Defense Date

2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Pharmaceutical Sciences

First Advisor

Dr. Huang, Rong

Abstract

The methylation at the α-N-terminal amines of proteins that start with a canonical motif X-P-K (X=A/P/S) has been a known modification for nearly four decades. In 2010, protein α-N-terminal methyltransferase 1 (NTMT1/NRMT1) was identified as the first enzyme responsible for this modification. NTMT2 was discovered as a second member belonging to this family, but it was reported as a mono-methylase. The identification of RCC1, retinoblastoma (Rb) protein, centromere protein-A/B (CENP-A/B), and DNA damaged-binding protein 2 (DDB2) as new NTMT1 substrates revealed NTMT1’s biological significance in mitosis, cell-cycle regulation, centromere formation, and damaged DNA repair, respectively. Although significant progress had been made, a clear understanding of how NTMT1 recognizes substrates remains to be determined. Also, there is no specific small molecule inhibitor for NTMT1.

To fill these gaps, we first established a fluorescence-based assay for kinetic characterization of NTMT1. Subsequently, ternary complex crystal structures of NTMT1 were obtained to illustrate the structural basis for enzyme-substrate interactions. The structures of the enzyme-substrate complex coupled with mutagenesis, binding, and enzymatic studies demonstrated the key elements involved in interaction with its substrates. In the meantime, we utilized computational studies and fluorescence assays for novel small molecule discovery. Lastly, we closely monitored the substrates’ methylation progression by NTMT1 and NTMT2 in parallel using a MALDI-MS based assay.

Our results indicated that NTMT1 follows a Bi-Bi mechanism, and its methylation proceeds in a distributive pattern. Furthermore, NTMT1 was identified has broad substrate specificity beyond its canonical motif X-P-K (X=A/P/S), since X can be any amino acid except D/E and the third amino acids can also be R. We had also discovered an inhibitor that targets the substrate binding site of NTMT1 with IC50 = 7 µM. Lastly, our methylation progression studies has demonstrated that NTMT2 can also di-, tri-methylate certain substrates although its methylation rate is lower than NTMT1.

Overall, this project has laid the foundation for further investigation of N-terminal methylation in terms of functions, mechanisms, and inhibitor design.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

8-4-2016

Available for download on Tuesday, August 03, 2021

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