Fig 1.
Mapping protein-ligand interactions by HDXMS.
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 (E) would exist in multiple conformations in the absence of ligand. Here a representative target protein is shown containing two sites- an orthosteric (O) site forming the ligand binding pocket (sites 1–4 are represented) and an allosteric (A) 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 (E:L) would result in decreased exchange while changes at the A-site (E*:L) could be reflected as decreases or increases in deuterium exchange.
Fig 2.
(A) A modified mirror plot of relative deuterium uptake values (y-axis) for every pepsin digest peptide analyzed, listed from the N to C terminus (x-axis) of Hsp90 for each time point of deuterium exchange. Reporter Regions were determined according to Eq 1 and boxed in teal. (B, C) Regions showing high dynamics were mapped onto the surface representation of Hsp90 (PDB Id: 4EGK) in blue bound to Radicicol (green sticks).
Fig 3.
(A) 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. The top panel shows regions showing differences upon Radicicol binding and the bottom panel shows differences upon 17-AAG binding. Regions showing significant differences above a threshold of 0.5 Da (red dashed line) are compared with structural data to identify orthosteric regions. Peptides spanning these ligand binding sites which mediate orthosteric interactions with ligands are marked in blue boxes and are divided into four orthosteric regions marked O1 to O4.Time points of deuterium exchange are colored according to key. (B) Regions showing decreased exchange upon ligand binding (either Radicicol or 17-AAG) are mapped onto the structure of Hsp90 in blue. Radicicol bound at the ligand binding pocket is shown as sticks (PDB ID: 4EGK).
Fig 4.
(A) Orthosteric regions identified by structural and HDXMS analysis overlap with changes in both ligands and fragments. 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 form of Hsp90. The panels A to D show regions showing differences upon binding Radicicol, 17-AAG, fragments 1 and 2, respectively. Regions showing significant differences above a threshold of 0.5 Da (red dashed line) are correlated with structural data to define orthosteric sites. Peptides spanning these ligand binding sites which make orthosteric interactions with fragments are marked in blue boxes are divided into the same four orthosteric regions O1 to O4, observed in Radicicol and 17-AAG. Fragment 2 shows minimal protection in the region O2 as it is oriented away in this region, consistent with deuterium exchange data.
Fig 5.
Distinguishing orthosteric and allosteric effects in Hsp90.
(A) 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) 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).
Fig 6.
Fragments 1 and 2 differ in the nature of the allosteric effect in Hsp90.
(A) 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 2 does not show any changes in region A4, similar to 17-AAG, while fragment 1 shows differences, similar to Radicicol. In addition, fragment 1 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,C) The identified orthosteric (blue) and allosteric regions (red) for fragments are mapped on to the structure of Hsp90 in blue. (C) The allosteric site A5 in Hsp90, which is observed only fragment 2 is highlighted in orange. Radicicol bound at the ligand binding pocket is shown as sticks (PDB ID: 4EGK).