Signaling mechanisms for activation of extracytoplasmic function (ECF) sigma factors

Abstract

A variety of mechanisms are used to signal extracytoplasmic conditions to the cytoplasm. These mechanisms activate extracytoplasmic function (ECF) sigma factors which recruit RNA-polymerase to specific genes in order to express appropriate proteins in response to the changing environment. The two best understood ECF signaling pathways regulate sigma(E)-mediated expression of periplasmic stress response genes in Escherichia coli and FecI-mediated expression of iron-citrate transport genes in E. coli. Homologues from other Gram-negative bacteria suggest that these two signaling mechanisms and variations on these mechanisms may be the general schemes by which ECF sigma factors are regulated in Gram-negative bacteria.

Figure 1

Mechanism of σ^E activation. 1. The PDZ domain of DegS recognizes the CTD of an unfolded outer membrane protein. 2. The activated DegS protease domain cleaves RseA at Site 1, thus relieving inhibition imposed by RseB on RseP activity through the glutamine rich domains of RseA and the PDZ domain of RseP. 3. RseP acts as RIP protease and cuts RseA near the cytosolic side of the membrane (Site 2). 4. ClpXP and other ATP driven proteases remove RseA from σ^E. 5. σ^E binds the β′ subunit of RNA polymerase and directs it to the appropriate promoter sites. Figures 1, 2, 7, and 8 were prepared with Adobe Illustrator.

Figure 2

Comparison of the domain structures of ECF (Group IV) sigma factors and Group I sigma factors. The 1.1 region, non-conserved region (NCR), and much of σ₃ are missing form ECF sigma factors. The ECF sigma factors only have two globular domains compared to the three of Group I sigma factors [121, 39].

Figure 3

X-ray crystal structure illustrating the interaction between RseA (yellow) and σ^E (blue (σ₂) and red (σ₄)) [39]. RseA helices α3 and α4 clash with portions of RNA polymerase to inhibit binding to σ^E. Figures 3, 4, 5, 6, 9, and 10 were prepared with PYMOL [129].

Figure 4

Crystal structure of DegS with OmpC_CTD peptide [52]. A subunit of the DegS trimer is shown in purple (protease domain) and light blue (PDZ domain). The interacting OmpC_CTD is shown in orange. The remaining subunits are shown in green.

Figure 5

Illustration of loop movements that cause protease domain activation on the binding of a peptide mimicking the OmpC C-terminus (orange). DegS (apo DegS in yellow and peptide bound DegS in green) loops 2 and 3 move towards the protease active site serine (S201) on ligand binding. These movements cause a number of changes in other loops including L1 and LD. The resultant changes remove obstructing residues from the substrate binding pocket and improve the geometry of the active site residues.

Figure 6

Comparison of the two states of DegS to β-trypsin with a monoisopropyl phosphate (MIP) modified active site serine. The modifying moiety occupies the substrate binding site and catalytic site. A. Leucine 218 blocks the substrate binding site in the Apo DegS (pink) structure as shown by clashing with the MIP moiety on β-trypsin (yellow with black text). The carbonyl of DegS (pink) His198 points into the active site as compared to the N192 carbonyl of β-trypsin that is in a catalytically active position. B. When DegS has bound an OmpC C-terminal peptide, L218 no longer interferes with substrate binding and the carbonyl of H198 is in a catalytically active orientation.

Figure 7

Cartoon representation of RseP. The periplasmic PDZ domain plays a role in the regulation of the activity of the protease. It may bind a glutamate rich region of RseA. The HEXXH and LDG residues are part of the conserved metalloprotease active site.

Figure 8

Illustration of the FecARI signaling mechanism. Iron-citrate binding to FecA at the outer membrane allows TonB to recognize the TonB box region of FecA. TonB is required for signaling the presence of iron-citrate sensed by FecA to FecR in the inner membrane. Activated FecR in turn activates FecI, which is bound to the N-terminus of FecR. RNA polymerase is then recruited to the FecA promoter after binding activated FecI.

Figure 9

X-ray crystal structures of FecA with and without iron-citrate indicate a path of structural change which may signal the presence of iron-citrate [90, 91]. The β-barrel is shown in grey and is transparent in the front. The plug domain is shown in darker grey. Changes in loop movements are indicated by the differences in the loop structures of the apo (blue) and iron citrate bound (magenta) FecA crystal structures.

Figure 10

NMR structure of the signaling domain of the N-terminus of FecA [92, 93]. Mutagenesis indicates that interaction with FecR is likely to occur at the shallow binding site proximal to the N-terminus and helices α1 and α3 (Site 1) or along the β3 (Site 2). However, some residues that are outside of these regions, such as T70, are important for activity.