天然质谱和结构研究揭示了脂质对MsbA-核苷酸相互作用的调节-动脉网

Native mass spectrometry and structural studies reveal modulation of MsbA–nucleotide interactions by lipids

Nature 等信源发布 2024-07-15 23:29

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AbstractThe ATP-binding cassette (ABC) transporter, MsbA, plays a pivotal role in lipopolysaccharide (LPS) biogenesis by facilitating the transport of the LPS precursor lipooligosaccharide (LOS) from the cytoplasmic to the periplasmic leaflet of the inner membrane. Despite multiple studies shedding light on MsbA, the role of lipids in modulating MsbA-nucleotide interactions remains poorly understood.

摘要ATP结合盒（ABC）转运蛋白MsbA通过促进LPS前体脂多糖（LOS）从细胞质向内膜周质小叶的转运，在脂多糖（LPS）的生物发生中起着关键作用。尽管多项研究揭示了MsbA，但脂质在调节MsbA核苷酸相互作用中的作用仍然知之甚少。

Here we use native mass spectrometry (MS) to investigate and resolve nucleotide and lipid binding to MsbA, demonstrating that the transporter has a higher affinity for adenosine 5’-diphosphate (ADP). Moreover, native MS shows the LPS-precursor 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)2-lipid A (KDL) can tune the selectivity of MsbA for adenosine 5’-triphosphate (ATP) over ADP.

在这里，我们使用天然质谱（MS）来研究和解析核苷酸和脂质与MsbA的结合，证明该转运蛋白对腺苷5'-二磷酸（ADP）具有更高的亲和力。此外，天然MS显示LPS前体3-脱氧-D-甘露基-oct-2-果糖酸（Kdo）2-脂质A（KDL）可以调节MsbA对ADP上腺苷5'-三磷酸（ATP）的选择性。

Guided by these studies, four open, inward-facing structures of MsbA are determined that vary in their openness. We also report a 2.7 Å-resolution structure of MsbA in an open, outward-facing conformation that is not only bound to KDL at the exterior site, but with the nucleotide binding domains (NBDs) adopting a distinct nucleotide-free structure.

在这些研究的指导下，确定了MsbA的四个开放的向内结构，它们的开放性各不相同。我们还报告了MsbA的2.7Å分辨率结构，该结构呈开放的向外构象，不仅在外部位点与KDL结合，而且核苷酸结合域（NBD）采用独特的无核苷酸结构。

The results obtained from this study offer valuable insight and snapshots of MsbA during the transport cycle..

从这项研究中获得的结果为运输周期中的MsbA提供了有价值的见解和快照。。

IntroductionGram-negative bacteria, including E. coli, possess a complex envelope consisting of an inner membrane and an outer membrane separated by the periplasm1,2,3. The inner membrane forms a typical phospholipid bilayer surrounding the cytoplasm of bacteria, while the outer membrane adopts an asymmetric structure, with phospholipids comprising the inner leaflet and lipopolysaccharides (LPS) as the major component of the outer leaflet1,2,3.

引言革兰氏阴性细菌，包括大肠杆菌，具有复杂的包膜，由内膜和由周质隔开的外膜组成1,2,3。内膜在细菌细胞质周围形成典型的磷脂双层，而外膜采用不对称结构，磷脂包含内小叶和脂多糖（LPS）作为外小叶的主要成分1,2,3。

LPS plays a vital role in maintaining an effective outer membrane barrier, providing resistance against antibiotics and various environmental stresses4,5. Notably, MsbA, a member of the ATP-binding cassette superfamily, plays a crucial role in LPS biosynthesis by facilitating the flipping of the LPS-precursor lipooligosaccharide (LOS) from the cytoplasmic side of the inner membrane to the periplasmic side6,7.

LPS在维持有效的外膜屏障，提供对抗生素和各种环境压力的抗性方面起着至关重要的作用4,5。值得注意的是，作为ATP结合盒超家族成员的MsbA通过促进LPS前体脂寡糖（LOS）从内膜的细胞质侧翻转到周质侧，在LPS生物合成中起着至关重要的作用6,7。

The essentiality of E. coli MsbA is evident from studies reporting that MsbA knockouts are lethal7,8, making this transporter an attractive target for developing antibiotics that inhibit function thereby combating multidrug-resistant infections.Numerous investigations have shed light on the function, structure, and mechanism of MsbA9,10,11,12,13,14,15,16,17.

大肠杆菌MsbA的重要性从报道MsbA敲除是致命的7,8的研究中显而易见，这使得该转运蛋白成为开发抑制功能从而对抗多药耐药性感染的抗生素的有吸引力的靶标。大量研究揭示了MsbA9,10,11,12,13,14,15,16,17的功能，结构和机制。

MsbA forms a homodimer and exhibits a topology similar to other ABC transporters, comprising two NBDs and two transmembrane domains (TMDs) containing 12 transmembrane helices18,19. More specifically, the NBDs contain a RecA-type ATP binding core (RecAcore), composed of six beta sheets and four alpha helices (helices A-D), that is decorated with three additional beta sheets and an alpha-helical subdomain (ABCα)20,21.

MsbA形成同型二聚体，并表现出与其他ABC转运蛋白相似的拓扑结构，包括两个NBD和两个跨膜结构域（TMD），其中包含12个跨膜螺旋18,19。更具体地说，NBD包含一个RecA型ATP结合核心（RecAcore），由六个β折叠和四个α螺旋（螺旋a-D）组成，并用三个额外的β折叠和一个α螺旋子域（ABCα）20,21。

Other conserved motifs of the NBDs of MsbA include the hydrophobic residue of the A-loop (residue 351), Walker A or P-loop (GxxGxGK(S/T), where x denotes any.

MsbA的NBD的其他保守基序包括A环（残基351），Walker A或P环（GxxGxGK（S/T）的疏水残基，其中x表示任何。

(1)

[P]total represents total protein concentration:$${\left[P\right]}_{{total}}=\left[P\right]+\,{\varSigma }_{{{{{{\rm{i}}}}}}=1}^{n}\left[P{L}_{i}\right]=\left[P\right]+\,{\varSigma }_{{{{{{\rm{i}}}}}}=1}^{n}\left[P\right]{[L]}^{i}{\varPi }_{j=1}^{i}{K}_{{Aj}}$$

[P] total表示总蛋白质浓度：$${\左[P \右]}}{{总}}=\左[P \右]+\，{\ varSigma}{{{{\ rm{i}}}}}=1}^{n}\左[P{L}_{i} \right]=\left[P\right]+\，{\varSigma}}u{{{{{\ rm{i}}}}=1}^{n}\left[P\right]{[L]}^{i}{\varPi}}uj=1}^{i}{K}_{{Aj}}$$

(2)

The above equation can be rearranged to calculate the mole fraction (Fn) of PLn:$${F}_{n}=\frac{\left[P{L}_{n}\right]}{{\left[P\right]}_{{total}}}=\frac{{\left[L\right]}_{{free}}^{n}{\prod }_{j=1}^{n}{K}_{{Aj}}}{1+{\sum }_{i=1}^{n}{\left[L\right]}_{{free}}^{i}{\prod }_{j=1}^{i}{K}_{{Aj}}}$$.

可以重新排列上述方程式以计算PLn的摩尔分数（Fn）：$${F}_{n} =\frac{\left[P{L}_{n} \right]}{{\left[P\right]}}{{{total}}=\frac{{\left[L\right]}}{{free}}^{n}{\prod}}{uj=1}^{n}{K}_{{Aj}}}{1+{\ sum}}{i=1}^{n}{\左[右]}}}{{自由}}^{i}{\ prod}}{j=1}^{i}{K}_{{{Aj}}$$。

(3)

where [L]free is the free ligand concentration at equilibrium, which can be calculated with known [P]total:$${\left[L\right]}_{{free}}={\left[L\right]}_{{total}}-{\left[P\right]}_{{total}}{\sum }_{i=1}^{n}i{F}_{i}$$

其中[L]free是平衡时的游离配体浓度，可以用已知的[P]总量计算：$${\左[L \右]}{{自由}={\左[L \右]}{{{总}-{\左[P \右]}{{总}{\和}{i=1}^{n}i{F}_{我}$$

(4)

To obtain KAn, the sequential ligand binding model was globally fit to the mole fraction data by minimization of pseudo-${\chi }^{2}$ function:$${\chi }^{2}=\,{\sum }_{i=0}^{n}{\sum }_{j=1}^{d}{({F}_{i,\, j,\exp }-{F}_{i,\, j,{calc}})}^{2}$$

为了获得KAn，通过最小化伪函数：$${{chi}^{2}=\，{sum}{i=0}^{n}{sum}{j=1}^{d}，将顺序配体结合模型全局拟合到摩尔分数数据{({F}_{i，\，j，\ exp}-{F}_{i，\，j，{计算}}^{2}$$

(5)

where n is the number of bound ligands and d is the number of the experimental mole fraction data points.Sample preparation for single-particle cryoEMTo prepare samples for cryoEM studies, MsbA was pre-saturated with copper(II). Excess copper and glycerol were removed using a desalting column.

其中n是结合配体的数量，d是实验摩尔分数数据点的数量。单颗粒冷冻电镜的样品制备为了制备用于冷冻电镜研究的样品，将MsbA用铜（II）预饱和。使用脱盐柱除去过量的铜和甘油。

Peak fractions were pooled and concentrated to 10 mg ml−1. Vitrification was performed using a Vitrobot Mark IV (Thermo Fisher) operating at 8 °C and 100% humidity. A total of 3.5 μL of sample in cryoEM buffer (150 mM NaCl, 20 mM TRIS, 0.065% C10E5, pH 7.4) incubated with 1 mM MgCl2, 1 mM ATP and 194 µM KDL at 4 °C for 6 h was applied to holey carbon grids (Quantifoil 300 mesh Cu 1.2/1.3) glow-discharged for 30 s.

合并峰级分并浓缩至10mg/ml。使用在8℃和100%湿度下操作的Vitrobot Mark IV（Thermo Fisher）进行玻璃化冷冻。将在cryoEM缓冲液（150mM NaCl，20mM TRIS，0.065%C10E5，pH 7.4）中的总共3.5μL样品与1mM MgCl 2,1mM ATP和194μMKDL在4℃下孵育6小时，应用于多孔碳栅（Quantifoil 300目Cu 1.2/1.3）辉光放电30秒。

The grids were blotted for 5 s at blotting force 1 using standard Vitrobot filter paper (Ted Pella, 47000-100), and then plunged into liquid ethane.Data collection for single-particle cryoEMData collection was performed at the Advanced Electron Microscopy Facility at the University of Chicago. The dataset was collected as movie stacks with a Titan Krios electron microscope operating at 300 kV, equipped with a K3 direct detector camera.

使用标准Vitrobot滤纸（Ted Pella，47000-100）在吸墨力1下将网格吸干5秒，然后浸入液体乙烷中。单粒子cryoEMData收集的数据收集是在芝加哥大学的高级电子显微镜设备上进行的。该数据集被收集为电影堆栈，其中Titan Krios电子显微镜在300 kV下运行，配备有K3直接探测器相机。

Images were recorded at a nominal magnification of 81,000× at super-resolution counting mode by image shift. The total exposure time was set to 4 s with a frame recorded every 0.1 s, resulting in 40 frames in a single stack with a total exposure around 50 electrons/Å2. The defocus range was set at −1.0 to −2.5 μm.

通过图像移位，在超分辨率计数模式下以81000倍的标称放大率记录图像。总曝光时间设置为4秒，每0.1秒记录一帧，导致单个堆栈中有40帧，总曝光量约为50个电子/Å2。散焦范围设置为-1.0至-2.5μm。

See Supplementary Table 4 for the details of data collection parameters.Image processing for single-particle cryoEMCollected movies were processed using CryoSPARC59 and RELION60. The detailed data processing flow is shown in Supplementary Fig. 13. Briefly, stage drift and anisotropic motion of the stack images wer.

有关数据收集参数的详细信息，请参见补充表4。单粒子冷冻的图像处理使用CryoSPARC59和RELION60处理收集的电影。详细的数据处理流程如补充图13所示。简要介绍了叠加图像的阶段漂移和各向异性运动。

Data availability

数据可用性

MsbA cryoEM structures and maps have been deposited in the PDB and EMDB as follows: 8TSO and EMD-41596, 8TSP and EMD-41597, 8TSQ and EMD-41598, 8TSS and EMD-41560; and 8TSR and EMD-41599. Previously reported protein structures used in this study are: 3B5W (open, inward-facing MsbA), 8DMO (open, inward-facing MsbA), 6BL6 (open, inward-facing Salmonella typhimurium MsbA), 8DMM (vanadate-trapped MsbA bound to KDL), 6BPL (MsbA in complex with LPS and G907), and 7BCW (vanadate-trapped MsbA).

MsbA cryoEM结构和图谱已保存在PDB和EMDB中，如下所示：8TSO和EMD-41596、8TSP和EMD-41597、8TSQ和EMD-41598、8TSS和EMD-41560；和8TSR和EMD-41599。本研究中使用的先前报道的蛋白质结构是：3B5W（开放的，向内的MsbA），8DMO（开放的，向内的MsbA），6BL6（开放的，向内的鼠伤寒沙门氏菌MsbA），8DMM（钒酸盐捕获的MsbA与KDL结合），6BPL（与LPS和G907复合的MsbA）和7BCW（钒酸盐捕获的MsbA）。

Native MS data has been deposited at Zenodo (https://doi.org/10.5281/zenodo.10845033). Source data are provided with this paper..

本机MS数据已保存在Zenodo(https://doi.org/10.5281/zenodo.10845033)。本文提供了源数据。。

Code availability

代码可用性

Python code to determine individual equilibrium binding constants is available at https://github.com/LaganowskyLab (https://doi.org/10.5281/zenodo.11040823).

用于确定单个平衡结合常数的Python代码可在https://github.com/LaganowskyLab（笑声）(https://doi.org/10.5281/zenodo.11040823)。

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Download referencesAcknowledgementsThis work was supported by National Institutes of Health (NIH) under grant numbers (R01GM121751, R01GM139876, R01GM138863, and RM1GM145416 to A.L.; and R35GM143052 to M.Z.). We thank the staff at the University of Chicago Advanced Electron Microscopy (RRID: SCR_019198) for the help with cryo-EM data collection.

下载参考文献致谢这项工作得到了美国国立卫生研究院（NIH）的资助（A.L.的R01GM121751，R01GM139876，R01GM138863和RM1GM145416；M.Z.的R35GM143052）。我们感谢芝加哥大学高级电子显微镜（RRID:SCR\U 019198）的工作人员在低温电磁数据收集方面的帮助。

We thank the Research Computing Center at the University of Chicago for the support of this work by providing the computing resources of the Beagle3 HPC cluster funded by NIH (S10OD028655).Author informationAuthors and AffiliationsDepartment of Chemistry, Texas A&M University, College Station, TX, USATianqi Zhang, Jixing Lyu, Sangho D.

我们感谢芝加哥大学研究计算中心通过提供由NIH资助的Beagle3 HPC集群的计算资源（S10OD028655）对这项工作的支持。作者信息作者和附属机构德克萨斯农工大学化学系，德克萨斯州大学城，USATianqi Zhang，Jixing Lyu，Sangho D。

Yun, Elena Scott & Arthur LaganowskyDepartment of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USABowei Yang & Minglei ZhaoAuthorsTianqi ZhangView author publicationsYou can also search for this author in.

Yun，Elena Scott＆Arthur Laganowsky芝加哥大学生物化学与分子生物学系，伊利诺伊州芝加哥，USABowei Yang＆Minglei ZhaoAuthorsTianqi ZhangView作者出版物您也可以在中搜索这位作者。

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PubMed Google ScholarContributionsT.Z. and A.L. designed the research. T.Z. and J.L. expressed and purified MsbA. S.Y. and E.S. helped prepare MS samples. T.Z. performed mass spectrometry experiments. T.Z. and A.L. analyzed the data. B.Y., M.Z., and A.L. collected and processed cryo-EM data and built and refined atomic models.

PubMed谷歌学术贡献者。Z、 A.L.设计了这项研究。T、 Z.和J.L.表达并纯化了MsbA。S、 Y.和E.S.帮助准备了MS样品。T、 Z.进行了质谱实验。T、 Z.和A.L.分析了数据。B、 Y.，M.Z。和A.L.收集并处理了低温电磁数据，建立并改进了原子模型。

T.Z. and A.L. wrote the manuscript with input from the other authors.Corresponding authorCorrespondence to.

T、 Z.和A.L.在其他作者的意见下撰写了手稿。对应作者对应。

Arthur Laganowsky.Ethics declarations

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The authors declare no competing interests.

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Reprints and permissionsAbout this articleCite this articleZhang, T., Lyu, J., Yang, B. et al. Native mass spectrometry and structural studies reveal modulation of MsbA–nucleotide interactions by lipids.

转载和许可本文引用本文Zhang，T.，Lyu，J.，Yang，B。等人。天然质谱和结构研究揭示了脂质对MsbA-核苷酸相互作用的调节。

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生物物理脂质质谱结构生物学

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