Heptameric structures of two ?-hemolysin mutants imaged with in situ atomic force microscopy

Malghani, M. S.; Ye, Fang; Cheley, Stephen; Bayley, Hagan; Yang, Jie

doi:10.1002/(sici)1097-0029(19990301)44:5<353::aid-jemt6>3.0.co;2-0

Cited by 14 publications

(8 citation statements)

References 14 publications

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“…2). Similar entrance diameters were also deduced from lowresolution EM images [55,56], or atomic force microscopy [43,44], or from the di¡erential permeability of sugar molecules of di¡erent sizes through pores reconstituted in arti¢cial membranes [57]. In the EM, leukotoxin lesions appear remarkably similar to K-hemolysin, with an entrance diameter between 20 and 30 A î [46,47].…”

Section: The ¢Nal Porementioning

confidence: 57%

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Ion channels and bacterial infection: the case of β‐barrel pore‐forming protein toxins of Staphylococcus aureus

et al. 2003

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Staphylococcus aureus strains causing human pathologies produce several toxins, including a pore-forming protein family formed by the single-component K K-hemolysin and the bicomponent leukocidins and Q Q-hemolysins. The last comprise two protein elements, S and F, that co-operatively form the active toxin. K K-Hemolysin is always expressed by S. aureus strains, whereas bicomponent leukotoxins are more speci¢cally involved in a few diseases. X-ray crystallography of the K K-hemolysin pore has shown it is a mushroom-shaped, hollow heptamer, almost entirely consisting of L L-structure. Monomeric F subunits have a very similar core structure, except for the transmembrane stem domain which has to refold during pore formation. Large deletions in this domain abolished activity, whereas shorter deletions sometimes improved it, possibly by removing some of the interactions stabilizing the folded structure. Even before stem extension is completed, the formation of an oligomeric pre-pore can trigger Ca 2+ -mediated activation of some white cells, initiating an in£ammatory response. Within the bicomponent toxins, Q Q-hemolysins de¢ne three proteins (HlgA, HlgB, HlgC) that can generate two toxins: HlgA+HlgB and HlgC+HlgB. Like K K-hemolysin they form pores in planar bilayers with similar conductance, but opposite selectivity (cation instead of anion) for the presence of negative charges in the ion pathway. Q Q-Hemolysin pores seem to be organized as K K-hemolysin, but should contain an even number of each component, alternating in a 1:1 stoichiometry.

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Section: The ¢Nal Porementioning

confidence: 57%

“…Evidence from electron microscopy (EM), molecular and structural biology, indicated the formation of heptamers by α‐hemolysin [35,43], though the presence of hexamers was also noted [44]. During oligomerization, monomer–monomer interactions are established, which involve His35.…”

Section: Mode Of Actionmentioning

confidence: 99%

Ion channels and bacterial infection: the case of β‐barrel pore‐forming protein toxins of Staphylococcus aureus

et al. 2003

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“…All of these Staphylococcal toxins are secreted as water‐soluble monomers, and assemble on target cell membranes to form oligomeric membrane pores. αHL is a homoheptamer and assembles on erythrocyte and egg phosphatidylcholine membranes, and in deoxycholate micelles (Bhakdi et al 1981; Gouaux et al 1994; Song et al 1996; Fang et al 1997; Malghani et al 1999; Krasilnikov et al 2000). Under some conditions, however, Shao and colleagues contend that a hexamer may also be formed, as determined by atomic force microscopy (Czajkowsky et al 1998).…”

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confidence: 99%

Arresting and releasing Staphylococcal α‐hemolysin at intermediate stages of pore formation by engineered disulfide bonds

Kawate¹,

Gouaux

2003

Protein Science

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Abstract␣-Hemolysin (␣HL) is secreted by Staphylococcus aureus as a water-soluble monomer that assembles into a heptamer to form a transmembrane pore on a target membrane. The crystal structures of the LukF water-soluble monomer and the membrane-bound ␣-hemolysin heptamer show that large conformational changes occur during assembly. However, the mechanism of assembly and pore formation is still unclear, primarily because of the difficulty in obtaining structural information on assembly intermediates. Our goal is to use disulfide bonds to selectively arrest and release ␣HL from intermediate stages of the assembly process and to use these mutants to test mechanistic hypotheses. To accomplish this, we created four double cysteine mutants, D108C/K154C (␣HL-A), M113C/K147C (␣HL-B), H48C/ N121C (␣HL-C), I5C/G130C (␣HL-D), in which disulfide bonds may form between the pre-stem domain and the ␤-sandwich domain to prevent pre-stem rearrangement and membrane insertion. Among the four mutants, ␣HL-A is remarkably stable, is produced at a level at least 10-fold greater than that of the wild-type protein, is monomeric in aqueous solution, and has hemolytic activity that can be regulated by the presence or absence of reducing agents. Cross-linking analysis showed that ␣HL-A assembles on a membrane into an oligomer, which is likely to be a heptamer, in the absence of a reducing agent, suggesting that oxidized ␣HL-A is halted at a heptameric prepore state. Therefore, conformational rearrangements at positions 108 and 154 are critical for the completion of ␣HL assembly but are not essential for membrane binding or for formation of an oligomeric prepore intermediate.

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“…Not only is the apparent stability of the active CPAF dimers important for chlamydiae to be able to constantly modify host cells during chlamydial intravacuolar persistence, but it also helps CPAF avoid being processed by host proteasomes in the cytosol. Finally, many bacterium-secreted protein toxins, some of which have proteolytic activities, can undergo posttranslational modification, including dimerization or oligomerization, to become active (2,9,13,18). Although CPAF is not a toxin, chlamydiae may have preserved a parallel structure or function for interacting with host cells.…”

Section: Discussionmentioning

confidence: 99%

Intramolecular Dimerization Is Required for the Chlamydia-Secreted Protease CPAF To Degrade Host Transcriptional Factors

et al. 2004

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We previously identified a chlamydial protein designated CPAF (chlamydia protease/proteasome-like activity factor) that is secreted into host cell cytosol for degrading host transcription factors required for major histocompatibility complex antigen expression. Here we report that CPAF, synthesized as a 70-kDa proprotein, is processed into two fragments (designated CPAFn and CPAFc) to form intramolecular dimers that are much more stable than the naïve CPAF. Precipitation with antibodies that recognized CPAF dimers removed the proteolytic activity responsible for degrading host transcription factor RFX5 from chlamydia-infected host cell cytosol, while precipitation with antibodies that recognized free CPAF fragments alone did not remove this activity. Separation of CPAFn from CPAFc resulted in a loss of proteolytic activity. Furthermore, neither expressed full-length CPAF that was not processed nor coexpressed CPAFn and CPAFc fragments that failed to form dimers degraded RFX5. These observations demonstrate that intramolecular dimerization is required for CPAF to degrade host transcription factors, a strategy that is utilized by an obligate intracellular bacterial species to evade host defenses.

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Heptameric structures of two ?-hemolysin mutants imaged with in situ atomic force microscopy

Cited by 14 publications

References 14 publications

Ion channels and bacterial infection: the case of β‐barrel pore‐forming protein toxins of Staphylococcus aureus

Ion channels and bacterial infection: the case of β‐barrel pore‐forming protein toxins of Staphylococcus aureus

Arresting and releasing Staphylococcal α‐hemolysin at intermediate stages of pore formation by engineered disulfide bonds

Intramolecular Dimerization Is Required for the Chlamydia-Secreted Protease CPAF To Degrade Host Transcriptional Factors

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