RESUMO
Bacterial topoisomerase IV (ParE) is essential for DNA replication and serves as an attractive target for antibacterial drug development. The X-ray structure of the N-terminal 24 kDa ParE, responsible for ATP binding has been solved. Due to the accessibility of structural information of ParE, many potent ParE inhibitors have been discovered. In this study, a pyridylurea lead molecule against ParE of Escherichia coli (eParE) was characterized with a series of biochemical and biophysical techniques. More importantly, solution NMR analysis of compound binding to eParE provides better understanding of the molecular interactions between the inhibitor and eParE.
Assuntos
Trifosfato de Adenosina/metabolismo , DNA Topoisomerase IV/metabolismo , DNA Topoisomerase IV/farmacologia , Escherichia coli/enzimologia , Trifosfato de Adenosina/antagonistas & inibidores , Sequência de Aminoácidos , Antibacterianos/farmacologia , Ligação Competitiva , DNA Topoisomerase IV/antagonistas & inibidores , DNA Topoisomerase IV/química , Desenho de Fármacos , Dados de Sequência Molecular , Ressonância Magnética Nuclear BiomolecularRESUMO
The N-terminal ATP binding domain of the DNA gyrase B subunit is a validated drug target for antibacterial drug discovery. Structural information for this domain (pGyrB) from Pseudomonas aeruginosa is still missing. In this study, the interaction between pGyrB and a bis-pyridylurea inhibitor was characterized using several biophysical methods. We further carried out structural analysis of pGyrB using NMR spectroscopy. The secondary structures of free and inhibitor bound pGyrB were obtained based on backbone chemical shift assignment. Chemical shift perturbation and NOE experiments demonstrated that the inhibitor binds to the ATP binding pocket. The results of this study will be helpful for drug development targeting P. aeruginosa.
Assuntos
Domínio Catalítico , DNA Girase/química , DNA Girase/metabolismo , Pseudomonas aeruginosa/enzimologia , Inibidores da Topoisomerase II/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacologia , Ureia/química , Ureia/metabolismo , Ureia/farmacologiaRESUMO
The hERG (human ether-a-go-go related gene) potassium channel is a voltage-gated potassium channel containing an N-terminal domain, a voltage-sensor domain, a pore domain and a C-terminal domain. The transmembrane segment 4 (S4) is important for sensing changes of membrane potentials through positively charge residues. A construct containing partial S2-S3 linker, S3, S4 and the S4-S5 linker of the hERG channel was purified into detergent micelles. This construct exhibits good quality NMR spectrum when it was purified in lyso-myristoyl phosphatidylglycerol (LMPG) micelles. Structural study showed that S3 contains two short helices with a negatively charged surface. The S4 and S4-S5 linker adopt helical structures. The six positively charged residues in S4 localize at different sides, suggesting that they may have different functions in channel gating. Relaxation studies indicated that S3 is more flexible than S4. The boundaries of S3-S4 and S4-S4-S5 linker were identified. Our results provided structural information of the S3 and S4, which will be helpful to understand their roles in channel gating.