Structural characterization of OM proteins from Gram-negative pathogens
革兰氏阴性病原体 OM 蛋白的结构表征
基本信息
- 批准号:9549803
- 负责人:
- 金额:$ 163.9万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
- 来源:
- 关键词:AcinetobacterAnimal Disease ModelsAntibioticsAreaBacteriaBacterial Outer Membrane ProteinsBacteriophagesBindingBinding ProteinsBiochemistryCationsCellsCitratesClinicalCodeColicin IaCommunicable DiseasesComplexComputer SimulationCrystallizationDetectionDown-RegulationDrug TargetingElectron MicroscopyElectrophysiology (science)EngineeringEnterobactinEscherichia coliExhibitsFamilyGram-Negative BacteriaHumanHuman GenomeImmuneInfectionIntegral Membrane ProteinIonsIronKlebsiella pneumonia bacteriumLeadLengthLower OrganismMembraneMembrane ProteinsMembrane Transport ProteinsMetalsMolecular ConformationMotorMulti-Drug ResistanceNatureNeisseriaNeisseria meningitidisNosocomial InfectionsPathogenicityPathway interactionsPenetrationPesticinPharmacologic SubstancePlaguePlasma ProteinsPreclinical Drug EvaluationProtein EngineeringProtein ImportProteinsPublishingRegulationResolutionRoentgen RaysSerumSiderophoresSignal TransductionSpectrum AnalysisStructureSurfaceSystemThickToxinTransferrinTransition ElementsUnited States National Institutes of HealthVaccinesVirulenceWorkX-Ray CrystallographyYersinia pestisZincaerobactinantimicrobialcapsulecolicincombatcrosslinkexperimental studyinterestlysinmolecular dynamicsnovelnovel therapeuticspathogenperiplasmpreventprotein complexprotein structurereceptorreceptor bindingsmall moleculesuccessuptakezinc-binding protein
项目摘要
Our early crystal structures showed how iron transporters specifically recognize Fe3+ bound to small molecules such as enterobactin (a siderophore synthesized by Escherichia coli) and citrate. Each transporter has a unique binding pocket for its preferred small molecule. When the correct substrate binds, the transporter undergoes conformational changes that send a signal across the outer membrane and prepare the system for transport.
We expanded our studies in this area to determine how Neisseria meningitidis binds to human serum transferrin and extracts the iron for import into the bacterial cell. These bacteria require iron for survival and obtain it directly from human proteins. Neisseria have an outer membrane protein, TbpA, and a co-receptor protein, TbpB, which together can extract the iron from a human plasma protein called transferrin. We used a combined approach of X-ray crystallography, electron microscopy, small angle X-ray scattering, biochemistry, and molecular dynamics simulations to elucidate the iron-scavenging mechanism. This was the first atomic resolution structure of a bacterial outer membrane protein bound to its full-length human target protein.
We broadened our focus on transition metal transport to Acinetobacter baumanni zinc uptake. A. baumannii is a hospital-acquired infection demonstrating multidrug resistance. It is of great interest to the NIH clinical center. Zinc correlates with virulence in A. baumannii and there are three putative TonB-dependent zinc transporters in this bacterium. When deprived of zinc, A. baumannii becomes much more sensitive to existing antibiotics, so inhibition of zinc uptake may lead to novel therapies against this Gram-negative bacterium. We just solved the structure of an A. baumannii zinc transporter with zinc bound. We are using the structure to perform in silico small molecule drug screening and to investigate the zinc transport pathway.
In our search for novel antimicrobial therapies, we extended our work on small-molecule transporters to ask how proteins are ferried across the outer membrane. Some of the metal transporters that we study also facilitate the uptake of large protein toxins called colicins. For example, we determined the structure of an outer membrane iron transporter from Yersinia pestis (which causes plague) that is required for virulence. We also determined the structure of a colicin, called pesticin, which uses this transporter to cross the outer membrane. The two structures showed us how to engineer a novel antibiotic that is the first example of phage therapy for any Gram-negative bacterium, and our antibiotic was demonstrated to be effective on clinical isolates Guided by this success, we will continue this type of protein engineering for other bacterial pathogens.
Interestingly, for all of these transition metal transporters, how the metal gets into the periplasm is not well understood. We know that transport involves an inner membrane protein complex (TonB-ExbB-ExbD) and energy in the form of protonmotive force. We recently determined the structure of a subcomplex of this motor, consisting of ExbB and ExbD. We used a combined approach of X-ray crystallography, electron microscopy, DEER spectroscopy, crosslinking, and electrophysiology to show that the Ton subcomplex forms pH sensitive, cation selective channels that couple ion flow to energy transduction at the outer membrane.
Another hospital-acquired infection of great importance to the NIH clinical center is Klebsiella pneumoniae. This bacterium exhibits multidrug resistance and some strains have shown hypervirulence. In an effort to identify new ways to combat infection, we are collaborating with Susan Gottesman, NCI, to investigate proteins involved in regulation of capsule. K. pneumoniae can escape immune detection and prevent penetration of antibiotics with its thick capsule layer that surrounds the outer membrane. Our hypothesis is that down-regulation of capsule synthesis might make K. pneumoniae more sensitive to available antibiotics, and thus more treatable than is currently the case. Structural and functional experiments on this system are in progress.
In a separate project targeting Klebsiella pneumoniae, we recently determined four structures of the Kp areobactin transporter, which is a TonB dependent transporter that correlates with virulence in hypervirulent K. pneumoniae. We are currently using in silico drug screening to identify small molecules that compete for binding with aerobactin, with plans to explore these compounds in an animal model of the disease. This work will be finalized and published within the coming year.
References
Buchanan, S.K., Smith, B.S., Venkatramani, L., Xia, D., Palnitkar, M., Chakraborty, R., van der Helm, D. & Deisenhofer, J. (1999). Crystal structure of the outer membrane active transporter FepA from Escherichia coli. Nat. Struct. Biol. 6, 56-63.
Yue, W.W., Grizot, S. & Buchanan, S.K. (2003). Structural evidence for iron-free citrate and ferric citrate binding to the TonB-dependent outer membrane transporter FecA. J. Mol. Biol. 332, 353-368.
Buchanan, S.K., Lukacik, P., Grizot, S., Ghirlando, R., Ali, M.M.U., Barnard, T.J., Jakes, K.S., Kienker, P.K. & Esser, L. (2007). Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import. EMBO J. 26, 2594-2604. PMCID: PMC1868905
Noinaj, N., Easley, N.C., Oke, M., Mizuno, N., Gumbart, J., Boura, E., Steere, A., Zak, O., Aisen, P., Tajkhorshid, E.M., Evans, R., Gorringe, A., Mason, A.B., Steven, A. & Buchanan, S.K. (2012). Structural basis for iron piracy by pathogenic Neisseria. Nature 483, 53-58. PMCID: PMC3292680
Lukacik, P., Barnard, T.J., Keller, P.W., Chaturvedi, K., Seddiki, N., Fairman, J.W., Noinaj, N., Kirby, T.L., Henderson, J.P., Steven, A.C., Hinnebusch, B.J. & Buchanan, S.K. (2012). Structural engineering of a phage lysin that targets Gram-negative pathogens. Proc. Natl. Acad. Sci. USA, 109, 9857-9862. PMCID: PMC3382549
我们的早期晶体结构显示了铁转运体如何特异性识别结合在小分子上的Fe3+,如肠杆菌蛋白(由大肠杆菌合成的铁载体)和柠檬酸盐。每个转运体都有一个独特的结合袋,用于其首选的小分子。当正确的底物结合时,转运体发生构象变化,向外膜发送信号,为运输系统做好准备。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Susan Buchanan其他文献
Susan Buchanan的其他文献
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{{ truncateString('Susan Buchanan', 18)}}的其他基金
Structural characterization of OM proteins from Gram-negative pathogens
革兰氏阴性病原体 OM 蛋白的结构表征
- 批准号:
8741336 - 财政年份:
- 资助金额:
$ 163.9万 - 项目类别:
structural characterization of iron uptake from human transferrin
人转铁蛋白吸收铁的结构特征
- 批准号:
8741420 - 财政年份:
- 资助金额:
$ 163.9万 - 项目类别:
structural characterization of iron uptake from human transferrin
人转铁蛋白吸收铁的结构特征
- 批准号:
8553451 - 财政年份:
- 资助金额:
$ 163.9万 - 项目类别:
Structural characterization of OM proteins from Gram-negative pathogens
革兰氏阴性病原体 OM 蛋白的结构表征
- 批准号:
8939481 - 财政年份:
- 资助金额:
$ 163.9万 - 项目类别:
Structural characterization of outer membrane proteins from Yersinia pestis
鼠疫耶尔森氏菌外膜蛋白的结构表征
- 批准号:
7733943 - 财政年份:
- 资助金额:
$ 163.9万 - 项目类别:
structural characterization of bacterial secretion channels
细菌分泌通道的结构特征
- 批准号:
10248132 - 财政年份:
- 资助金额:
$ 163.9万 - 项目类别:
structural characterization of bacterial secretion channels
细菌分泌通道的结构特征
- 批准号:
10000710 - 财政年份:
- 资助金额:
$ 163.9万 - 项目类别:
structural characterization of bacterial secretion channels
细菌分泌通道的结构特征
- 批准号:
7593557 - 财政年份:
- 资助金额:
$ 163.9万 - 项目类别:
structural characterization of bacterial secretion channels
细菌分泌通道的结构特征
- 批准号:
8148751 - 财政年份:
- 资助金额:
$ 163.9万 - 项目类别:
structural characterization of bacterial secretion channels
细菌分泌通道的结构特征
- 批准号:
8741419 - 财政年份:
- 资助金额:
$ 163.9万 - 项目类别:
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