Substrate-Induced Conformational Changes of the Perplasmic N-Terminus of an Outer-Membrane Transporter by Site-Directed Spin Labeling

Abstract

The structure and dynamics of the N-terminal and core regions of BtuB, an outer membrane vitamin B(12) transporter from Escherichia coli, were investigated by site-directed spin labeling. Cysteine mutants were generated by site-directed mutagenesis to place spin labels in the N-terminal region (residues 1-17), the core region (residues 25-30), and double labels into the Ton box (residues 6-12). BtuB mutants were expressed, spin labeled, purified, and reconstituted into phosphatidylcholine. In the presence of substrate (vitamin B(12)), EPR spectroscopy demonstrates that there is a conformational change in the Ton box similar to that seen previously for BtuB in intact outer membranes. The Ton box is positioned within the beta-barrel of BtuB in the absence of substrate (docked configuration) but becomes unfolded and increases its aqueous exposure upon substrate binding (undocked configuration). This conformational change and the similarity in the EPR spectra between reconstituted and native membranes indicate that BtuB is correctly folded and functional in the reconstituted system. The protein segment on the N-terminal side of the Ton box is highly mobile, and it becomes more mobile in the presence of substrate. Side chains in the region C-terminal to the Ton box also show increases in mobility with substrate addition, but position 16 appears to define a hinge point for this conformation change. EPR line shapes and relaxation data indicate that residues 25-30 form a beta-strand structure, which is analogous to the first beta-strand in the cores of the homologous iron transporters. When substrate binds to BtuB, this first beta-strand remains folded. The EPR spectra of double-nitroxide labels within the Ton box are broadened because of dipolar and collisional exchange interactions. The broadening pattern indicates that the Ton box is not helical but is in an extended or beta-strand structure.

Osmolytes modulate conformational exchange in solvent‐exposed regions of membrane proteins

“…The X-band EPR spectra from spin labels placed at positions [1][2][3][4][5] in BtuB are shown in Figure 3, along with the position of these sites in the BtuB meso structure. These spectra are similar to those published previously, 22 and are composed of two components resulting from populations of spin labels that have different dynamics. The mobile component (labeled ''m'') in these spectra is similar to that expected for R1 attached to an unstructured protein segment; and as a result, it was concluded previously that this segment was flexible.…”

“…16,22 As indicated earlier, this transition is modulated by osmolytes. Several other sites within BtuB also yield EPR spectra that are dependent on substrate addition.…”

“…An earlier report on BtuB using SDSL indicated that the N-terminal segment (residues 1-5) was dynamic, 22 which is apparently inconsistent with the mesophase crystal structure. 15 However, while the mobile component in these spectra cannot be reconciled with this structure, the immobilized components in these EPR spectra ( Fig.…”

“…The mobile component (labeled ''m'') in these spectra is similar to that expected for R1 attached to an unstructured protein segment; and as a result, it was concluded previously that this segment was flexible. 22 However, the spectra also contain a component resulting from an immobilized nitroxide in tertiary contact with the protein (labeled ''i''). For the nitroxide label attached to the very N-terminus of BtuB (Q1R1), the difference between the immobile and mobile components is pronounced, and the immobile component is near the rigid limit of nitroxide motion at X-band (correlation times longer than 30-50 ns).…”

Substrate-dependent transmembrane signaling in TonB-dependent transporters is not conserved

“…These proteins have homologous structures based on a 22-stranded ␤-barrel, extracellular binding loops, and an N-terminal region that is folded into the barrel. Substrate binding occurs in the extracellular binding loops and triggers conformational changes in the N-terminal region near the periplasmic surface, thus transducing a signal across the outer membrane (11).…”