamic gate in the last stage of the conformational transition due to the extensive hydrophobic interactions at this region, consistent with the functional requirement of unidirectional transport of the substrates. The reverse inward-facing to outward-facing (IRO) transition was found to exhibit intrinsic diversity of the conformational transition pathways and significant structural asymmetry, suggesting that the asymmetric crystal structure of BtuCD-F is a

Range Gate Pull-Off Mainlobe Jamming Suppression Approach with FDA-MIMO Radar: Theoretical Formalism and Numerical Study

With the development of an electronic interference technique, the self-defense jammer can generate mainlobe jamming using the range gate pull-off (RGPO) strategy, which brings serious performance degradation of target tracking for the ground-based warning radar. In this paper, a RGPO mainlobe jamming suppression approach is proposed, with a frequency diverse array using multiple-input multiple-output (FDA-MIMO) radar. The RGPO mainlobe jamming differs from the true target in slant range, thus it is possible to identify the true target from the RGPO mainlobe jammings by exploiting the transmit beampattern diversity of FDA-MIMO radar. A RGPO mainlobe jamming suppression approach is devised by using joint transmit–receive beamforming for a group of range sectors. The jamming suppression performance is studied, in consideration of practical time-delay of RGPO jamming. Simulation examples are provided to verify the effectiveness of the proposed approach

ATP Hydrolysis Induced Conformational Changes in the Vitamin B₁₂ Transporter BtuCD Revealed by MD Simulations

<div><p>ATP binding cassette (ABC) transporters utilize the energy of ATP hydrolysis to uni-directionally transport substrates across cell membrane. ATP hydrolysis occurs at the nucleotide-binding domain (NBD) dimer interface of ABC transporters, whereas substrate translocation takes place at the translocation pathway between the transmembrane domains (TMDs), which is more than 30 angstroms away from the NBD dimer interface. This raises the question of how the hydrolysis energy released at NBDs is “transmitted” to trigger the conformational changes at TMDs. Using molecular dynamics (MD) simulations, we studied the post-hydrolysis state of the vitamin B₁₂ importer BtuCD. Totally 3-μs MD trajectories demonstrate a predominantly asymmetric arrangement of the NBD dimer interface, with the ADP-bound site disrupted and the ATP-bound site preserved in most of the trajectories. TMDs response to ATP hydrolysis by separation of the L-loops and opening of the cytoplasmic gate II, indicating that hydrolysis of one ATP could facilitate substrate translocation by opening the cytoplasmic end of translocation pathway. It was also found that motions of the L-loops and the cytoplasmic gate II are coupled with each other through a contiguous interaction network involving a conserved Asn83 on the extended stretch preceding TM3 helix plus the cytoplasmic end of TM2/6/7 helix bundle. These findings entail a TMD-NBD communication mechanism for type II ABC importers.</p></div

Opioid-free anesthesia with ultrasound-guided quadratus lumborum block in the supine position for lower abdominal or pelvic surgery: a randomized controlled trial

Abstract In the past, quadratus lumborum block (QLB) was mostly used for postoperative analgesia in patients, and few anesthesiologists applied it during surgery with opioid-free anesthesia (OFA). Consequently, it is still unclear whether QLB in the supine position can provide perfect analgesia and inhibit anesthetic stress during surgery under the OFA strategy. To observe the clinical efficacy of ultrasound-guided quadratus lumborum block (US-QLB) in the supine position with OFA for lower abdominal and pelvic surgery. A total of 122 patients who underwent lower abdominal or pelvic surgery in People’s Hospital of Wanning between March 2021 and July 2022 were selected and divided into a quadratus lumborum block group (Q) (n = 62) and control group (C) (n = 60) according to the random number table method. Both groups underwent general anesthesia combined with QLB in the supine position. After sedation, unilateral or bilateral QLB was performed via the ultrasound guided anterior approach based on images resembling a “human eye” and “baby in a cradle” under local anesthesia according to the needs of the operative field. In group Q, 20 ml of 0.50% lidocaine and 0.20% ropivacaine diluted in normal saline (NS) were injected into each side. In group C, 20 ml of NS was injected into each side. The values of BP, HR, SPO2, SE, RE, SPI, NRS, Steward score, dosage of propofol, dexmedetomidine, and rocuronium, the number of patients who needed remifentanil, propofol, or diltiazem, puncture point, block plane, duration of anesthesia, catheter extraction, and wakefulness during the operation were monitored. There were no significant differences in the general data, number of cases requiring additional remifentanil, propofol, or diltiazem treatment, as well as puncture point and puncture plane between the two groups (P > 0.05). HR, SBP, and DBP values were higher in group Q than in group C at T1; HR, SPI, and SE, while RE values were lower in group Q than in group C at T3, SE, and RE; the Steward score was higher in group Q than in group C at T4 and T5, and the difference was statistically significant (P < 0.05). The extubation and awake times were lower in group Q than in group C, and the difference was statistically significant (P < 0.05). The SE, RE, and SPI values were lower at T1, T2, T3, and T4 than at T0. The Steward scores at T4 and T5 were higher in group Q than in group C, and were lower than at T0, with a statistically significant difference (P < 0.05). There were significant differences in the effectiveness of postoperative analgesia between the two groups at t1, t3 and t4 (P < 0.05). US-QLB in the supine position with OFA is effective in patients undergoing lower abdominal or pelvic surgery with stable intraoperative vital signs, complete recovery and better postoperative analgesia

Creating Conformational Entropy by Increasing Interdomain Mobility in Ligand Binding Regulation: A Revisit to N-Terminal Tandem PDZ Domains of PSD-95

The two N-terminal PDZ domains of postsynaptic density protein-95 (PDS-95 PDZ1 and PDZ2) are closely connected in tandem by a conserved peptide linker of five amino acids. The interdomain orientation between PDZ1 and PDZ2 of the ligand-free PDZ12 tandem is restrained, and this conformational arrangement facilitates the synergistic binding of PDZ1 2 to multimeric targets.' The interdomain orientation of the target-bound state of PDZ12 is not known. Here, we have solved the structure of PDZ1 2 in complex with its binding domain from cypin. Both chemical shift data and residual dipolar coupling measurements showed that the restrained interdomain orientation disappeared upon cypin peptide binding. NMR-based relaxation experiments revealed slow interdomain motions in the PDZ12/cypin peptide complex. Molecular dynamics simulations also showed that the PDZ12/cypin complex has larger conformational flexibility than the ligand-free PDZ12. This dramatic change of protein dynamics provides extra conformational entropy upon ligand binding, thus enhancing the ligand binding affinity of the PDZ1 2 tandem. Modulation of ligand binding affinity through concerted interdomain structural and dynamic rearrangements may represent a general property of multidomain scaffold proteins

Conformational Dynamics and Protein–Substrate Interaction of ABC Transporter BtuCD at the Occluded State Revealed by Molecular Dynamics Simulations

ATP-binding cassette (ABC) transporters are ubiquitous in all three kingdoms of life and are implicated in many clinically relevant physiological processes. They couple the energy released by ATP hydrolysis to facilitate substrate translocation across cell membranes. The crystal structures of type II ABC importers have revealed their unique transmembrane domain architecture consisting of 10 transmembrane helices and their structurally conserved nucleotide-binding domains among all ABC transporters. However, molecular details of the interactions between the importers and their substrate remain largely elusive. Taking vitamin B₁₂ importer BtuCD as an exemplar of type II importers, we investigated the dynamics of its occluded state and the detailed protein–substrate interactions using molecular dynamics simulation. Our trajectories show that the importer accommodates the substrate through a nonspecific binding mode as the substrate undergoes evident vertical and tilt motions inside the translocation cavity. Extensive hydrogen bond and hydrophobic interactions were observed between the substrate and the importer; however, most of these interactions are weak, with <38% occurrence. The presence of substrate leads to enlargement of the translocation cavity, especially at its cytoplasmic end, which may activate cytoplasmic regions and probably facilitate the transportation. The perturbations caused by periplasmic binding protein and nucleotides were also investigated. The study provides deeper insight into the translocation mechanism of BtuCD

Conformational changes at the cytoplasmic side of the translocation pathway.

<p>(a) Close-up bottom view from the cytoplasmic side of the translocation pore. Hydrophobic residues are shown with stick model. (b) Evolution of d_pairs of residues at the cytoplasmic side along the simulation trajectory of O→I transition. (c) Cartoon representation of the hydrophobic network at the cytoplasmic gate in outBtuCD and inBtuCD. (d) Evolution of minimal distances between residues L90-V150 and L85-L146 during the O→I transition.</p