Fig 1.
Group four capsule operon and GfcD.
(a) The Escherichia coli group 4 operon consists of seven genes. The genes gfcA, gfcB, gfcC, and gfcD encode proteins of an unknown function. Genes gfcE, etp, and etk share homology with proteins of Group 1 capsule export systems. Distant homologs are also found in Vibrio and Burkholderia species. (b) Membrane topology of GfcD as predicted by HHOMP and classified as a potential outer membrane β-barrel protein. The central region (273–427) is devoid of transmembrane strands and may be an independently folded domain with the predicted secondary structure shown. The signal sequence at the N-terminus (shaded blue) shows a putative signal peptidase II cleavage and acylation site (arrow 19) as previously annotated, and the peptidase I cleavage site (arrow 22) revealed in the present work by MALDI-TOF and N-terminal sequencing.
Fig 2.
(a) SDS-PAGE gel showing the purification of GfcD, encoded from pBH31 vector. The lysates (lane 1) were separated by ultracentrifugation into soluble (lane 2) and membrane (lane 3) fractions. Lane 4 is the molecular weight ladder (Invitrogen Benchmark). After solubilizing GfcD with 5% Elugent, it was affinity purified on TALON (lane 5). His-tag was removed by TEV cleavage (lane 6), and then GfcD was purified by ionic exchange (lane 7) followed by size exclusion purification (lane 8). Boiling the sample results in a migration shift (native vs. denatured) common to β-barrel proteins upon unfolding (lane 9). (b) Circular dichroism spectra of GfcD protein compared against the known β-barrel protein OmpW. (c) The GfcD protein was exchanged into a buffer of 20mM Hepes pH 7, 150mM NaCl, 0.02% DDM over a Superdex 200 10/300 size exclusion column and demonstrated a symmetrical peak.
Table 1.
Secondary structure of GfcD.
Fig 3.
Localization and protein interactions of GfcD.
GfcD-6His dominantly localizes to outer membrane fractions. (a) NADH oxidase activity was measured for each fraction and (b) fractions were blotted for known markers of the inner (Etk) and outer membrane (OmpA) and the His-tag of GfcD to define inner and outer membrane fractions (original blot scans in S1 Raw images). (c) GfcB-His6, GfcC, and GfcD were co-expressed in EPEC cells and pulled-down with NiNTA beads. GfcC and GfcD proteins were detected on Western blots. Right, Absence of a His-tagged GfcB results in loss of pull-down of GfcC or GfcD. Abbreviations: Mem: membrane fraction; W4: fourth wash of pull-down, Elute: pull-down product. (d) Over-expressing GfcC or GfcD alone inhibits cell growth of EPEC; no inhibition is observed during co-expression of GfcB, GfcC, and GfcD (BCD). (e) Anti-GfcD blot comparing levels of GfcD when expressed alone or with GfcB and GfcC. Full length GfcD is at 60 kDa on this blot. GfcD rapidly undergoes proteolysis when expressed alone.
Fig 4.
GfcB and GfcC form a tight complex.
(a) Isothermal calorimetry. 24 injections of GfcB into GfcC at 25°C were measured (upper) and the heat released (μJ) at each injection was subtracted by the heats released for the control injections in buffer (middle) and were fit (lower). GfcB-GfcC have a 1:1 interaction with KD of 0.1 μM in solution. (b) Biotinylated GfcB was anchored to an SA (streptavidin) sensor and the real-time binding response of GfcC at concentrations of 200 nM to 1.6 μM were measured by BLI. (c) Biotinylated GfcC on the sensor and binding of GfcB (with C-terminal His6-tag expressed from pET-BLUE2) at concentrations of 100 nM to 1.6 μM were measured. The BLI data in (b) and (c) were fit with either a one-site binding model (top) or one-site binding followed by conformation change model (bottom).
Table 2.
Affinities of interactions of GfcB, GfcC, and GfcD.
Fig 5.
Interaction of GfcD with GfcB and GfcC.
(a) Purified GfcB-6XHis, GfcC, and/or GfcD were cross-linked by DSP and run on SDS-PAGE. Western blotting identified 150 kDa complexes of GfcC-GfcD (lane 4), 50 kDa heterodimers with GfcB-GfcC (lane 6) and large complexes of GfcB, GfcC, and GfcD (lane 7). (b) Isothermal titration calorimetry of GfcBC into GfcD measured the heats of each injection (top) which were subtracted by heats of control injections of GfcBC into buffer (middle) and fit (bottom) to measure the affinity near 28 μM KD for GfcBC. (c) BLI for the interactions of immobilized biotinylated GfcD with GfcB, GfcC, or the GfcBC complex at concentrations from 3.75–60 μM. Traces for GfcD-GfcB and GfcD-BC are a moving average over 20 data points to aid comparison against the fit.
Fig 6.
Possible scheme for the formation of the GfcB-GfcC heterodimer and complex with GfcD.
The lipoprotein GfcB (possibly a dimer based on PDB entry 2IN5 and the crosslinking experiment in Fig 5A) is anchored to the inner leaflet of the outer membrane and forms a heterodimer with GfcC before binding to GfcD as suggested by the experiment shown in Fig 5A and the relative binding affinities to GfcD (Fig 5C). Domain labeled p is the predicted periplasmic domain of GfcD (see also S3 Fig) which may make interactions with GfcC and/or GfcB, and/or gate the opening of the GfcD barrel.