Understanding the structural, dynamical, and functional effects of serine versus threonine phosphorylation
Date
2024
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Phosphorylation, a prevalent post-translational modification, intricately regulates cellular processes through modifications on serine, threonine, and tyrosine residues. This study focuses on the profound impact of phosphorylation on the cAMP-dependent protein kinase A catalytic subunit (PKAc), focusing notably on the extremely well conserved activation site, threonine 197 (T197). Our research stems from the hypothesis that the phosphate-amide hydrogen bond, specific to phosphorylated threonine (pThr), plays a crucial role in dictating PKAc's structural stability and enzymatic activity. We aim to comprehensively compare the wild-type (WT) , T197S, and T197P variants of PKAc to elucidate their respective characteristics and functional implications. ☐ Initial efforts involved the purification of recombinant WT, T197S and T197P PKAc, followed by structural and stability assessments. Mass spectrometry analysis revealed a 4% reduction in phosphorylation at site-197 after the T197S mutation. Conversely, phosphorylation percentages at the other three sites - S10, S139, and S338 - exhibited minor increases. These findings suggest that the phosphorylation status of the activation site, T197, has minimal impact on the phosphorylation levels of the other three sites. Through circular dichroism (CD) and thermal denaturation measurements, we observed a ranking of stability: WT > T197S > T197P, implicating pThr’s ability to provide higher stability over pSer through intra-residue hydrogen bonding with the backbone amide. And despite mimicking the intra-residue hydrogen bond, proline’s inability in forming hydrogen bonds with neighboring residues still ☐ causes significant loss in stability. In X-ray crystallography, we successfully acquired high quality WT crystals and managed to improve the resolution of the structure of WT PKAc to a resolution of 1.37Å. But obtaining crystals for the T197S and T197P PKAc variants posed significant challenges, likely due to crystal packing disruptions caused by the mutations. A single mutation T197S within PKAc's activation loop can disrupt its conformation, thus impeding proper packing of protein crystals. Kinetic enzyme characterization of PKAc variants was performed with Kemptide as substrate using an ADP-Glo assay. The activity results showed a ranking of WT＞T197S＞T197P where WT exhibited a 10-fold greater activity compared to T197S, and T197P exhibited no activity. The results fit well to a model of substrate inhibition. However, further experiments were performed to optimize ATP concentration, which led to the conclusion that previously published substrate inhibition interpretation was due to a a limiting concentration of ATP during assays. In molecular dynamics (MD) simulations, we observed Root Mean Square Deviation (RMSD) ranking T197S＞T197P＞WT. Suggesting T197S has induced a notable structural and dynamical change to the protein’s activation loop which has a greater structural deviation from the original coordinates. Additionally, we successfully used proline to mimic the local constrained geometry provided by phosphorylated threonine and the backbone amide. MD simulations further elucidated local structure alterations in both WT and T197S. Results showed pSer has multiple Ch1 and Ch2 peaks while pThr only has one single peak. This implies that pSer has higher local flexibility around the activation loop and thus leads to a decreased enzyme activity. ☐ Our analysis revealed that phosphorylation at threonine-197 in PKAc establishes a constrained backbone geometry, unlike serine phosphorylation. And the consequences of lacking a constrained backbone is a disturbed activation loop, which will result in a significant loss of activity. Our findings underscore the importance of threonine phosphorylation at PKAc's activation site over millions of years of evolution. It unveils the intricate molecular mechanisms governing kinase regulation, while emphasizing the essential role of specific phosphorylation events in modulating enzyme function and cellular signaling pathway.
Description
Keywords
Phosphorylation, Catalytic subunits, Phosphorylated threonine, Cellular signaling, Molecular dynamics