Using HDXMS to Study Low Affinity Protein Fragment Interactions
0:00 Introduction to HDXMS: The presentation focuses on using Hydrogen Deuterium Exchange Mass Spectrometry (HDXMS) to study low affinity protein-fragment interactions. HDXMS is based on the exchange of hydrogen atoms in a protein with deuterium from the surrounding solution.
0:00 HDXMS Experiment Setup: An HDXMS experiment compares two samples: an apo protein and the same protein incubated with a ligand of interest.
0:01 Deuterium Uptake Over Time: Both samples are incubated in a D2O buffer, and deuterium uptake is measured at various time points. Short time points reveal solvent-exposed and flexible regions, while longer time points show exchange in internal structures.
0:01 Quenching and Digestion: The exchange reaction is quenched by lowering pH and temperature, followed by proteolytic digestion to generate peptides.
0:02 Uptake Plots: Uptake plots show the percentage of deuterium uptake over time for each peptide. Peptides involved in binding are protected from deuterium exchange in the bound sample, leading to decreased uptake.
0:03 HDXMS for Fragment-Based Drug Discovery: While HDXMS is established for characterizing strong interactions, this project applies it to low affinity fragments, relevant to fragment-based drug discovery.
0:04 Challenge with Crystallography: Obtaining crystal structures for low-affinity fragments is difficult, so HDXMS is used to fill this information gap.
0:04 Case Study: Copyin D (CPD): The research uses Copyin D, a mitochondrial protein, as a model. CPD's dysfunction is linked to diseases like multiple sclerosis and cardiovascular disease.
0:05 Protein Production and Purification: CPD is produced in E. coli, yielding a pure, stable, and homogeneous sample.
0:05 Known Binding Sites: Crystal structures of CPD have revealed three binding pockets: prolin, analine, and P old pocket.
0:05 Low-Affinity Fragments: High affinity ligands and low affinity fragments were synthesized to target these pockets. Obtaining crystal structures was not always possible, making CPD a good model for assessing HDXMS.
0:06 Targeting In Pocket: An initial experiment involves a fragment targeting the in pocket. A differential heat map visually represents differences in deuterium uptake between the apo and bound samples.
0:07 Statistical Analysis: Statistical analysis, including a two-layer filtering process (global significance level and a P value set at 99% confidence), identifies statistically significant peptides.
0:07 Volcano Plots: Volcano plots visualize statistical significance. Significant peptides are then mapped onto the CPD crystal structure.
0:08 Confirmation with Crystal Structure: HDXMS data corresponds with existing crystal structures confirming that this approach works.
0:09 Targeting Purol Pocket: A second experiment involves a fragment with KV of 20M targeting the purol pocket of CPD. Results from statistical analysis shows uptake decreasing in both the pural and analin pockets of the protein.
0:10 Proline Pocket and Overlapping Pockets: A third experiment involves a fragment targeting the pring pocket, same statistical analysis was performed. HDXMS detected interactions in both the proline and analine pockets of the protein, highlighting the close proximity of pockets.
0:10 Fragments Without Crystal Structures: The study also used HDXMS to characterize the binding of two fragments for which crystal structures were unavailable. Both fragments showed protection in the an in pocket, and hdxms determined they target this protein through different binding modes.
0:11 Conclusions: HDXMS is a useful tool to characterize distinct Hdxms profiles across differing pockets. Even if not crystalized, fragments with millimolar affinities are detectable.
0:11 Acknowledgments: Kina Mal acknowledges supervisors Alesio Yorr and Thiago, the mer Healthcare team, the structor biology for drug Discovery group, and Mar Portugal for funding.