Structural Dynamics of the Heterodimeric ABC Transporter TM287/288 Induced by ATP and Substrate Binding

TM287/288 is a heterodimeric ATP-binding cassette (ABC) transporter, which harnesses the energy of ATP binding and hydrolysis at the nucleotide-binding domains (NBDs) to transport a wide variety of molecules through the transmembrane domains (TMDs) by alternating inward- and outward-facing conformations. Here, we conducted multiple 100 ns molecular dynamics simulations of TM287/288 in different ATP- and substrate-bound states to elucidate the effects of ATP and substrate binding. As a result, the binding of two ATP molecules to the NBDs induced the formation of the consensus ATP-binding pocket (ABP2) or the NBD dimerization, whereas these processes did not occur in the presence of a single ATP molecule or when the protein was in its apo state. Moreover, binding of the substrate to the TMDs enhanced the formation of ABP2 through allosteric TMD–NBD communication. Furthermore, in the apo state, α-helical subdomains of the NBDs approached each other, acquiring a conformation with core half-pockets exposed to the solvent, appropriate for ATP binding. We propose a “core-exposed” model for this novel conformation found in the apo state of ABC transporters. These findings provide important insights into the structural dynamics of ABC transporters

Helical Foldamer Containing a Combination of Cyclopentane-1,2-diamine and 2,2-Dimethylmalonic Acid

We have developed new helical oligomers using a combination of (1<i>S</i>,2<i>S</i>)-cyclopentane-1,2-diamine [(<i>S</i>,<i>S</i>)-CPDA] and 2,2-dimethylmalonic acid (DMM) residues as building blocks. In solution, the preferred secondary structure of the (<i>S</i>,<i>S</i>) tetramer <b>6</b> was a right-handed (<i>P</i>) helix, and that of the (<i>R</i>,<i>R</i>) tetramer <i>ent</i>-<b>6</b> was a left-handed (<i>M</i>) helix. In the crystalline state, both <b>6</b> and the (<i>S</i>,<i>S</i>) pentamer <b>7</b> folded into (<i>P</i>) 11-helices, and <i>ent</i>-<b>6</b> folded into an (<i>M</i>) 11-helix with hydrogen bonds that were oriented in alternating directions

Helical Foldamer Containing a Combination of Cyclopentane-1,2-diamine and 2,2-Dimethylmalonic Acid

We have developed new helical oligomers using a combination of (1<i>S</i>,2<i>S</i>)-cyclopentane-1,2-diamine [(<i>S</i>,<i>S</i>)-CPDA] and 2,2-dimethylmalonic acid (DMM) residues as building blocks. In solution, the preferred secondary structure of the (<i>S</i>,<i>S</i>) tetramer <b>6</b> was a right-handed (<i>P</i>) helix, and that of the (<i>R</i>,<i>R</i>) tetramer <i>ent</i>-<b>6</b> was a left-handed (<i>M</i>) helix. In the crystalline state, both <b>6</b> and the (<i>S</i>,<i>S</i>) pentamer <b>7</b> folded into (<i>P</i>) 11-helices, and <i>ent</i>-<b>6</b> folded into an (<i>M</i>) 11-helix with hydrogen bonds that were oriented in alternating directions

Twisted Structure of a Cyclic Hexapeptide Containing a Combination of Alternating l‑Leu‑d‑Leu-Aib Segments

We designed and synthesized a <i>C</i>₂-symmetric cyclic hexapeptide, <i>cyclo</i>(l-Leu-d-Leu-Aib)₂ (<b>2</b>), which contains l- and d-amino acids and achiral Aib residues. The conformation of <b>2</b> was analyzed in the crystalline state and in solution, which was a unique figure-eight-shaped conformation

Twisted Structure of a Cyclic Hexapeptide Containing a Combination of Alternating l‑Leu‑d‑Leu-Aib Segments

We designed and synthesized a <i>C</i>₂-symmetric cyclic hexapeptide, <i>cyclo</i>(l-Leu-d-Leu-Aib)₂ (<b>2</b>), which contains l- and d-amino acids and achiral Aib residues. The conformation of <b>2</b> was analyzed in the crystalline state and in solution, which was a unique figure-eight-shaped conformation

Oligopeptides with Equal Amounts of l- and d‑Amino Acids May Prefer a Helix Screw Sense

We investigated the preferred conformations of two nonapeptides, Boc-(l-Leu-d-Leu-Aib)₃-OMe (<b>2</b>) and its enantiomer Boc-(d-Leu-l-Leu-Aib)₃-OMe (<i>ent</i>-<b>2</b>), four dodecapeptides, Boc-(l-Leu-d-Leu-Aib)₄-OMe (<b>3</b>), Boc-(l-Leu-Aib-d-Leu)₄-OMe (<b>4</b>), Boc-(Aib-l-Leu-d-Leu)₄-OMe (<b>5</b>), and Boc-(l-Leu-Aib-d-Leu-Aib)₃-OMe (<b>6</b>), and a decapeptide, Boc-l-Leu-(d-Leu-l-Leu-Aib)₃-OMe (<b>7</b>), in solution and in the crystalline state. The nonapeptide <b>2</b> formed a right-handed (<i>P</i>) α-helix, and its enantiomer <i>ent</i>-<b>2</b> formed a left-handed (<i>M</i>) α-helix. The dodecapeptides <b>3</b> and <b>5</b> were folded into (<i>P</i>) helices, and <b>4</b> formed an (<i>M</i>) helical structure. As for <b>6</b>, roughly equivalent amounts of (<i>P</i>) and (<i>M</i>) helices were observed in solution, and two (<i>M</i>) α-helices were detected in the crystalline state. Furthermore, the decapeptide <b>7</b>, which possesses four l-Leu residues and three d-Leu residues, was folded into an (<i>M</i>) α-helix

Effect of integrin extension on the binding of the anti-αVβ3 mAbs.

<p>Wild-type αV or αV carrying the Q589NAT mutation was transiently expressed together with wild-type β3 in CHO cells. The binding of function-blocking mAbs to these cells was examined by FACS. The MFI obtained from the whole cell population with each mAb was normalized by the MFI obtained with an anti-αV mAb 17E6 that represents the αV expression. There was no significant difference in the binding of leg-binding mAbs in cells expressing Q589NAT (solid column) as compared with cells expressing wild-type αV (hatched column).</p

MAb binding to cells expressing human-to-mouse αV mutants.

<p>The numbers represent the percentage of the cell population stained with each mAb. Bindings significantly lower than those for 17E6 or SZ21 are marked in red.</p

Effect of anti-αVβ3 mAbs on fibrinogen binding.

<p>A. FITC-fibrinogen binding to cells expressing αVβ3 in the presence of 1 mM Ca²⁺ and 1 mM Mg²⁺ (open column) or in the presence of 2 mM Mg²⁺ and 5 µM EGTA (hatched column) is shown. The ratio of the MFI (FL1) to the MFI (FL2) in the gated cell population was used to normalize the binding with the expression of αVβ3 on the cell surface. B. FITC-fibrinogen binding to cells expressing wild-type αVβ3 in the presence of 2 mM Mg²⁺ and 5 µM EGTA was examined. Binding in the presence of 100 µg/mL of the indicated mAb is shown in the hatched column. An equivalent volume of PBS, instead of the mAb solution, was included to obtain the control binding. The asterisks indicate statistically different binding abilities, compared with the control (*<i>P</i><0.01, **<i>P</i><0.05).</p

Schematic representation of αV/αIIb chimeras.

<p>The abbreviations P, T, C1, C2, and TC in the figure stand for β-propeller, thigh, calf-1, calf-2, and transmembrane-cytoplasmic domain, respectively. The numbers indicate the domain boundaries used to create the chimeras.</p