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1.
Proteins ; 90(1): 282-298, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34414607

RESUMO

Disruptor of telomeric silencing 1-like (DOT1L) is the only non-SET domain histone lysine methyltransferase (KMT) and writer of H3K79 methylation on nucleosomes marked by H2B ubiquitination. DOT1L has elicited significant attention because of its interaction or fusion with members of the AF protein family in blood cell biology and leukemogenic transformation. Here, our goal was to extend previous structural information by performing a robust molecular dynamic study of DOT1L and its leukemogenic partners combined with mutational analysis. We show that statically and dynamically, D161, G163, E186, and F223 make frequent time-dependent interactions with SAM, while additional residues T139, K187, and N241 interact with SAM only under dynamics. Dynamics models reveal DOT1L, SAM, and H4 moving as one and show that more than twice the number of DOT1L residues interacts with these partners, relative to the static structure. Mutational analyses indicate that six of these residues are intolerant to substitution. We describe the dynamic behavior of DOT1L interacting with AF10 and AF9. Studies on the dynamics of a heterotrimeric complex of DOT1L1-AF10 illuminated describe coordinated motions that impact the relative position of the DOT1L HMT domain to the nucleosome. The molecular motions of the DOT1L-AF9 complex are less extensive and highly dynamic, resembling a swivel-like mechanics. Through molecular dynamics and mutational analysis, we extend the knowledge previous provided by static measurements. These results are important to consider when describing the biochemical properties of DOT1L, under normal and in disease conditions, as well as for the development of novel therapeutic agents.


Assuntos
Carcinogênese , Histona-Lisina N-Metiltransferase , Leucemia/metabolismo , Carcinogênese/química , Carcinogênese/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Simulação de Dinâmica Molecular , Nucleossomos/química , Nucleossomos/metabolismo , Proteínas de Fusão Oncogênica/química , Proteínas de Fusão Oncogênica/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo
2.
bioRxiv ; 2023 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-37207265

RESUMO

Current capabilities in genomic sequencing outpace functional interpretations. Our previous work showed that 3D protein structure calculations enhance mechanistic understanding of genetic variation in sequenced tumors and patients with rare diseases. The KRAS GTPase is among the critical genetic factors driving cancer and germline conditions. Because KRAS-altered tumors frequently harbor one of three classic hotspot mutations, nearly all studies have focused on these mutations, leaving significant functional ambiguity across the broader KRAS genomic landscape observed in cancer and non-cancer diseases. Herein, we extend structural bioinformatics with molecular simulations to study an expanded landscape of 86 KRAS mutations. We identify multiple coordinated changes strongly associated with experimentally established KRAS biophysical and biochemical properties. The patterns we observe span hotspot and non-hotspot alterations, which can all dysregulate Switch regions, producing mutation-restricted conformations with different effector binding propensities. We experimentally measured mutation thermostability and identified shared and distinct patterns with simulations. Our results indicate mutation-specific conformations which show potential for future research into how these alterations reverberate into different molecular and cellular functions. The data we present is not predictable using current genomic tools, demonstrating the added functional information derived from molecular simulations for interpreting human genetic variation.

3.
Comput Struct Biotechnol J ; 21: 4790-4803, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37841325

RESUMO

Current capabilities in genomic sequencing outpace functional interpretations. Our previous work showed that 3D protein structure calculations enhance mechanistic understanding of genetic variation in sequenced tumors and patients with rare diseases. The KRAS GTPase is among the critical genetic factors driving cancer and germline conditions. Because KRAS-altered tumors frequently harbor one of three classic hotspot mutations, nearly all studies have focused on these mutations, leaving significant functional ambiguity across the broader KRAS genomic landscape observed in cancer and non-cancer diseases. Herein, we extend structural bioinformatics with molecular simulations to study an expanded landscape of 86 KRAS mutations. We identify multiple coordinated changes strongly associated with experimentally established KRAS biophysical and biochemical properties. The patterns we observe span hotspot and non-hotspot alterations, which can all dysregulate Switch regions, producing mutation-restricted conformations with different effector binding propensities. We experimentally measured mutation thermostability and identified shared and distinct patterns with simulations. Our results indicate mutation-specific conformations, which show potential for future research into how these alterations reverberate into different molecular and cellular functions. The data we present is not predictable using current genomic tools, demonstrating the added functional information derived from molecular simulations for interpreting human genetic variation.

4.
Comput Struct Biotechnol J ; 20: 2200-2211, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35615018

RESUMO

The histone demethylase KDM6A has recently elicited significant attention because its mutations are associated with a rare congenital disorder (Kabuki syndrome) and various types of human cancers. However, distinguishing KDM6A mutations that are deleterious to the enzyme and their underlying mechanisms of dysfunction remain to be fully understood. Here, we report the results from a multi-tiered approach evaluating the impact of 197 KDM6A somatic mutations using information derived from combining conventional genomics data with computational biophysics. This comprehensive approach incorporates multiple scores derived from alterations in protein sequence, structure, and molecular dynamics. Using this method, we classify the KDM6A mutations into 136 damaging variants (69.0%), 32 tolerated variants (16.2%), and 29 variants of uncertain significance (VUS, 14.7%), which is a significant improvement from the previous classification based on the conventional tools (over 40% VUS). We further classify the damaging variants into 15 structural variants (SV), 88 dynamic variants (DV), and 33 structural and dynamic variants (SDV). Comparison with variant scoring methods used in current clinical diagnosis guidelines demonstrates that our approach provides a more comprehensive evaluation of damaging potential and reveals mechanisms of dysfunction. Thus, these results should be taken into consideration for clinical assessment of the damaging potential of each mutation, as they provide hypotheses for experimental validation and critical information for the development of mutant-specific drugs to fight diseases caused by KDM6A dysfunctions.

5.
Orphanet J Rare Dis ; 16(1): 66, 2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33546721

RESUMO

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.


Assuntos
Doenças Hematológicas , Histona Desmetilases/genética , Doenças Vestibulares , Anormalidades Múltiplas , Face/anormalidades , Doenças Hematológicas/genética , Humanos , Simulação de Dinâmica Molecular , Mutação , Doenças Vestibulares/genética
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