Unveiling Allosteric Regulation and Binding Mechanism of BRD9 through Molecular Dynamics Simulations and Markov Modeling

Abstract

Bromodomain-containing protein 9 (BRD9) is a key player in chromatin remodeling and gene expression regulation, and it is closely associated with the development of various diseases, including cancers. Recent studies have indicated that inhibition of BRD9 may have potential value in the treatment of certain cancers. Molecular dynamics (MD) simulations, Markov modeling and principal component analysis were performed to investigate the binding mechanisms of allosteric inhibitor POJ and orthosteric inhibitor 82I to BRD9 and its allosteric regulation. Our results indicate that binding of these two types of inhibitors induces significant structural changes in the protein, particularly in the formation and dissolution of α-helical regions. Markov flux analysis reveals notable changes occurring in the α-helicity near the ZA loop during the inhibitor binding process. Calculations of binding free energies reveal that the cooperation of orthosteric and allosteric inhibitors affects binding ability of inhibitors to BRD9 and modifies the active sites of orthosteric and allosteric positions. This research is expected to provide new insights into the inhibitory mechanism of 82I and POJ on BRD9 and offers a theoretical foundation for development of cancer treatment strategies targeting BRD9.

Keywords: BRD9; Markov models; binding free energy; molecular dynamics simulations.

Figure 1

Molecular structures: (A) structural superimposition of inhibitor-bound BRD9 complex, (B) binding pocket of orthosteric site and allosteric site, (C) orthosteric inhibitor 82I, (D) allosteric inhibitor POJ, (E) orthosteric inhibitor compound 9 (named as LIG) and (F) orthosteric inhibitor P8Z. In this figure, the boxes illustrate the differences among molecular groups. The compound 9 was taken from the work of Clegg et al [22].

Figure 2

Secondary structure evolution of BRD9 in four systems as the simulation time: (A) the APO-BRD9, (B) the 82I-BRD9, (C) the POJ-BRD9 and (D) the ALL-BRD9.

Figure 3

Structural flexibility of the APO-, 82I-, POJ-, ALL-BRD9: (A) the RMSFs of BRD9 calculated using the coordinates of the Cα atoms, (B) structural regions with obvious alterations of RMSFs and (C–F) corresponding to the structural flexibility of APO-BRD9, 82I-BRD9, POJ-BRD9 and 82I/POJ-BRD9. The tendency from blue to red indicates the increase in structural flexibility which is scaled in B-factor.

Figure 4

DCCMs of BRD9 calculated by using the coordinates of the Cα atoms: (A) the APO-BRD9, (B) the 82I-BRD9, (C) the POJ-BRD9 and (D) the ALL-BRD9. The red-colored R1, R2, and R3 regions represent the primary differences among the four systems.

Figure 5

Concerted motions of structural domains in four systems: (A) the APO-BRD9, (B) the 82I-BRD9, (C) the POJ-BRD9 and (D) the ALL-BRD9. In this figure, BRD9 is shown in cartoon modes and inhibitors are displayed in stick modes.

Figure 6

Free energy surface of four systems arising from the TICA: (A) the results of k-means diagrams of the APO-BRD9, (B) the results of k-means diagrams of the 82I-BRD9, (C) the results of k-means diagrams of the POJ-BRD9 and (D) the results of k-means diagrams of the ALL-BRD9.

Figure 7

Lag time determination of four systems: (A) the APO-BRD9, (B) the 82I-BRD9, (C) the POJ-BRD9 and (D) the ALL-BRD9. In the figure, each curve’s color indicates a different implied timescale, and the black line with the gray shading below shows the model’s resolution limit.

Figure 8

Flux analysis of four simulation systems: (A) the APO-BRD9, (B) the 82I-BRD9, (C) the POJ-BRD9 and (D) the ALL-BRD9.

Figure 9

Inhibitor–residue interactions: (A) the 82I-residue interactions in the 82I-BRD9, (B) the POJ-residue interactions in the POJ-BRD9, (C) the 82I-residue interactions in the ALL-BRD9, (D) the POJ-residue interactions in the ALL-BRD9 and (E) the orthosteric and allosteric binding pockets.

Figure 10

The flowchart of this study, which is used to describe the flow that we performed this study.