Analysis of binding pocket variants of p38 to understand the binding of p38 and NOS3
Disciplines
Biochemistry, Biophysics, and Structural Biology
Abstract (300 words maximum)
The p38 mitogen-activated protein kinase (MAPK) pathway is a significant player in cellular reactions to stress and inflammation. Our lab is interested in understanding the binding of p38 alpha with nitric oxide synthase 3 (NOS3, aka eNOS). NOS3 and p38 alpha bind with nM affinity and using peptides we have previously shown that p38 binds similarly with portions of the N-terminus (NT) and the autoinhibitory (AI) loop. There are currently two known binding areas on p38 the CD groove and the hydrophobic patch; it is unknown which of these is used to bind the two regions of NOS3 (NT, AI). Two variants of p38 were created previously to demonstrate when p38 MAPK uses the CD groove vs the hydrophobic patch, the first is D316N, which sits within a section commonly used for CD groove interactions and significantly impacts substrate binding. The second is Y258A which disrupts substrates and proteins that use the hydrophobic patch. Previous research showed that the D316 mutation lowers binding interaction with MAPK-activated protein kinase 2 (MK2), which binds to the CD groove to be phosphorylated p38. We hypothesize that the Y258A mutation would not impact binding to MK2 and that activation would be similar to wild type active p38, while in contrast D316 which alters the CD groove is predicted to have lowered ability to bind and activate MK2. We have purified wild type, D316N and Y258A in active phosphorylated forms and are testing how all of them activate MK2, ultimately to test how they differentially bind and phosphorylate NOS3. These insights are important for exploring how p38 binds NOS3.
Academic department under which the project should be listed
CSM - Molecular and Cellular Biology
Primary Investigator (PI) Name
Carol Chrestensen
Analysis of binding pocket variants of p38 to understand the binding of p38 and NOS3
The p38 mitogen-activated protein kinase (MAPK) pathway is a significant player in cellular reactions to stress and inflammation. Our lab is interested in understanding the binding of p38 alpha with nitric oxide synthase 3 (NOS3, aka eNOS). NOS3 and p38 alpha bind with nM affinity and using peptides we have previously shown that p38 binds similarly with portions of the N-terminus (NT) and the autoinhibitory (AI) loop. There are currently two known binding areas on p38 the CD groove and the hydrophobic patch; it is unknown which of these is used to bind the two regions of NOS3 (NT, AI). Two variants of p38 were created previously to demonstrate when p38 MAPK uses the CD groove vs the hydrophobic patch, the first is D316N, which sits within a section commonly used for CD groove interactions and significantly impacts substrate binding. The second is Y258A which disrupts substrates and proteins that use the hydrophobic patch. Previous research showed that the D316 mutation lowers binding interaction with MAPK-activated protein kinase 2 (MK2), which binds to the CD groove to be phosphorylated p38. We hypothesize that the Y258A mutation would not impact binding to MK2 and that activation would be similar to wild type active p38, while in contrast D316 which alters the CD groove is predicted to have lowered ability to bind and activate MK2. We have purified wild type, D316N and Y258A in active phosphorylated forms and are testing how all of them activate MK2, ultimately to test how they differentially bind and phosphorylate NOS3. These insights are important for exploring how p38 binds NOS3.
