1. Distinct Structural and Functional Properties of the ATPase Sites in an Asymmetric ABC Transporter 
Erik Procko, Ian Ferrin-O'Connell, Sze-Ling Ng, Rachelle Gaudet 
Molecular Cell 
Volume 24, Issue 1, Pages (October 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 Construct Design and ATPase Activity
(A) Important TAP1 and TAP2 motifs are aligned with ABC transporter consensus sequences (x, any amino acid; Φ, hydrophobic). Deviations from consensus are shaded.
(B) Schematic diagrams of different TAP-NBD dimers. The TAP1 (white)/TAP2 (gray) heterodimer has a consensus (lower) and degenerate (upper) active site. The TAP1 homodimer has hybrid sites and was mutated to provide constructs with consensus or degenerate sites.
(C) ATPase activity was measured for the listed NBD constructs using a coupled-reaction assay at room temperature. Values are the mean ± SD from three recordings.
(D) Graphical representation of ATPase activity ± SD at 1.0 mM ATP.
(E) The ADP and ATP affinities of TAP-NBD mutants were qualitatively compared by nucleotide-agarose precipitation. Bound protein was detected on Coomassie-stained SDS gels.
Molecular Cell  , 51-62DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

3. Figure 2 NBD Dimerization Properties
TAP-NBDs with 1 mM ATP were injected (250 μl at 0.4–0.7 mM) onto a S200 16/60 size exclusion chromatography column and eluted in the presence of 1 mM ADP or ATP. The peak UV absorbance (280 nm) values have been scaled for clarity. For reference, vertical dashed and solid gray lines are overlaid on the TAP1-NBD plots to indicate WT-TAP1-NBD elution volumes in the presence of ADP and ATP, respectively. Individual panels are referred to in the text. Data are representative of two to five experiments.
Molecular Cell  , 51-62DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

4. Figure 3 Crystal Structures of Three TAP1-NBD Constructs with ATP
(A) TAP1-NBD D→N·ATP is a dimer with two ATP-Mg2+ at the interface. The two NBDs, colored light and dark blue, are viewed from the TMDs looking down onto the NBDs. Important functional motifs are highlighted in one active site.
(B) NBDs from the three structures were superimposed via their ATPase subdomains (lighter shades). This demonstrates rigid-body motions of the helical subdomain (darker shades).
(C and D) σA-weighted 2Fo–Fc map, contoured at 1.3 σ, for the consensus (TAP1-NBD D→Q/Q→H) (C) and hybrid (TAP1-NBD D→N) (D) active sites. The putative hydrolytic water is labeled.
(E) The degenerate TAP1-NBD SG→AV/D→N signature motif structure (magenta) superimposed onto the consensus active site (green). Polar contacts from S621 of the consensus signature motif are shown.
(F) Stereoview of the superposition in (E), zooming in on the two residues that differ between the consensus (green) and degenerate (magenta) signature motifs. The van der Waals radii of V622 and a Mg2+-coordinated water are shown with a dotted surface, demonstrating a steric clash.
Molecular Cell  , 51-62DOI: ( /j.molcel )
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5. Figure 4 Peptide Transport Activity of Full-Length TAP Mutants
(A) Sf21 cells were coinfected with baculovirus encoding both TAP subunits and baculovirus encoding HLA-B27 with β2-microglobulin. Surface class I MHC was measured by flow cytometry and normalized against the mean fluorescence of wild-type TAP-expressing cells. Graphed data are the average ± SD from three separate experiments. Above the graph are representative Western blots showing equivalent protein expression levels of TAP2 and MHC heavy chain (gel lanes match the graph). Numbers above the lanes are referred to in the text.
(B) The level of HLA-B27 surface expression was compared in the absence (gray bars) or presence (black bars) of tapasin. Data are normalized against the maximum recorded mean fluorescence, and the average ± SD from three experiments is shown.
(C) Same as (B), except that the class I MHC allele was HLA-B44.
Molecular Cell  , 51-62DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

6. Figure 5 Position of the Consensus Site Is Not Critical
A TAP mutant with swapped ATPase motifs was compared to wild-type, consensus, and degenerate TAP in an insect-cell-based class I (HLA-B27) loading assay. Representative flow cytometry plots and western blots are shown from three experiments.
Molecular Cell  , 51-62DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

7. Figure 6 Model of ATP-Dependent Peptide Transport
Two opposing models are discussed. In model 1, the preferred model, peptide binding stimulates a conformational change in TAP2-NBD, facilitating ATP binding and NBD dimerization. This is coupled to peptide transport. In model 2, the NBDs have instrinsic ability to form an ATP-dependent dimer, and peptide binding stimulates ATP hydrolysis and NBD dissociation, which drives peptide translocation. TAP1 is green, TAP2 is blue, and peptide is red.
Molecular Cell  , 51-62DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions