The cytoplasm is the location of translation of all proteins. Papanikou et al. list the major tasks of the cell that are required for successful extracytoplasmic positioning of membrane proteins.[107] These tasks are to identify proteins that need to be exported and to keep polypeptide chains from folding. Further, the cell must eciently and accurately target proteins to, into and across the plasma membrane. This also includes the release of α-helical membrane proteins into the plasma membrane while translo-cating proteins destined for other locations, such as integral Omps, across the plasma membrane.
An important role in the targeting of non-cytoplasmic proteins to their destinations is played by the Sec translocation complex which acts as both, protein insertase and translocase. The SecYEG translocon is part of the Sec translocation complex whose task is insertion of α-helical proteins into the plasma membrane. In this process the subunit SecY forms a channel that can open laterally and release transmembrane re-gions of α-helical proteins into the membrane.[108] The insertion process requires the presence of hydrophobic transmembrane segments.[109]
2. CURRENT STATE OF RESEARCH 2.2. GRAM-NEGATIVE BACTERIA
Table 2.1: Selection of known Outer membrane proteins and their function.
Name Organism β-strands Function
OmpA E.coli 8 Porin, permeable for small solutes[91,92]
PagP E. coli 8 Palmityltransferase[68]
TtoA T. thermophilus 8 Unknown[93]
Nalp N. meningitidis 12 Autotransporter[94]
TbuX R. pickettii 14 Toluene transporter[95]
α-hemolysin S. aureus 2x7 Toxin[64]
BamA E. coli 16 Insertion of Omps into the Omp[96]
FhaC B. pertussis 16 Secretion of
lamentous hemagglutinin (FHC)[97]
OmpF E. coli 16 Porin, transport of
small hydrophilic molecules[98,99]
TtOmp85 T. thermophilus 16 Insertase[100]
MspA M. smegmatis 2x8 Porin[78]
VDAC1 M. musculus 19 Metabolite channel[57]
FhuA E. coli 22 Ferrichrome-iron receptor[70,71]
FepA E. coli 22 Ferric enterobactin receptor[101]
FecA E. coli 22 Ferric citrate transporter[73,102]
FptA P. aeruginosa 22 Ferric-pyochelin receptor[103]
PapC E. coli 24 Pilus assembly and secretion[13,104]
FimD E. coli 24 Pilus assembly and secretion[74]
LptD E. coli 26 Assembly of LPS into the outer
mem-brane[105,106]
2. CURRENT STATE OF RESEARCH 2.2. GRAM-NEGATIVE BACTERIA
Figure 2.4: The transport of unfolded outer membrane proteins (black) from the cytoplasm to the outer membrane. The signal sequence on the N-terminal end of the un-folded Omp is important for the transport of the Omp to the SecYEG translocon by the chaperone SecB and the ATPase SecA. The signal sequence is cleaved o in the periplasm by SPase. Chaperones like Skp, FkpA and SurA keep the Omp unfolded. An insertase (here: YaeT) is responsible for insertion of the client Omp into the OM. For further details see text. Adapted from[52,112]
Proteins that span the outer membrane do not possess hydrophobic stretches like inner membrane proteins[110] However, a highly hydrophobic N-terminal sequence of around 20 amino acids has been identied that is thought to act as a signal sequence for the transport of precursor Omps to the plasma membrane by the chaperone SecB and to the SecYEG translocon for subsequent transport through the plasma membrane into the periplasm. Cleavage of the N-terminal signal sequence by a leader peptidase follows translocation across the plasma membrane in the periplasm[111] (SPase in Figure 2.4).
The unfolded Omps cross the periplasm to the outer membrane. Periplasmic isomerases (SurA[113] and FkpA,[114] both also have chaperone function), proteases (DegS,[115]
DegP[116]) and chaperones (DegP[116]PpiD[117] and Skp[118]) are involved in the periplas-mic transport of the Omps to the outer membrane. These proteins function in absence of ATP which is not available in the periplasm. The production of these proteins is upregulated by the ρ(E) stress response system which is discussed in detail by Alba and Gross and by Barchinger and Ades.[119,120]
The insertion of Omps into the OM, once they have reached their destination, is still poorly understood. However, Omp folding and insertion into the OM must be tightly
2. CURRENT STATE OF RESEARCH 2.2. GRAM-NEGATIVE BACTERIA
controlled because a big increase of pores in the membrane would result in loss of pro-ton motor force and, thus, in cell death. It is known that most Omps need the help of insertases for ecient membrane insertion in vivo, due to the lipid composition of the outer membrane's inner leaet. No PC (phosphatidylcholine) is present. Instead, in case of E. coli for example, PE (phosphoethanolamine) and PG (phosphatidylglycerol) are found here. While PE acts as a strong inhibitor for Omp folding, the eect of PG can kinetically inhibit or promote Omp folding, depending on the individual Omp.[96]
The family of proteins involved in the insertion and secretion of substrates into and across the OM is classied as Omp85/BamA.[87,121] The members of this family share several common structures. These include a 16-stranded up-and-down transmembrane β-barrel, periplasmic polypeptide-transport-associated (POTRA) domains which are thought to interact with the unfolded target protein[86,122,123] and a lid-lock structure at the extracellular barrel opening.[124,125] Omp85 stands for Outer membrane protein 85 kD. Bam stands forβ-barrel assembly machine, and BamA is its central component, with accessory proteins BamB, BamC, BamD and BamE. TamA and TpsB are also among the members of this family. Tam stands for Translocation and assembly module and Tps for Two-partner-secretion. In the crystal structure the POTRA domains of two BamA molecules form dimers via β-augmentation.[126,127] Kim et al. report that BamA POTRA domains consist of a three-strandedβ-sheet and two α-helices.[126] De-lattre et al.[128] found that the two POTRA domains of FhaC, an outer membrane secretion protein of the Omp85 family from Bordetella pertussis, interact with the am-phipathic strands of its substrate, the adhesin FHA, via electrostatic and hydrophobic interactions. These interactions lead toβ-augmentation as aβ-sheet from the POTRA domain forms intermolecular bonds with the FHA-β helix. Upon introduction of cys-teine residues in the conserved FHA secretion domain and FhaC, crosslinks between FHA and the POTRA domains could be detected.[129]
Several hypothetic models propose TtOmp85-assisted folding and insertion mechanism of Omps into the outer membrane. One model assumes translocation of an unfolded Omp through the channel of Omp85. Insertion follows from the extracellular side of the outer membrane.[130] In the case of the secretory Omp85 FhaC in a lipid bilayer,
2. CURRENT STATE OF RESEARCH 2.2. GRAM-NEGATIVE BACTERIA
channels with a conductivity of 1200 pS were found, and topological rearrangements of FhaC in presence of FHA were observed.[131,132] The extended substrate FHA might pass through the pore of the FhaC transmembrane barrel.[121] Although FhaC is a translocase and not an insertase, similar mechanism might exist in case of Omp85 family members and their target Omps. Another model suggests that Omp85 destabilizes the lipid bilayer and primes it for spontaneous insertion of Omps. In E. coli, the exterior rim of the assembly machinery subunit BamA has a narrowed hydrophobic surface and causes local disturbances in the periplasmic layer of the outer membrane which might favor Omp insertion at the Omp85/membrane interface.[90,133135] Noinaj et al.
suggest that this mechanism is relevant for less complex Omps, which might fold before membrane insertion and subsequently insert via the Omp85/membrane interface.[133]
A third model pictures the Omp85 assembly machinery as a pore-forming multimer.
The target Omp might enter the pore and then move into the membrane through a lateral opening of the Omp85 pore.[121] A further model proposes that the formation of an Omp β-barrel takes place via a process called β-augmentation, which is the mechanism by which POTRA domains and unfolded substrates interact.[100,126] In this model, the Omp85 barrel opens laterally to form a gradually growing hybrid barrel with its substrate. Eventually, the newly formed barrel buds o from the Omp85 barrel. Estrada-Mallarino et al. performed black lipid membrane conductance studies of TtOmp85 in absence and presence of short peptides from the client Omp TtoA and found increased conductivity of TtOmp85 when peptide was present, indicating the presence of enlarged hybrid barrels.[100] Evidence for the insertion mechanism via β-augmentation was also found by Noinaj et al. who solved the crystal structure of BamA from N. gonorrhoeae and found a weak hydrogen bond link between strands one and sixteen of the transmembrane barrel[133]and an exit pore above this lateral opening site.[136] Molecular Dynamics simulations showed that this pore is opened by a ipping motion of loop 1 of the barrel, and cross-linking of the loop 1 with loop 6 or of strands one and sixteen both hindered bacterial growth.[136] The assumption is that barrel-strand 1 could be a nucleation template for nascent Omps. While transmembrane segments of a client Omp are inserted into the OM by lateral opening of the BamA
2. CURRENT STATE OF RESEARCH 2.2. GRAM-NEGATIVE BACTERIA
barrel the pore is an exit for extracellular loops and other structural elements. Noinaj et al. suggest that this mechanism applies for more complex client Omps, whereas simpler Omps insert into the OM at the Omp85/membrane interface.[133] The structure TamA, a close homolog of BamA, suggests a similar mechanism.[124] It should be mentioned, however, that the crystal structure from BamA of H. ducreyi does not show a weak link between two strands of the transmembrane barrel.[133] Dierent insertion mechanisms might apply to dierent members of the Omp85/BamA family. For an overview of the dierent folding and insertion models see.[121,130]
This thesis strives to conrm that, out of the existing models for Omp folding and inser-tion in the OM by Omp85, the most likely one for the pair TtOmp85/TtoA is the one that involves the transient formation of a hybrid barrel via β-augmentation.[124,133,137]
A schematic example of the process is depicted in Figure 2.5. For the insertion of the eight-stranded Omp TtoA from Thermus thermophilus via TtOmp85, Estrada-Mallarino et al. propose that unfolded TtoA is recognized by TtOmp85 via TtoA's C-terminal signal sequence, which is essential for all Omp85 substrates.[93,100,122] We assume that some TtoA β-structure forms and interacts with one of the six TtOmp85 POTRA domains and is thus kinetically able to insert between strands β1 and β16 of the TtOmp85 barrel. The interaction with the longer strand β1 will be stronger.
Here, subsequent TtoAβ-strands are transferred to the barrel. The POTRA-domain might help to slide the subsequent strands into the hybrid barrel[128] until a straining threshold is reached and the rest of the TtoA strands will insert spontaneously and bud o from the TtOmp85 barrel. This mode of interaction of TtOmp85 and TtoA via β-augmentation was tested before by conductance measurements in black lipid mem-brane experiments.[100] The study by Estrada-Mallarino et al. agrees that the TtoA barrel separates from the TtOmp85 barrel when the accumulating strain energy gets too high[100] Noinaj et al. propose that a separation occurs because the anity be-tween the rst and last barrel-strands of the newly formed client Omp is higher than the anity of these strands to the Omp85 host barrel.[136]
2. CURRENT STATE OF RESEARCH 2.2. GRAM-NEGATIVE BACTERIA
N
1 8 16
N C PO C
TRA-Domain
Figure 2.5: A hypothetical mechanism for the interaction of TtOmp85 and TtoA via β -augmentation. Unfolded TtoA (green, left panel) recognizes TtOmp85 POTRA domains (blue oblongs) via a signal sequence. The TtOmp85 barrel opens up between strands β1 and β16 and allows the formation of a hybrid barrel with the sequentially forming strands of TtoA. After the process is complete, TtoA buds o and inserts laterally into the membrane. Adapted from[100]