Molecular mechanics and dynamic simulations of well-known Kabuki syndrome-associated KDM6A variants reveal putative mechanisms of dysfunction

Abstract

Background: Kabuki syndrome is a genetic disorder that affects several body systems and presents with variations in symptoms and severity. The syndrome is named for a common phenotype of faces resembling stage makeup used in a Japanese traditional theatrical art named kabuki. The most frequent cause of this syndrome is mutations in the H3K4 family of histone methyltransferases while a smaller percentage results from genetic alterations affecting the histone demethylase, KDM6A. Because of the rare presentation of the latter form of the disease, little is known about how missense changes in the KDM6A protein sequence impact protein function.

Results: In this study, we use molecular mechanic and molecular dynamic simulations to enhance the annotation and mechanistic interpretation of the potential impact of eleven KDM6A missense variants found in Kabuki syndrome patients. These variants (N910S, D980V, S1025G, C1153R, C1153Y, P1195L, L1200F, Q1212R, Q1248R, R1255W, and R1351Q) are predicted to be pathogenic, likely pathogenic or of uncertain significance by sequence-based analysis. Here, we demonstrate, for the first time, that although Kabuki syndrome missense variants are found outside the functionally critical regions, they could affect overall function by significantly disrupting global and local conformation (C1153R, C1153Y, P1195L, L1200F, Q1212R, Q1248R, R1255W and R1351Q), chemical environment (C1153R, C1153Y, P1195L, L1200F, Q1212R, Q1248R, R1255W and R1351Q), and/or molecular dynamics of the catalytic domain (all variants). In addition, our approaches predict that many mutations, in particular C1153R, could allosterically disrupt the key enzymatic interactions of KDM6A.

Conclusions: Our study demonstrates that the KDM6A Kabuki syndrome variants may impair histone demethylase function through various mechanisms that include altered protein integrity, local environment, molecular interactions and protein dynamics. Molecular dynamics simulations of the wild type and the variants are critical to gain a better understanding of molecular dysfunction. This type of comprehensive structure- and MD-based analyses should help develop improved impact scoring systems to interpret the damaging effects of variants in this protein and other related proteins as well as provide detailed mechanistic insight that is not currently predictable from sequence alone.

Keywords: Epigenetic regulators; Genomic variation; Histone demethylase; KDM6A; Kabuki syndrome; Molecular dynamics; Mutational impact analysis; Protein structure.

Fig. 1

Catalytic mechanism, architecture, key functional sites, and disease-causing missense mutations of KMD6A. a Domain structure and the schematic of its catalytic mechanism. Relative positions of KDM6A Kabuki variants identified from the patients are highlighted in red on top of the domain diagram. b The catalytic domain structure of KDM6A in complex with the H3K27me3 peptide, metal ions and the cofactor 2OG (PDB access code 3AVR). The catalytic domain is composed of the jumonji domain flanked by two additional sub-domains and a long flexible linker. The bound substrate is shown as ball-and-sticks while the catalytic domain is shown as ribbons. The color codes are identical to the ones used in Fig. 1A. (c–e) Zoomed views of the active site, substrate binding interface, and the zinc ion binding site. two damaging control residues are labeled in red. H3 histone residues are labeled in orange. f Mapping of Kabuki syndrome missense variants onto its molecular structure. None of the Kabuki variants are found right at the critical molecular interaction sites. Figures were made using PyMOL (The PyMOL Molecular Graphics System Version 2.3.0., Schrödinger, LLC)

Fig. 2

Intrinsic mobility of the KDM6A catalytic domain and the essential dynamic motions. a Linear RMSF plot of the KDM6A catalytic domain during MD simulation. The protein regions of greater than 1.0 Å displacements (fluctuations) are indicated by a red dotted line. The secondary structure elements and the domain structures are shown above. b Dynamic fluctuations (RMSF) of each residue during MD simulation are also plotted on the molecular structure surface, which is in good agreement of the temperature factor plot of its crystal structure (c). They are color-coded according the ranges indicated by the horizontal bar at the bottom of each panel. (d) Porcupine plot of trajectories representing the essential dynamic in each principal component during MD simulation. The length of cone represents the motion magnitude and the pointing of the arrow indicates the direction. The catalytic core jumonji domain movements mostly involve PC1 and PC2 while the substrate binding dynamics involve all three main PCs. This plot shows only the enzyme movement

Fig. 3

Representative impact analyses. a A heatmap representation of the global pK_a shifts due to each mutation. pK_a shifts of the residues that are directly affected by the substitution (e.g., H1060 of the H1060L mutant or H1146A, E1148A, R1255W, and R1351Q original residues and newly introduced charged residues such as C1135R, Q1212R, and Q1248R) are not shown and only indirect effects are represented. Shift amounts in pH unit are color-coded (indicator bar on right). The vertical axis shows the list of titratable residues in a descending order from the top. Noticeably affected residues are indicated on both sides; key functional residues on left and potential allosteric residues on right. b, c MD-based evaluation of global dynamic alterations, such as b the comparison of RMSD distribution and median values and c the absolute average RMSF differences. Wild type (blue), benign (green), damaging controls (red) and Kabuki variants (orange) are shown. Various comparative values have been tested and ‘Median Difference’ for RMSD and Avg. (|WT-variant|)’ per residue were chosen as the best metrics and plotted for variant impact assessment. All Kabuki variants displayed elevated global motions that deviate from the wild type

Fig. 4

Close-up views of Kabuki variants under study and their category-wise assessment. Further descriptions on the alterations of local environment and chemical interactions by these mutations are provided in the main text. At the bottom of each panel, colored boxes represent overall assessment of each category for each Kabuki syndrome variant; sequence-, structure-, and MD-based, respectively from left to right. The intensity of redness indicates the level of damaging impacts at each layer light to dark. Although N910S and R1351Q are predicted to be non-damaging by sequence-based analysis, they are predicted to be damaging by static structure- and dynamics-based mechanistic analyses. There is a set of indicators for C1153 representing C1153R (top) and C1153Y Kabuki variants