Fragments 1 and 2 differ in the nature of the allosteric effect in Hsp90.

<p><b>(A)</b> The absolute difference in numbers of deuterons (inferred from difference in mass in Daltons (Da) (y-axis) between the free and ligand bound state is plotted for each pepsin digest fragment listed from the N to C terminus (x-axis) of Hsp90 for each deuterium exchange time point (t = 0.5, 2, 5, 10 min) in a ‘difference plot’. Shifts in the positive scale represent decreases in deuterium exchange and shifts in the negative scale represent increases in deuterium exchange when compared to the apo-Hsp90. Regions showing significant differences above a threshold of 0.5 Da (red dashed line) are compared with orthosteric sites (blue boxes) to establish allosteric regions (red boxed). Fragment <b>2</b> does not show any changes in region A4, similar to 17-AAG, while fragment <b>1</b> shows differences, similar to Radicicol. In addition, fragment <b>1</b> shows an allosteric response at the regions A5 (residues 201–213 shown in orange box), which is not observed in the other three ligands. Time points are colored according to key. <b>(B,C)</b> The identified orthosteric (blue) and allosteric regions (red) for fragments are mapped on to the structure of Hsp90 in blue. <b>(C)</b> The allosteric site A5 in Hsp90, which is observed only fragment <b>2</b> is highlighted in orange. Radicicol bound at the ligand binding pocket is shown as sticks (PDB ID: 4EGK).</p

Distinguishing orthosteric and allosteric effects in Hsp90.

<p><b>(A)</b> The absolute difference in numbers of deuterons (y-axis) between the free and ligand bound state is plotted for each pepsin digest fragment listed from the N to C terminus (x-axis) of Hsp90 for each deuterium exchange time point (t = 0.5, 2, 5, 10 min) in a ‘difference plot’. Shifts in the positive scale represent decreases in deuterium exchange and shifts in the negative scale represent increases in deuterium exchange when compared to apo-Hsp90. Regions showing significant differences above a threshold of 0.5 Da (red dashed line) are compared with orthosteric sites (blue boxes) to predict allosteric regions. Peptides highlighted in red show regions showing differences in distal allosteric regions, not involved in orthosteric binding. Peptides spanning these regions are marked in red boxes and divided into four allosteric regions A1 to A4. Radicicol and 17-AAG shows differences in A1 and A2, while only radicicol showed changes in A3 and A4. Time points are colored according to key. <b>(B)</b> Predicted allosteric regions are mapped on to the structure of Hsp90 (red), together with the orthosteric regions, in blue. Radicicol bound at the ligand binding pocket is shown as sticks (PDB ID: 4EGK).</p

Mapping protein-ligand interactions by HDXMS.

<p>Protein-ligand interactions can be analyzed by HDXMS by comparing deuterium exchange of the unliganded state of the protein with that bound to ligand (shown in yellow sticks). An ensemble view entails that the target protein <b>(E)</b> would exist in multiple conformations in the absence of ligand. Here a representative target protein is shown containing two sites- an orthosteric <b>(O)</b> site forming the ligand binding pocket (sites 1–4 are represented) and an allosteric <b>(A)</b> site. Deuterium exchange at the orthosteric site (O-site) (blue) is then governed by ligand binding kinetic parameters: kon, koff, concentration of ligand as well as the observed rate of HDX exchange, kex, which varies across different regions of the protein. The HDXMS output encompasses changes at the orthosteric O-site and long range conformational changes (red) at the allosteric A-site. Binding of ligand at the O-site <b>(E:L)</b> would result in decreased exchange while changes at the A-site <b>(E*:L)</b> could be reflected as decreases or increases in deuterium exchange.</p