The Different Effects of Substrates and Nucleotides on the Complex Formation of ABC Transporters

Abstract

The molybdate importer (ModBC-A of Archaeoglobus fulgidus) and the vitamin B₁₂ importer (BtuCD-F of Escherichia coli) are members of the type I and type II ABC importer families. Here we study the influence of substrate and nucleotide binding on complex formation and stability. Using native mass spectrometry we show that the interaction between the periplasmic substrate-binding protein (SBP) ModA and the transporter ModBC is dependent upon binding of molybdate. By contrast, vitamin B₁₂ disrupts interactions between the transporter BtuCD and the SBP BtuF. Moreover, while ATP binds cooperatively to BtuCD-F, and acts synergistically with vitamin B₁₂ to destabilize the BtuCD-F complex, no effect is observed for ATP binding on the stability of ModBC-A. These observations not only highlight the ability of mass spectrometry to capture these importer-SBP complexes but allow us to add molecular detail to proposed transport mechanisms.

Keywords: ABC importers; ATP hydrolysis; BtuCD-F; ModBC-A; cooperativity; molybdate; native mass spectrometry; vitamin B(12).

Graphical abstract

Figure 1

Structures of BtuCD-F and ModBC-A and Their Respective Mass Spectra (A) X-ray crystal structures of BtuCD-F (left, PDB: 2QI9) and ModBC-A (right, PDB: 2ONK) visualized using PyMol (Schrödinger). (B) Mass spectrum of BtuCD-F reveals a charge state series consistent with the mass of the tetrameric transporter (BtuC₂D₂) bound to the SBP (BtuF) (BtuC₂D₂-F) a smaller population of uncomplexed transporter (BtuC₂D₂ red peaks) is also apparent. Different lipid species, primarily LPS, are bound to the transporter both in the presence and the absence of the SBP (light blue peaks). (C) Mass spectrum of ModBC-A shows a charge state series consistent with the tetrameric transporter bound to SBP and molybdate with different lipid-bound species primarily LPS (light blue peaks). Theoretical and observed molecular masses of all species are presented in Table 1.

Figure 2

BtuCD-F Is Destabilized by Vitamin B₁₂ and Cyanocobinamide (A) Mass spectrum of BtuCD-F recorded after addition of 2 mM vitamin B₁₂ to BtuF before buffer exchange. An increase in the intensity of the charge states corresponding to uncomplexed BtuCD (red peaks 45% with respect to BtuCD-F 100%) is observed in the presence of vitamin B₁₂ (purple peak) compared with in the absence of ligand (cf. Figure 1B). The inset shows the mass spectrum of BtuF-vitamin B₁₂ complex after addition of 2 mM vitamin B₁₂. (B) Mass spectrum of BtuCD-F after addition of 2 mM dicyanocobinamide to BtuF before buffer exchange. The ratio of BtuCD (red peaks 44% with respect to BtuCD-F 100%) is closely similar to the extent of complex formation in the presence of vitamin B₁₂. The inset shows the mass spectrum of BtuF-cyanocobinamide complex after addition of 2mM dicyanocobinamide. (C) Mass spectrum of BtuCD-F after addition of 2 mM vitamin B₉ to BtuF before buffer exchange. The ratio of BtuCD (red peaks 15% with respect to BtuCD-F 100%) in the presence of vitamin B₉ is closely similar to the extent of complex formation in the absence of ligand. Data are represented as means ± SD (n = 3). (D) Chemical structures of the molecules used for the binding experiments.

Figure 3

Effect of Nucleotides on BtuCD-F and ModBC-A Complex Formation (A) Deconvoluted masses of the spectrum of BtuCD-F recorded after addition of 5 mM ATP to BtuCD before buffer exchange. The presence of ADP-bound species of both BtuCD-F and BtuCD are observed with similar ratios. The relative abundance of BtuCD in this spectrum is 40% of the BtuCD-F signal. (B) Deconvoluted masses of the spectrum of BtuCD-F recorded after addition of 5 mM AMP-PNP to BtuCD before buffer exchange. Also in this case, AMP-PNP-bound species are detected. The relative abundance of BtuCD in this spectrum is 43% of the BtuCD-F signal. (C) Deconvoluted masses of the spectrum of ModBC-A recorded following the addition of 10 μM ATP to ModBC after buffer exchange. ModBC-A species bound to zero, one and two ADP molecules are observed and no ModBC alone is detected.

Figure 4

Transport Models for Type I Importer ModBC-A and TYPE II IMPorter BtuCD-F (A) Transport model for ModBC-A. The formation of the complex in the absence of molybdate is unfavorable (state I). Binding of molybdate triggers the docking of ModA onto ModBC (state II). Following a rearrangement of the TMDs, molybdate moves toward the SBS (state III). ATP binding and hydrolysis triggers a further conformational change that translocate the substrate into the cytoplasm (state IV). The release of ADP resets the transporter to the resting state (state I). (B) Transport model for BtuCD-F. BtuCD and BtuF form a stable complex even in the absence of vitamin B₁₂ (state I). ATP binds cooperatively, leading to a partial displacement of BtuF from BtuCD (state II). When vitamin B₁₂ is available, a transient BtuCD-F⋅2ADP⋅B₁₂ complex is formed (state III). This unstable conformation is relaxed through ADP and Pi dissociation, followed by vitamin B₁₂ release (state IV) leading to the resetting of the transporter.