NMR-Based and Automated Docking Characterization of Protein Structure, Dynamics, and Ligand Binding

NMR-based methods used in conjunction with a technique called docking are used to characterize ligand binding to proteins. Standard NMR methods were used to study the backbone dynamics of substrate binding to phosphomevalonate kinase (PMK) and it was observed that ligand binding caused PMK to undergo large conformational changes. These changes were reflected by the appearance of many chemical shift changes upon binding of the natural substrates of PMK (both the binary and ternary complexes) in 1H-15N HSQC NMR titration experiments. The same process was used to characterize the effect ligand binding has on the many arginines in the active site (and distal to the active site) to determine the effect of long-range coulombic interactions on ligand binding. While studying the backbone dynamics of PMK it was discovered that the N-terminal tail of PMK consisting of 10 residues was very disordered which is unlike every other monphosphate kinase. The function of this N-terminal tail was investigated by attempting to find other proteins in human liver cells that bind this peptide, monitored by ESI mass spectrometry. The thioredoxin system of Mycobacterium Tuberculosis consists of a thioredoxin reductase and three thioredoxins. To help facilitate the understanding of this mechanism the solution structures of the oxidized and the reduced forms of thioredoxin C (TrxC) were solved by NMR and modeled with the crystal structure of the thioredoxin reductase complex. The two redox states of TrxC are very similar to each other with most of the differences coming from subtle changes in the active site of TrxC. Automated docking is the process of computationally determining how a ligand binds to a protein and the correct orientation. A large scale docking study, termed virtual screening, was carried out by docking 10,590 compounds into three proteins to find inhibitors for each protein, and those predicted to bind best were tested experimentally. For each protein there were 3 compounds found to bind with reasonable affinity. When ligands bind to a protein they can undergo dynamic changes. To explore this phenomenon, 15N labeled NAD+ cofactor (and other derivatives) was synthesized and bound to oxidoreductases. Relevant binding motions were monitored using CPMG relaxation NMR experiments