Structural analysis of substrate binding by the TatBC component of the twin-arginine protein transport system

Abstract

The Tat system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. In Escherichia coli substrate proteins initially bind to the integral membrane TatBC complex which then recruits the protein TatA to effect translocation. Overproduction of TatBC and the substrate protein SufI in the absence of TatA led to the accumulation of TatBC-SufI complexes that could be purified using an affinity tag on the substrate. Three-dimensional structures of the TatBC-SufI complexes and unliganded TatBC were obtained by single-particle electron microscopy and random conical tilt reconstruction. Comparison of the structures shows that substrate molecules bind on the periphery of the TatBC complex and that substrate binding causes a significant reduction in diameter of the TatBC part of the complex. Although the TatBC complex contains multiple copies of the signal peptide-binding TatC protomer, purified TatBC-SufI complexes contain only 1 or 2 SufI molecules. Where 2 substrates are present in the TatBC-SufI complex, they are bound at adjacent sites. These observations imply that only certain TatC protomers within the complex interact with substrate or that there is a negative cooperativity of substrate binding. Similar TatBC-substrate complexes can be generated by an alternative in vitro reconstitution method and using a different substrate protein.

Fig. 1.

Purification of a complex between TatBC and the Tat substrate SufI. TatBC and a modified version of the SufI precursor protein possessing a C-terminal hexa-histidine tag were coordinately overexpressed in the ΔtatABCDΔtatE strain DADE. Membranes were isolated, solubilized in digitonin, and subjected to Ni²⁺-affinity chromatography. The peak TatBC-containing fractions from the Ni²⁺ affinity column were pooled, concentrated, and applied to a size exclusion chromatography column. (Top) The 280 nm absorbance profile of the eluant. The void volume (V₀) and elution positions of water soluble standard proteins are indicated below the profile. (Bottom) The proteins present in successive indicated fractions eluting from the size exclusion column are analyzed by SDS/PAGE and Coomassie Blue staining. The molecular masses in kDa of standard proteins are given to the left of each gel and the positions of SufI, TatB and TatC to the right of each gel. SufI degradation products are indicated by (*).

Fig. 2.

Biochemical analysis of purified TatBC-substrate complexes. (A) Blue native-PAGE analysis of the TatBC-SufI_His preparation and of purified TatBC_His. (B) The TatBC-SufI_His preparation was applied to a Ni²⁺-affinity chromatography column and bound proteins eluted with washes at 150 mM and 300 mM imidazole. SDS/PAGE analysis of proteins that did not bind to the column (FT) and of bound proteins that were eluted from the column by the 150 mM and 300 mM imidazole steps. (C) Blue native-PAGE analysis of the TatBC-SufI_His preparation that was applied to the Ni²⁺ affinity column (Applied) and the protein that did not bind to the column (FT). (D) Blue native-PAGE analysis of the TatBC-SufI_His preparation that was applied to the Ni²⁺ affinity column (Applied) and of the bound proteins eluted from the column by the 150 mM and 300 mM imidazole steps. (E) SDS/PAGE analysis of the TatBC-CueO_His preparation. (F) Bue native-PAGE analysis of the TatBC-CueO_His preparation and purified TatBC_His. All gels were stained with Coomassie Brilliant Blue. The molecular masses (kDa) of standard proteins are given to the left of each gel. Protein complexes resolved by blue native-PAGE are labeled by molecular mass (kDa) to the right of the gels.

Fig. 3.

Class averages of the TatBC complex with and without bound substrate. Class averages obtained by multireference alignment of the untilted images of TatBC_His, TatBC-SufI_His, and TatBC-CueO_His samples. (A) The small TatBC_His complex. (B) The large TatBC_His complex. (C) The TatBC complex with no SufI_His bound. (D) The TatBC complex with 1 SufI_His bound. (E) The TatBC complex with 2 SufI_His bound. (F) The TatBC complex with 1 CueO_His bound. There were 15–25 images per class in each case. (Scale bar, 10 nm.)

Fig. 4.

3D maps of TatBC_His and TatBC-SufI_His complexes. (A) The small TatBC_His complex (light blue). (B) The large TatBC_His complex (blue). (C) TatBC (green) with 1 SufI_His (yellow) bound. (D) TatBC (green) with 2 SufI_His (yellow) bound. Each map is shown in 2 orientations related by a 90 ° rotation about the horizontal axis. All maps are shown in the same orientation relative to the support film which is at the bottom in the right hand views. The gray box in A shows the possible location of the membrane. The X-ray structure of SufI (yellow) is fitted into the 3-D maps (gray) for the TatBC complex with 1 (E) or 2 (F) bound SufI_His molecules. (G) From left to right, views of the small and large TatBC_His complexes, and the TatBC complex with 1 and 2 bound SufI_His molecules with the front surface cut away to reveal the internal cavity. The complexes are shown in the same orientation as the right hand views in panels (A–D). The map surfaces are shown as wire mesh with the back surface represented in darker colors. (Scale bars, 10 nm; larger scale bar applies to E and F only.)