Conformational dynamics of the nucleotide binding domains and the power stroke of a heterodimeric ABC transporter
- Vanderbilt University, United States
- University of Illinois, United States
- Center for Biophysics and Computational Biology, University of Illinois, United States
- The Beckman Institute for Advanced Science and Technology, University of Illinois, Unites States
Figures
Figure 1 with 5 supplements
Conformational states of BmrCD NBDs.
(A) Ribbon representation of BmrCD homology model showing the spin label pairs at symmetric sites across the NBD dimer interface (the transporter is rotated 90° and NBD interface is viewed from …
Figure 1—figure supplement 1
Sequence alignments.
(A) Sequence alignment of BmrC with TM287. (B) Sequence alignment of BmrD with TM288. These sequence alignments were used as input for BmrCD homology modeling. (C) Multiple sequence alignment to …
Figure 1—figure supplement 2
The ATPase activity of BmrCD-WT (wild type BmrCD), BmrCD-WT* (Cysteine-less BmrCD) and its spin-labeled mutants is stimulated by Hoechst33342 (hereafter referred as Hoechst) (10 µM) and inhibited by vanadate (1 mM).
(A) Substitution of the native cysteines with alanines marginally reduces the Vmax of ATP turnover. The solid line is a non-linear least-squares fit that yields Vmax (48.52 ± 1.85 nmol/min/mg), Km …
Figure 1—figure supplement 3
Predicted distance distributions between spin-labeled pairs monitoring the NBD interface.
The distributions were calculated using the MMM package (Polyhach et al., 2011) for the BmrCD homology model and nucleotide-bound TM287/TM288 crystal structure.
Figure 1—figure supplement 4
CW-EPR and DEER data analysis for spin-labeled pairs in the NBDs.
(A) Close up view of BmrCD homology model highlighting the location of the spin-labeled pairs in the NBDs. (B) Superposition of the CW-EPR spectra demonstrates minimal changes in the lineshape, and …
Figure 1—figure supplement 5
Conformational states of MsbA NBDs.
(A) Ribbon representation of MsbA (PDB:3B60) showing the spin label pair 561 at symmetric sites across the NBD dimer interface (the transporter is turned 90° and NBD interface is viewed from …
Figure 2 with 1 supplement
Structural asymmetry of the NBSs.
(A–C) Close up side view of the NBD dimer and distance distributions for spin label pairs monitoring the consensus NBS. The overlapping distributions in the AMP-PNP bound and ADP-Vi trapped …
Figure 2—figure supplement 1
DEER data analysis for spin-labeled pairs in the NBSs.
Close up view of BmrCD homology model highlighting the location of the pairs (A) (440/441), (B) (348/532) along with the baseline-corrected DEER decays and the corresponding distance distributions. …
Figure 3 with 1 supplement
The inward- to outward-facing transition of the TMD requires ATP hydrolysis.
(A) Ribbon representation of the BmrCD homology model showing the spin-labeled pairs designed to monitor the conformation of the TMD. Residue 147 is shown since 146 was not included in the homology …
Figure 3—figure supplement 1
CW-EPR and DEER data analysis for spin-labeled pairs in the TMDs.
(A) Close up view of BmrCD homology model highlighting the location of the spin-labeled pairs at the extracellular (upper panel) and intracellular (lower panel) side. * residue 146 was not modeled …
Figure 4 with 4 supplements
Conformational states of BmrCD in lipid bilayers.
(A) Close up view from cytoplasmic side for spin label pairs monitoring the NBD dimer, the consensus NBS and the degenerate NBS along with the corresponding distance distributions. (B) Close up view …
Figure 4—figure supplement 1
Reconstitution of BmrCD-WT* (Cysteine-less BmrCD) and its spin-labeled mutants in PC/PA nanodiscs.
(A) Size-exclusion chromatography of BmrCD in nanodiscs and a cartoon depicting BmrCD-WT* assembly in nanodiscs. Similar size-exclusion chromatographs were obtained for spin-labeled mutants. (B) …
Figure 4—figure supplement 2
The ATPase activity of BmrCD-WT (wild type BmrCD), BmrCD-WT* (Cysteine-less BmrCD) and its spin-labeled mutants in PC/PA nanodiscs is stimulated by Hoechst (10 µM) and inhibited by vanadate (5 mM).
(A) Reconstitution of BmrCD-WT, BmrCD-WT* and its spin-labeled mutants in PC/PA nanodiscs stimulates the basal ATPase activity. The solid line is a non-linear least-squares fit that yields Vmax …
Figure 4—figure supplement 3
CW-EPR and DEER data analysis for BmrCD spin-labeled pairs reconstituted in nanodiscs.
(A) Close up view of BmrCD homology model highlighting the location of the spin-labeled pairs in the NBDs and NBSs. (B) Superposition of the CW-EPR spectra demonstrates minimal changes in the …
Figure 4—figure supplement 4
CW-EPR and DEER data analysis for BmrCD spin-labeled pairs reconstituted in nanodiscs.
(A) Close up view of BmrCD homology model highlighting the location of the spin-labeled pairs at the extracellular (upper panel) and intracellular (lower panel) side. * residue 146 was not modeled …
Figure 5 with 1 supplement
Structural asymmetry of the NBSs under turnover conditions.
(A) Ribbon diagram of BmrCD NBDs showing the location of spin label pairs monitoring the consensus and the degenerate NBS (the NBD dimer is viewed from the cytoplasm, along the membrane normal). …
Figure 5—figure supplement 1
DEER data analysis for spin-labeled pairs in the NBSs under turnover conditions.
Panels from left to right: location of spin-labeled pairs on BmrCD homology model, baseline-corrected DEER decays along with the fits and the resulting distance distributions.
Figure 6 with 1 supplement
In the presence of excess ATP, the inward-facing state of BmrCD is favored.
Distance distributions of spin label pairs monitoring the (A) extracellular and (B) intracellular side demonstrate that in the presence of 10 mM ATP the predominant population is inward-facing.
Figure 6—figure supplement 1
DEER data analysis for spin-labeled pairs in the TMDs under turnover conditions.
Panels from left to right: location of spin-labeled pairs on BmrCD homology model, baseline-corrected DEER decays along with the fits and the resulting distance distributions.
Figure 7 with 2 supplements
MsbA conformational dynamics under turnover conditions reveal a two-state equilibrium.
(A) Ribbon representation of the AMP-PNP structure of MsbA (PDB:3B60) showing the spin-labeled pairs. (B) Binding of ATP induces the formation of the closed NBD dimer and concomitantly drives the …
Figure 7—figure supplement 1
ATPase activity of MsbA.
(A) Hoechst33342 (hereafter referred to as Hoechst) does not stimulate, but vanadate inhibits the ATPase activity of cysteine-less MsbA (MsbA-WT*) and (B) spin label mutants. ATPase activity of …
Figure 7—figure supplement 2
DEER data analysis for spin-labeled pairs.
(A) Ribbon representation of the TMDs and NBDs of MsbA showing the spin label pairs (B) CW-EPR spectra (C) baseline-corrected DEER signals along with the fits and (D) the resulting distance …
Figure 8
Distinct mechanisms of ABC heterodimers and homodimers.
(A) Asymmetric hydrolysis of ATP in an ABC heterodimer. The conformation of the transporter and a top view of the NBD dimer are shown for each catalytic state. Substrate binding can precede or …
