Purification and characterization of IcmQ, IcmR and IcmR-IcmQ.
A. A domain diagram is shown for IcmQ.
B. (left) Purified IcmQ is shown on an SDS gel (lane 1). Crosslinking with DTSSP reveals an IcmQ dimer (lane 2). Trypsinization of IcmQ overnight at a 1:1000 ratio (wt/wt) generates Qn and Qc domains (lane 3). (right) A CD spectrum of IcmQ shows that the protein is mostly α-helical.
C. (left) Purified IcmR is shown in lane 1 and the moderately trypsin resitant product is shown in lane 2. (right) IcmR is strongly α-helical based on its CD spectrum.
D. (left) Purified IcmR-IcmQ complex is shown on an SDS gel (lane 1) and DTSSP crosslinking reveals a simple heterodimer (lane 2). (right) IcmR-IcmQ retains the α-helicity of its components, based on a CD spectrum.
E. Molecular weights of IcmQ, IcmQ-domains, IcmR and their complexes were determined on a calibrated Superose-12 HR column. A plot of the log of molecular mass versus elution position is shown for standards and the purified proteins (also see S_Table 1).

Characterization of the Qn, Qc and Qcl domains.
A. (left) A trypsinization time course is shown for Qn(1-66)-6xHis to form Qn(1-57) (lanes 1-3). Crosslinking with DTSSP shows that Qn is a dimer (lanes 4, 5). (right) Qn is mostly α-helical, based on a CD spectrum.
B. (left) Purified Qc is shown on an SDS gel. (right) The Qc domain is only partly-folded.
C. (left) Purified Qc(49/50-191) is shown on an SDS gel (lane 1) and cross-linking does not generate a visible product (lane 2). (right) A CD spectrum shows that Qcl is well-folded and strongly α-helical.

Purification and characterization of the Rm-Qn complex.
A. Domain diagrams are shown for IcmR and Qn and the interacting regions are shaded.
B. Purified IcmR-Qn(1-66)-6xHis complex is shown on an SDS gel (lane 1). Trypsinization generates the Rm-Qn complex (lane 2) and the similarly sized bands are shown in an expanded view on the right.
C. A CD spectrum indicates that the Rm-Qn complex is strongly α-helical.

The structure of an Rm-Qn four helix bundle.
A. A rotation series of the Rm-Qn 4-helix bundle is presented with the molecules displayed as ribbons. This structure is from the derivative crystals.
B. Non-polar side chains in the Rm-Qn interface are shown as solid spheres and are color coded for those from Qn (blue) and from Rm (tan).
C. Side chains in the Rm-Qn interface are shown as stick models with CPK colors and are labeled.
D. A top view is shown of the Rm-Qn 4-helix bundle with α-helices displayed as ribbons and side chains as CPK stick models. The figure was made with Chimera.

Structure-based sequence alignments of the interacting regions of IcmQ and IcmR.
A. A sequence alignment is shown for Qn domains from 11 representative Legionella species and from C. burnetti. Hydrophobic residues are highlighted in green. The Qn α-helices (in blue) are aligned below the sequences and hydrophobic residues in the Rm-Qn interface are marked with blue dots.
B. A sequence alignment is shown for the middle regions of 24 FIR proteins. The Rm α-helices (red) are aligned with IcmR and FIR sequences. Residues in the Rm-Qn interface are marked with blue dots. Hydrophobic residues in the Rm-Rm interface are marked with red dots, polar residues are marked with yellow dots.

The Qn α-helices are amphipathic and show a strong charge segregation.
A. Qn α-helices are shown in the context of the Rm-Qn dimer (left) and are dissected out from the helix bundle (center, right). The Qn α-helices are amphipathic with a hydrophobic face (green arrows) and charged faces (blue arrow for basic and red arrow for acidic).
B. Helix wheel diagrams for the two Qn α-helices show the charge segregation between the two α-helices.
C. A possible model is shown for the hypothetical Qn-Qn 4-helix bundle that may mediate IcmQ dimerization.

Mutations in Qn disrupt IcmQ function.
A. The stability of four IcmQ mutants expressed in L. pneumophila was ascertained by blotting with an antibody to the M45 epitope on the tagged IcmQ molecules. Cells infected with the vector without IcmQ (ΔIcmQ) did not express the protein, while the 4 mutants were made as full length proteins, albeit at lower levels than wild type IcmQ.
B. A plaque forming assay indicated that the Δ40-42 and [P25L, S29F] mutations in IcmQ inhibited growth of L. pneumophila in mouse macrophages.
C. In a DotA secretion assay, the protein was monitored in bacterial pellets and in the culture media by immuno-blotting. All 4 mutations in IcmQ prevented DotA secretion into the media, as this process is very sensitive to alterations in components of the T4bSS.

A model for IcmR and IcmQ function.
(top left) IcmQ dimers are formed by pairwise interactions between the Qn domains and the resulting interface may be either parallel or anti-parallel (not shown). IcmQ also aggregates in the absence of IcmR which may be mediated by Qn. (top middle) IcmR may form a dimer using interactions between Rm regions in the absence of IcmQ. (top right) IcmR interacts with the Qn domain to form a 4-helix bundle which “disrupts” the IcmQ dimer and also dis-aggregates IcmQ. (bottom right) Possible IcmQ binding partners could displace IcmR and would potentially be targeted to the cell membrane by the Qcl region. (bottom middle) IcmR does not completely block membrane association of IcmQ which is mediated by binding of Qcl to the bilayer surface. (bottom left) IcmR must be displaced from IcmQ to allow Qn to interact with the membrane.