Ying Li

Conformational sampling remains a major challenge in molecular dynamics (MD) simulations of proteins with large-scale functional motions. Here, we use apo Escherichia coli adenylate kinase (AdK) as a benchmark for the open–closed transition and compare nine sampling protocols under a uniform explicit-solvent budget of 300 ns per method. The methods include unbiased MD, metadynamics, targeted MD (TMD), accelerated MD, simulated tempering, interpolation-based MD, and BioEmu. Closed-state sampling was evaluated relative to a reference-closed basin defined from ligand-stripped PDB 1AKE. TMD was the only method that both reached and maintained the closed reference, whereas unbiased MD did not enter this basin within 300 ns. Metadynamics increased sampling along the domain-distance coordinate but also promoted excessive LID opening, while simulated tempering showed a similar tendency. BioEmu generated closed-like conformations, but these states were not maintained during subsequent short MD simulations. Overall, the results show that collective-variable coverage does not necessarily correspond to stable target state occupancy and that different sampling methods provide different types of conformational information. These findings highlight the importance of selecting sampling strategies according to the specific scientific objective.

Bacterial cytochrome P450s are cornerstone monooxygenases that enable selective oxidation across the fields of metabolism and biocatalysis. Yet, for the orphan Bacillus subtilis enzyme CYP134A1 (CypX), how ligand identity governs conformational readiness remains unclear despite its solved structure. Here we combine atomistic simulations of apo CypX and four ligand-bound complexes with principal-component analysis, two-dimensional free energy surfaces, and an operational three-state classification based on two gating coordinates: d_mouth, the distance between the centers of geometry of the BC-loop and FG-loop Cα sets that reports opening at the channel entrance, and d_cover, the distance from the FG-loop Cα centroid to the heme Fe that reports the cover position over the distal pocket. These descriptors are complemented by reactive-geometry and hydrogen-bond analyses and by MM-PBSA component analysis as a supportive energetic readout. The simulations reveal a conserved mouth-cover landscape with three recurrent basins, open, intermediate, and closed. Ligands mainly redistribute populations across these pre-existing states and reshape gate geometry rather than generating new conformational states. Together, these findings support a conformational-selection mechanism linking ligand recognition, gating, and catalytic readiness in this orphan bacterial P450.

Berberine (BBR) is widely used as a botanical pesticide due to its broad-spectrum antibacterial and antifungal activities. However, BBR degradation pathway in soil microorganisms, which determines its impact on soil environment, remains poorly understood. Herein, a novel BBR-degrading bacterium Agrobacterium sp. V1 was isolated and characterized. Agrobacterium sp. V1 was able to utilize BBR as the sole carbon source for cell growth, and 50 μg/mL of BBR was completely degraded within 48 h. To reveal the possible BBR degradation pathway, whole genome sequencing of Agrobacterium sp. V1 was conducted, and proteins in Agrobacterium sp. V1 were aligned with enzymes involved in BBR biosynthesis in Rhizoma Coptidis. The results indicated that more than 60% of enzymes in BBR biosynthesis pathway had orthologs in Agrobacterium sp. V1. Combined with the primary mass spectra of BBR metabolites, a novel BBR degradation pathway in this bacterium was proposed. In summary, the proposed BBR degradation pathway offered new insights into the impact of BBR to the environment and also provided a reference for studying BBR metabolism in microorganisms.

Efflux pumps play important roles in bacterial detoxification and some of them are stress-response elements that are up-regulated when the host is treated with antibiotics. However, efflux pumps that are down-regulated by stimulations are rarely discovered. Herein, we analyzed multiple transcriptome data and discovered a special (Major Facilitator Superfamily) MFS efflux pump, KpsrMFS, from Klebsiella pneumoniae, which was down-regulated when treated with antibiotics or extra carbon sources. Interestingly, overexpression of kpsrmfs resulted in halted cell growth in normal conditions, while the viable cells were rarely affected. The function of KpsrMFS was further analyzed and this efflux pump was determined to be a proton-driven transporter that can reduce the intracellular tetracycline concentration. In normal conditions, the expression of kpsrmfs was at a low level, while artificial overexpression of it led to increased endogenous reactive oxygen species (ROS) production. Moreover, by comparing the functions of adjacent genes of kpsrmfs, we further discovered another four genes that can confer similar phenotypes, indicating a special regulon that regulates cell growth. Our work provides new insights into the roles of efflux pumps and suggests a possible regulon that may regulate cell growth and endogenous ROS levels.

Multidrug resistance poses a major challenge to antibiotic therapy. A principal cause of antibiotic resistance is through active export by efflux pumps embedded in the bacterial membrane. Major facilitator superfamily (MFS) efflux pumps constitute a major group of transporters, which are often related to quinolone resistance in clinical settings. Although a rocker-switch model is proposed for description of their conformational transitions, detailed changes in this process remain poorly understood. Here we used MdfA from E. coli as a representative MFS efflux pump to investigate factors that can affect its conformational transition in silico. Molecular dynamics (MD) simulations of MdfA’s inward and outward conformations revealed an intermediate state between these two conformations. By comparison of the subtle differences between the intermediate state and the average state, we indicated that conformational transition from outward to inward was initiated by protonation of the periplasmic side. Subsequently, hydrophilic interaction of the periplasmic side with water was promoted and the regional structure of helix 1 was altered to favor this process. As the hydrophobic interaction between MdfA and membrane was also increased, energy was concentrated and stored for the opposite transition. In parallel, salt bridges at the cytoplasmic side were altered to lower probabilities to facilitate the entrance of substrate. In summary, we described the total and local changes during MdfA’s conformational transition, providing insights for the development of potential inhibitors.