Modeling of Biomolecular Systems Interactions, Dynamics, and Allostery: Bridging Experiments and Computations - September 10-14, 2014, Istanbul, Turkey

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.

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