<div>Protein N-terminal methyltransferases (NTMTs) are a family of enzymes that methylate the α-N-terminus of a variety of protein substrates. Both NTMT1 and NTMT2 recognize a unique N-terminal X-P-K/R motif (X represents any amino acid other than D/E) to install 1-3 methyl group(s) on the substrates. NTMT1 plays important roles in mitosis regulation, chromatin interactions, and DNA damage repair. Another member NTMT2 shares ~50% sequence similarity and the same substrate recognition motif although NTMT2 was initially characterized as a mono-methyltransferase. To understand the molecular mechanism of NTMT2, we obtained the first co-crystal structure of NTMT2 in complex with its peptide substrate. After an extensive investigation of substrate recognition and methylated products of NTMT1/2, we found out that NTMT2 can fully methylate G/P-PKRIA peptides despite a predominant mono-methyltransferase. Moreover, we identified a gatekeeper N89 in NTMT2 that controls the substrate entry and the product specificity of NTMT2.</div><div>To elucidate the biological functions of NTMT1/2-catalyzed N-terminal methylation, we applied two different strategies to discover cell-potent inhibitors. Guided by the co-crystal structures of NTMT1 in complex with previously reported inhibitors, we designed and synthesized a series of new peptidomimetic inhibitors. By introducing more hydrophobic groups, the most cell-potent peptidomimetic inhibitor GD562 (IC50 = 0.93 ± 0.04 µM) exhibited over 2-fold increased inhibition on cellular N-terminal methylation levels with an IC50 value of ~50 µM compared to previously reported peptidomimetic inhibitor DC541. Meanwhile, we also discovered the first potent small molecule inhibitor Genz-682452 (IC50 = 0.5 ± 0.04 µM) after screening ~58,000 compounds. Subsequent structural modifications led to the discovery of GD433 (IC50 = 27 ± 0.5 nM) with a 20-fold increased potency compared to the initial hit Genz-682452. Inhibition mechanism indicated both inhibitors bind to peptide-binding pocket and co-crystal structures of both Genz-682452 and GD433 with NTMT1 confirmed their binding modes. Furthermore, GD433 shows over 7-fold selectivity over other major 40 protein methyltransferases and DNA methyltransferase and exhibits improved selectivity for NTMT1 over glucosylceramide synthase (GCS). GD433 significantly decreases the cellular N-terminal methylation level of NTMT1 substrates RCC1 and SET at 10 nM in both HEK293 and HCT116 cells, providing a valuable probe for cell-based studies in the future.<br></div><p><br></p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/15138216 |
| Date | 09 August 2021 |
| Creators | Guangping Dong (11250960) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/thesis/PRODUCT_SPECIFICITY_AND_INHIBITION_OF_PROTEIN_N-TERMINAL_METHYLTRANSFERASE_1_2/15138216 |
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