Unraveling the Behavior of Intrinsically Disordered Protein c-Myc: A Study Utilizing Gaussian-Accelerated Molecular Dynamics

Abstract

The protein c-Myc is a transcription factor that remains largely intrinsically disordered and is known to be involved in various biological processes and is overexpressed in various cancers, making it an attractive drug target. However, intrinsically disordered proteins such as c-Myc do not show funnel-like basins in their free-energy landscapes; this makes their druggability a challenge. For the first time, we propose a heterodimer model of c-Myc/Max in full length in this work. We used Gaussian-accelerated molecular dynamics (GaMD) simulations to explore the behavior of c-Myc and its various regions, including the transactivation domain (TAD) and the basic helix-loop-helix-leucine-zipper (bHLH-Zipper) motif in three different conformational states: (a) monomeric c-Myc, (b) c-Myc when bound to its partner protein, Max, and (c) when Max was removed after binding. We analyzed the GaMD trajectories using root-mean-square deviation (RMSD), radius of gyration, root-mean-square fluctuation, and free-energy landscape (FEL) calculations to elaborate the behaviors of these regions. The results showed that the monomeric c-Myc structure showed a higher RMSD fluctuation as compared with the c-Myc/Max heterodimer in the bHLH-Zipper motif. This indicated that the bHLH-Zipper motif of c-Myc is more stable when it is bound to Max. The TAD region in both monomeric and Max-bound states showed similar plasticity in terms of RMSD. We also conducted residue decomposition calculations and showed that the c-Myc and Max interaction could be driven mainly by electrostatic interactions and the residues Arg299, Ile403, and Leu420 seemed to play important roles in the interaction. Our work provides insights into the behavior of c-Myc and its regions that could support the development of drugs that target c-Myc and other intrinsically disordered proteins.

Figure 1

Structure of c-Myc and its interaction with its partner protein, Max. (a) The sequence of c-Myc consists of two important regions, which are the transactivation domain (TAD) region and the bHLH-Zipper motif. (b) The structure of full-length human c-Myc (the initial AlphaFold model is shown in Figure S1). The TAD and the bHLH-Zipper motif are colored green and blue, respectively. (c) The structure of the binding partner, Max. (d) c-Myc and Max bind to the bHLH-Zipper motif of c-Myc to form the c-Myc/Max heterodimer. (e) The c-Myc and Max interaction results in the heterodimer binding to DNA. The structures of c-Myc and Max proteins shown are speculative.

Figure 2

Comparison of (a) RMSD and (b) RGyr of the bHLH-Zipper motif of c-Myc in its monomeric state and Max-bound c-Myc. The c-Myc monomeric state replicates, and the Max-bound c-Myc replicates are shown as red and blue lines, respectively. (c) Time evolution of c-Myc in replicate 1 when in monomeric state up to 0.3 μs shows that bHLH-Zipper increased its compactness over the course of the simulation. The bHLH-Zipper motif and the TAD region are shown in blue and green, respectively.

Figure 3

Comparison of RMSD and RGyr of c-Myc in its monomeric state and Max-bound c-Myc. (a) RMSD and (b) RGyr of the TAD region for c-Myc in the monomeric state and Max-bound c-Myc. (c) RMSD and (d) RGyr of c-Myc as a whole in c-Myc in its monomeric state and in Max-bound c-Myc. The c-Myc monomeric state replicates and the Max-bound c-Myc replicates are shown in red and blue lines, respectively. The initial volume of the TAD region was reduced during the modeling of the c-Myc/Max heterodimer.

Figure 4

RMSD of c-Myc once Max was removed from the Max-bound state of c-Myc for an additional 0.5 μs. (a) RMSD of the bHLH-Zipper motif when Max was removed from c-Myc. (b) RMSD of the TAD region when Max was removed. (c) RMSD of c-Myc as a whole when Max was removed.

Figure 5

RGyr of c-Myc was removed from Max-bound c-Myc for an additional 0.5 μs. (a) RGyr of the bHLH-Zipper motif when Max was removed from the Max-bound c-Myc. (b) RGyr of the TAD region when Max was removed. (c) RGyr of c-Myc as a whole when Max was removed.

Figure 6

RMSF analysis of the residues of the bHLH-Zipper motif and the TAD region that show variation. (a) The residues of the bHLH-Zipper motif that show variation in RMSF. (b) The residues from the TAD region that show variation in RMSF. The monomeric c-Myc and Max-bound c-Myc are colored red and blue, respectively. The standard errors of mean (SEMs) are shown for all replicates in red bars for monomeric c-Myc and in blue bars for Max-bound c-Myc.

Figure 7

Combined FEL plot of the c-Myc in the monomeric state, Max-bound c-Myc, and when Max was removed. Basins marked as 1, 2, and 3 were achieved with the Max-bound c-Myc replicates. Meanwhile, basin 4 was achieved by replicate 1 of c-Myc in its monomeric state. The lowest energy basin (basin 1) was achieved by replicate 3 of the Max-bound c-Myc simulation.

Figure 8

Four conformations correspond to the energy basins of the combined FEL plot. (a) Conformation 1, (b) conformation 2, and (c) conformation 3 were all achieved by the replicates of the Max-bound c-Myc simulations. (d) Conformation 4 of panel (d) was achieved by c-Myc in its monomeric state. The bHLH-Zipper motif and the TAD region are shown in blue and green, respectively. The Max protein is shown as a gray surface.

Figure 9

Residues of c-Myc that show high contribution toward binding to Max. (a) The residues that show a high contribution toward binding to Max in replicate 1. (b) The residues that show a high contribution toward the binding of Max in replicate 2. (c) The residues that show a high contribution toward the binding of Max in replicate 3. The residues that are common to all three replicates are Arg299, Ile403, and Leu420. Residues Ile403 and Leu420 belong to the bHLH-Zipper motif of c-Myc. The positions of the residues in (d) replicate 1, (e) replicate 2, and (f) replicate 3 that show significant contribution toward binding to Max are shown in red. The bHLH-Zipper motif and the TAD region are shown in blue and green, respectively. The Max protein is shown as a gray surface.

Figure 10

Intramolecular H-bond-forming residues of c-Myc in its monomeric state and the c-Myc/Max heterodimer state. Number of H-bonds formed in the monomeric state in (a) replicate 1, (b) replicate 2, and (c) replicate 3. H-bonds formed within c-Myc when bound to Max in (d) replicate 1, (e) replicate 2, and (f) replicate 3.

Figure 11

Binding pockets of conformation 1, conformation 4, and conformation 3. (a) The binding pocket 1 is colored orange in conformation 1. (b) Binding pocket 2 is shown in yellow in conformation 4. (c) Binding pocket 3 is shown in pink in conformation 3. The TAD region and the bHLH-Zipper motif are colored green and blue, respectively. The protein surface for Max in panels (a) and (c) is shown as a gray surface. Inhibitors 10058-F4, 10074-G5, and L755507 that bind to the bHLH-Zipper motif region are shown in red, blue, and green, respectively.