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Modeling of Biomolecular Systems Interactions, Dynamics, and Allostery Poster Session I
8-POS
Board 8
Distinguishing Binding and Allostery in Protein-Ligand Interactions Using Amide H/D
Exchange Mass Spectrometry
Ganesh Anand
, Srinath Krishnamurthy.
National University of Singapore, Singapore.
Amide H/D exchange mass spectrometry (HDXMS) is a method that is highly suited for
monitoring protein-ligand interactions and has important advantages including small sample
sizes, automation and user-friendly visual interfaces, ability to map protein-ligand interactions in
solution with no size limits of target proteins. We tested suitability of HDXMS to map
interactions of ligands of varying affinity for Hsp90 (Radicicol, allylamino-
demethoxygeldanamycin (AAG) (KDs of 19, 33 nM respectively) to low affinity geldanamycin
derivatives with affinities of 490 and 570
M). Amide exchange was initiated by incubating
Hsp90 alone and in the presence of the above ligands in deuterated (D2O) buffer (pHread ~7.0)
for a time series (0.5-10 min) and the reaction quenched by lowering the pHread to 2.5. A
combination of LC-ESI mass spectrometry and pepsin proteolytic fragmentation was then carried
out.Our results reveal that HDXMS shows high sensitivity to map interactions mediated by low
affinity ligands (binding affinity ~490
microM) which show similar magnitude changes in
deuterium exchange in a subset of overlapping regions of Hsp90 as the high affinity inhibitor
Radicicol (binding affinity ~19 nM). More importantly analysis of deuterium exchange kinetics
enabled distinguishing between direct binding sites and allosteric sites distal to the predicted
binding site- based on the crystal structure of Hsp90 bound to the low affinity ligand. Ligand
interactions at the active site are reflected in decreased exchange at early time points and
allosteric changes at distal sites are reflected in deuterium exchange decreases at later time
points. This highlights a powerful application of HDXMS in protein-ligand interactions and
development of specific allosteric target molecules for drug discovery.
