Drug-dependent inhibition of nucleotide hydrolysis in the heterodimeric ABC multidrug transporter PatAB from Streptococcus pneumoniae.
Drug-dependent inhibition of nucleotide hydrolysis in the heterodimeric ABC multidrug transporter PatAB from Streptococcus pneumoniae.
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肺炎链球菌异二聚ABC多药转运蛋白PatAB中核苷酸水解的药物依赖性抑制
DOI：
10.1111/febs.16366
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发表时间：
2022-07
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影响因子：
--
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细菌异源二聚体ATP结合盒（ABC）多药输出蛋白PatAB在肺炎链球菌多重耐药感染中赋予抗生素耐药性方面具有关键作用。与其他异二聚体ABC输出者一样，PatAB含有两个跨膜结构域，形成药物转运途径，用于外排，以及两个结合ATP的核苷酸结合结构域，其中一个在转运过程中水解。异源二聚ABC多药出口商的结构和功能元件，确定与药物的相互作用和夫妇的药物结合核苷酸水解尚未完全理解。在这里，我们使用质谱技术来确定乳球菌表达系统中PatAB的亚基化学计量，并使用荧光药物模拟叠氮基乙锭研究药物结合的位置。令人惊讶的是，我们对叠氮基乙锭标记的PatAB肽的分析指出，乙锭结合在PatA核苷酸结合结构域中，叠氮基部分与简并核苷酸结合位点的H样环中的残基Q521交联。对该化合物和残基在核苷酸水解中的作用的研究表明，Q521A突变体的活性降低，突变体和野生型中的乙锭依赖性抑制。大多数转运药物不刺激或抑制PatAB在洗涤剂溶液或纳米盘中的核苷酸水解。然而，在丙啶、新生霉素和香豆霉素A1中发现了乙啶样抑制的进一步实例，它们都通过非竞争性机制抑制核苷酸水解。这些数据揭示了药物可以通过PatAB调节核苷酸水解的潜在机制，这可能涉及核苷酸结合结构域附近的新药物结合位点。异二聚体ABC多药转运蛋白是致病菌抗生素耐药性的重要贡献者。然而，决定与药物相互作用和偶联药物结合核苷酸水解的结构和功能元件尚未完全理解。使用质谱和生物化学方法，我们确定的亚基组成的PatAB从肺炎链球菌和探针药物结合的位置来解释抑制核苷酸水解的抗生素和细胞毒性剂的一个子类。
The bacterial heterodimeric ATP‐binding cassette (ABC) multidrug exporter PatAB has a critical role in conferring antibiotic resistance in multidrug‐resistant infections by Streptococcus pneumoniae. As with other heterodimeric ABC exporters, PatAB contains two transmembrane domains that form a drug translocation pathway for efflux and two nucleotide‐binding domains that bind ATP, one of which is hydrolysed during transport. The structural and functional elements in heterodimeric ABC multidrug exporters that determine interactions with drugs and couple drug binding to nucleotide hydrolysis are not fully understood. Here, we used mass spectrometry techniques to determine the subunit stoichiometry in PatAB in our lactococcal expression system and investigate locations of drug binding using the fluorescent drug‐mimetic azido‐ethidium. Surprisingly, our analyses of azido‐ethidium‐labelled PatAB peptides point to ethidium binding in the PatA nucleotide‐binding domain, with the azido moiety crosslinked to residue Q521 in the H‐like loop of the degenerate nucleotide‐binding site. Investigation into this compound and residue’s role in nucleotide hydrolysis pointed to a reduction in the activity for a Q521A mutant and ethidium‐dependent inhibition in both mutant and wild type. Most transported drugs did not stimulate or inhibit nucleotide hydrolysis of PatAB in detergent solution or lipidic nanodiscs. However, further examples for ethidium‐like inhibition were found with propidium, novobiocin and coumermycin A1, which all inhibit nucleotide hydrolysis by a non‐competitive mechanism. These data cast light on potential mechanisms by which drugs can regulate nucleotide hydrolysis by PatAB, which might involve a novel drug binding site near the nucleotide‐binding domains. Heterodimeric ABC multidrug transporters are important contributors to antibiotic resistance in pathogenic bacteria. However, the structural and functional elements determining interactions with drugs and coupling drug binding to nucleotide hydrolysis are not fully understood. Using mass spectrometry and biochemical approaches, we determine the subunit composition of PatAB from Streptococcus pneumoniae and probe drug binding locations to explain the inhibition of nucleotide hydrolysis by a subclass of antibiotics and cytotoxic agents.
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影响因子：
5.9
作者：
Guo D;Singh H;Shimoyama A;Guffick C;Tang Y;Rowe SM;Noel T;Spring DR;Fukase K;van Veen HW
通讯作者：
van Veen HW
DOI：
10.1073/pnas.0800191105
发表时间：
2008-04-01
影响因子：
11.1
作者：
Ernst, Robert;Kueppers, Petra;Schmitt, Lutz
通讯作者：
Schmitt, Lutz
DOI：
10.1093/jac/dku449
发表时间：
2015-03
期刊：
The Journal of antimicrobial chemotherapy
影响因子：
--
作者：
Baylay AJ;Piddock LJ
通讯作者：
Piddock LJ
DOI：
10.1073/pnas.1717044115
发表时间：
2018-02-27
影响因子：
11.1
作者：
Alam A;Küng R;Kowal J;McLeod RA;Tremp N;Broude EV;Roninson IB;Stahlberg H;Locher KP
通讯作者：
Locher KP
DOI：
10.1126/science.1157987
发表时间：
2008-07-11
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Kadaba NS;Kaiser JT;Johnson E;Lee A;Rees DC
通讯作者：
Rees DC