Crystal structures of multidrug binding protein TtgR in complex with antibiotics and plant antimicrobials.
Crystal structures of multidrug binding protein TtgR in complex with antibiotics and plant antimicrobials.
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DOI：
10.1016/j.jmb.2007.03.062
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发表时间：
2007-06-08
影响因子：
5.6
通讯作者：
Zhang, Xiaodong
中科院分区：
文献类型：
--
作者：
Alguel, Yilmaz;Meng, Cuixiang;Teran, Wilson;Krell, Tino;Ramos, Juan L.;Gallegos, Maria-Trinidad;Zhang, Xiaodong
抗生素耐药性是一种广泛传播的现象。细菌产生抗生素耐药性的一个主要机制是有毒化合物通过膜结合的外排泵主动排出，这些外排泵通常在转录水平上受到调控。TtgR抑制恶臭假单胞菌的关键外排泵TtgABC的转录，恶臭假单胞菌对抗生素、溶剂和有毒植物次生产物具有高度抗性。以前我们证明了TtgR是唯一已报道的与不同类别的天然抗菌化合物结合的抑制物，这些化合物也是通过外排泵排出的。我们报道了耐溶剂菌株DOT-T1E的TtgR的五种高分辨晶体结构，包括与常用抗生素和植物次生代谢产物形成的复合体中的TtgR。我们为TtgR独特的配体结合特性提供了结构基础。我们发现了两个不同的重叠的配体结合部位，第一个较宽，主要由疏水残基组成，第二个较深，含有更多的极性残基，包括Arg176，这是DOT-T1E菌株中存在的唯一残基，但在其他假单胞菌中不存在。植物抗菌剂Phloretin可以与两个结合部位结合，具有不同的结合亲和力和化学计量比。用等温滴定量热法对天然和突变TtgR蛋白的配体结合特性的研究结果证实了TtgR蛋白的结合亲和力和化学计量比，并表明这两个结合位点之间存在潜在的正协同作用。与天然蛋白相比，Arg176从结合的DNA中释放突变的TtgR的能力降低，进一步证实了Arg176在根癌蛋白结合中的重要性。这里提出的结果强调了调控系统在细菌抗生素耐药性中的重要性和多功能性，并为新的抗菌药开发开辟了新的途径。
Antibiotic resistance is a widely spread phenomenon. One major mechanism that underlies antibiotic resistance in bacteria is the active extrusion of toxic compounds through the membrane-bound efflux pumps that are often regulated at the transcriptional level. TtgR represses the transcription of TtgABC, a key efflux pump in Pseudomonas putida, which is highly resistant to antibiotics, solvents and toxic plant secondary products. Previously we showed that TtgR is the only reported repressor that binds to different classes of natural antimicrobial compounds, which are also extruded by the efflux pump. We report here five high-resolution crystal structures of TtgR from the solvent-tolerant strain DOT-T1E, including TtgR in complex with common antibiotics and plant secondary metabolites. We provide structural basis for the unique ligand binding properties of TtgR. We identify two distinct and overlapping ligand binding sites; the first one is broader and consists of mainly hydrophobic residues, whereas the second one is deeper and contains more polar residues including Arg176, a unique residue present in the DOT-T1E strain but not in other Pseudomonas strains. Phloretin, a plant antimicrobial, can bind to both binding sites with distinct binding affinities and stoichiometries. Results on ligand binding properties of native and mutant TtgR proteins using isothermal titration calorimetry confirm the binding affinities and stoichiometries, and suggest a potential positive cooperativity between the two binding sites. The importance of Arg176 in phloretin binding was further confirmed by the reduced ability of phloretin in releasing the mutant TtgR from bound DNA compared to the native protein. The results presented here highlight the importance and versatility of regulatory systems in bacterial antibiotic resistance and open up new avenues for novel antimicrobial development.
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影响因子：
5.8
作者：
Gouet, P;Courcelle, E;Métoz, F
通讯作者：
Métoz, F
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
14.9
作者：
Binkowski, TA;Naghibzadeh, S;Liang, J
通讯作者：
Liang, J
DOI：
10.1107/s0907444998003254
发表时间：
1998-09-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Brunger, AT;Adams, PD;Warren, GL
通讯作者：
Warren, GL
影响因子：
3.6
作者：
Duque, E;Segura, A;Ramos, JL
通讯作者：
Ramos, JL