The Effects of Amino Acid Substitutions on MCL-1 Protein Structure and Function Open Access

Kim, Suyeon (Spring 2021)

Permanent URL: https://etd.library.emory.edu/concern/etds/1c18dh048?locale=en
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Abstract

Multiple myeloma (MM) is a B-cell malignancy that makes up 1% of all diagnosed cancers. Recent studies have outlined MM molecular mechanisms, such as the intrinsic apoptotic pathway regulated by the BCL-2 family of proteins. In the pathway, MCL-1 is an essential anti-apoptotic protein vital for cell maintenance, survival, and regulated cell death. Further, the BH regions of the MCL-1 protein form a binding pocket in which BH3 sensitizers and targeted therapy drugs can bind to induce apoptosis. Specifically, BH3-mimetics are small, lipophilic molecules that induce apoptosis in cancer cells. BIM is a pro-apoptotic BH3-only activator protein that is sequestered by MCL-1 to inhibit the activation of pro-apoptotic proteins BAK & BAX, ultimately inhibiting MOMP and apoptosis. In this study, three BIM bound MCL-1 complexes (2PQK, 6QFI, & 2KBW) & four drug inhibitor bound MCL-1 complexes (Servier S63845 (5LOF), Servier 2019 (6QYP), preliminary AMG-176 compounds (6OQC & 6OQB), and AstraZeneca (6FS0)) were investigated. The purpose of this investigation is to analyze the effects of MCL-1 amino acid substitutions found in newly diagnosed MM patients (V249L, L267V, N223S, and R214Q) and various other malignancies (V216I, V216L, V258L, and L267F) on the protein’s structure and function.

The alignment feature on PyMOL was utilized to display changes in the amino acid side chains between the WT and mutant variants. In particular, the MCL-1L267V amino acid substitution displayed changes in the side chain that may have implications for the binding of drug inhibitors and BIM. Distance measurements revealed that Val267 associated with MCL-1L267V was found to be closer in proximity to all drug inhibitors (6OQC, 6OQB, 6FS0, 5LOF, and 6QYP); however, the Val267 side chain was also found to be further in distance to all the BIM molecules (2PQK, 6QFI, and 2KBW). These results may indicate that the MCL- 1L267V mutation prevents the binding of drug inhibitors to MCL-1 & BIM displacement from MCL-1. Although the side chain substitutions are conserved in size and property, the L267V mutation is not exempt from phenotypic implications as these mutations alter the space available between the binding pocket and drug inhibitors & BIM molecules.

Taken together, the data suggests that MCL-1 mutations may affect the protein’s interaction with drug inhibitors and alter the downstream intrinsic apoptotic pathway. As a protein that interacts directly with BH3 sensitizer drugs and is important for cell survival, it is imperative that MCL-1 protein targeting therapy and the molecular mechanism of MM is further investigated.

Table of Contents

Introduction.................................................................................................................................1

Figure 1...............................................................................................................................7

Figure 2...............................................................................................................................8

Figure 3...............................................................................................................................9

Figure 4.............................................................................................................................10

Figure 5.............................................................................................................................11

Figure 6..............................................................................................................................12

Figure 7..............................................................................................................................13

Methods & Materials.................................................................................................................14

Results..........................................................................................................................................17

Figure 8..............................................................................................................................24

Figure 9..............................................................................................................................25

Figure 10............................................................................................................................26

Table 1................................................................................................................................27

Table 2................................................................................................................................28

Figure 11............................................................................................................................29

Figure 12............................................................................................................................30

Figure 13............................................................................................................................31

Figure 14............................................................................................................................32

Figure 15............................................................................................................................33

Figure 16............................................................................................................................34

Discussion...................................................................................................................................35

Conclusion...................................................................................................................................38 

Supplementary Information.....................................................................................................39

References...................................................................................................................................41

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