Ashok K. Varma

Molecular Modeling of Differentially Phosphorylated Serine 10 and Acetylated lysine 9/14 of Histone H3 Regulates their Interactions with 14-3-3ζ, MSK1, and MKP1

Histone modifications occur in precise patterns, with several modifications known to affect the binding of proteins. These interactions affect the chromatin structure, gene regulation, and cell cycle events. The dual modifications on the H3 tail, serine10 phosphorylation, and lysine14 acetylation (H3Ser10PLys14Ac) are reported to be crucial for interaction with 14-3-3ζ. However, the mechanism by which H3Ser10P along with neighboring site-specific acetylation(s) is targeted by its regulatory proteins, including kinase and phosphatase, is not fully understood. We carried out molecular modeling studies to understand the interaction of 14-3-3ζ, and its regulatory proteins, mitogen-activated protein kinase phosphatase-1 (MKP1), and mitogen- and stress-activated protein kinase-1 (MSK1) with phosphorylated H3Ser10 alone or in combination with acetylated H3Lys9 and Lys14. In silico molecular association studies suggested that acetylated Lys14 and phosphorylated Ser10 of H3 shows the highest binding affinity towards 14-3-3ζ. In addition, acetylation of H3Lys9 along with Ser10PLys14Ac favors the interaction of the phosphatase, MKP1, for dephosphorylation of H3Ser10P. Further, MAP kinase, MSK1 phosphorylates the unmodified H3Ser10 containing N-terminal tail with maximum affinity compared to the N-terminal tail with H3Lys9AcLys14Ac. The data clearly suggest that opposing enzymatic activity of MSK1 and MKP1 corroborates with non-acetylated and acetylated, H3Lys9Lys14, respectively. Our in silico data highlights that site-specific phosphorylation (H3Ser10P) and acetylation (H3Lys9 and H3Lys14) of H3 are essential for the interaction with their regulatory proteins (MKP1, MSK1, and 14-3-3ζ) and plays a major role in the regulation of chromatin structure.

Targeting Pim-1 kinase for potential drug-development

Dysregulation of Pim-1 kinase has been implicated in several human cancers. Many potential inhibitors of PIM kinase have been reported, but potential bioactive compounds are still far from reach. Keeping this in mind, we have selected structurally known diverse Pim-1 kinase inhibitors to find novel small molecule drug-leads. A ligand-based pharmacophore model for Pim-1 kinase was developed using PHASE software. A four feature pharmacophoric hypothesis (AAHR) was used to develop atom-based 3D-QSAR model with the best regression coefficient of 0.9433 and Pearson-R of 0.9344. Compounds from Asinex platinum database were obtained whose pIC(50) values matched the 3D-QSAR model. Structural and molecular interaction studies on the training and test sets suggest that designing novel compounds hydrogen bond with Asp128 in the bioactive region of Pim-1 kinase would result in therapeutic success.

Structural basis to stabilize the domain motion of BARD1-ARD BRCT by CstF50

BRCA1 associated ring domain protein 1(BARD1) is a tumor suppressor protein having a wide role in cellular processes like cell-cycle checkpoint, DNA damage repair and maintenance of genomic integrity. Germ-line mutation Gln 564 His discovered in linker region of BARD1 leads to loss of binding to Cleavage stimulating factor (CstF50), which in turn instigates the premature mRNA transcript formation and apoptosis. We have studied the dynamics of ARD domain present in the BARD1 wild-type and mutant protein in association with CstF50 using biophysical, biochemical and molecular dynamics simulations. It has been observed that the ARD domain is relatively more flexible than the BRCT domain of BARD1. Further relative orientations of both the ARD and BRCT domains varies due to the highly flexible nature of the connecting linker region present between the domains. It has been observed that mutant ARD domain is more dynamic in nature compared to wild-type protein. Molecular docking studies between BARD1 Gln 564 His mutant and CstF50 shows the loss of interactions. Furthermore, domain motion of ARD present in BARD1 was stabilized when complexed with CstF50.

Pathogenicity of Mutations Discovered in BRCA1 BRCT Domains is Characterized by Destabilizing the Hydrophobic Interactions

Background: Breast and ovarian cancer are the most common cancers among hetrogenetically diversified women. It is quite difficult to categorize the population at a high risk of breast cancer using peer genetic information because one particular mutation can be found in the same or in different families. Several mutations have been discovered across the full length of BRCA1 gene, and categorizing their pathogenicity is a major challenge. Carriers of BRCA1 mutations have an increased risk of developing cancer. In the breast cancer database BIC, approximately 1,500 genetic variants have been reported. It is very difficult to characterize each of the reported mutations. Given the complexities in characterizing the mutations, we decided to investigate functional basis associated with the mutations, rather than looking at each mutation. Materials and methods: BRCA1 BRCT domains were cloned, expressed, and purified using e-coli bacterial expression system. Mutations were generated using site-directed mutagenesis techniques, and all the mutations were sequence verified. The secondary structure of the mutant was characterized by Circular dichroism (CD) and Fluorescence spectroscopy. Molecular dynamics simulations were performed using Desmond software. Hydrophobic interactions and hydrogen bonding of docked molecules were compared using the LigPlot program. Results: Genetic mutations were discovered throughout BRCA1, and most of the pathogenic mutations were buried in the hydrophobic core and destabilized the BRCA1 BRCT domain. This unstable BRCT domain destabilized the full-length BRCA1, resulting in a loss of function. We conclude that the pathogenicity of each of the mutations in the BRCT domain can be categorized on the basis of its ability to destabilize the hydrophobic interactions. Although such instability is not sufficient to predispose someone to cancer, it provides a basis for formulating a concept for genetic counseling and targeted therapy.

Biophysical evaluation to categorize pathogenicity of cancer-predisposing mutations identified in the BARD1 BRCT domain

The BRCT domain of BARD1 (BARD1 BRCT) is involved in many cellular processes such as DNA damage repair (DDR) and cell-cycle checkpoint regulation. BARD1 BRCT performs tumor suppressor function by recruiting BRCA1 at DNA damage site via interactions with other DNA damage repair (DDR) proteins. Considering the importance of the BRCT domain in genomic integrity, we decided to evaluate reported mutations of BARD1 BRCT Cys645Arg, Val695Leu, and Ser761Asn for their pathogenicity. To explore the effect of the mutation on the structure and function, BARD1 BRCT wild-type proteins and the mutant proteins were studied using different biochemical, biophysical and in silico techniques. Comparative fluorescence, circular dichroism (CD) spectroscopy and limited proteolysis studies demonstrate the well-folded structural conformation of wild-type and mutant proteins. However, thermal and chemical denaturation studies revealed similarity in the folding pattern of BARD1 BRCT wild-type and Cys645Arg mutant proteins, whereas there was a significant loss in the thermodynamic stability of Val695Leu and Ser761Asn mutants. Molecular dynamics (MD) simulation studies on wild-type and mutant protein structures indicate the loss in structural integrity of mutants compared with the wild-type protein.

Studies of protein–protein interactions in Fanconi anemia pathway to unravel the DNA interstrand crosslink repair mechanism

Fanconi anemia (FA), a cancer predisposition syndrome exhibits hallmark feature of radial chromosome formation, and hypersensitivity to DNA crosslinking agents. A set of FA pathway proteins mainly FANCI, FANCD2 and BRCA2 are expressed to repair the covalent crosslink between the dsDNA. However, FA, BRCA pathways play an important role in DNA ICL repair as well as in homologous recombination repair, but the presumptive role of FA-BRCA proteins has not clearly explored particularly in context to function associated protein-protein interactions (PPIs). Here, in-vivo, in-vitro and in-silico studies have been performed for functionally relevant domains of FANCI, FANCD2 and BRCA2. To our conclusion, FANCI ARM repeat interacts with FANCD2 CUE domain and BRCA2 C-terminal region. Interestingly, FANCD2 CUE domain also interacts strongly with BRCA2 C-terminal region. Interactions between BRCA2 CTR and functionally relevant mutations Ser222Ala (cell cycle checkpoint mutant) and Leu231Arg (DNA ICL repair mutant) present in FANCD2 CUE domain have been analysed. To our finding, these mutations abrogate the binding between FANCD2 CUE domain and BRCA2 CTR. Furthermore, (1) different domain of FANCI, FANCD2 and BRCA2 are playing important role in PPIs, (2) mutations cause the impairment in the PPIs which in turn may disrupt the DNA ICL repair mechanism.

Abstract 4546: Structural evaluation of mutations identified on Cue domain of FANCD2

Fanconi anemia complementation group D2 (FANCD2) protein plays pivotal role in DNA interstrand crosslink (ICL) repair and genome stability. FANCD2 comprises conserved CUE domain at N terminus, which is important for DNA ICL repair and protein stability. However, molecular mechanism associated to genetic alteration has not well characterized. Here, we have carried out multimodal approaches to explore folding pattern of FANCD2 Cue domain and functionally relevant mutations. Dynamic Light Scattering and Mass Spectrometry data of purified proteins could revealed that wild- type protein is predominantly monomeric, but mutated counterparts are exhibiting oligomeric properties. Secondary, tertiary structure assessment and their thermal stability of wild- type, mutants were studied by Circular Dichroism (CD) and Fluorescence spectroscopy. Furthermore, binding affinity and kinetics of Cue domain and mutants with monoubiquitin was evaluated by surface plasmon resonance (SPR). Molecular modelling, simulation, normal mode analysis, principal component analysis and docking studies were carried out to understand in-depth conformational dynamics and interactions. References:- 1. Regulation of the Fanconi anemia pathway by a CUE ubiquitin-binding domain in the FANCD2 protein. Rego, M. A. et al, Blood 120: 2109-2117,(2012). 2. Hypomorphic mutations in the gene encoding a key Fanconi anemia protein, FANCD2, sustain a significant group of FA-D2 patients with severe phenotype. Kalb et al, Am J Hum Genet 80: 895-910, (2007) Citation Format: Mohd Quadir Siddiqui, Ashok K. Varma. Structural evaluation of mutations identified on Cue domain of FANCD2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4546. doi:10.1158/1538-7445.AM2017-4546

Protein–protein interactions of Fanconi anemia proteins FANCI, FANCD2 and BRCA2

Fanconi anemia (FA), a cancer predisposition syndrome, exhibit hallmark feature of radial chromosome formation and hyper sensitivity to DNA crosslinking agents [1]. A set of FA pathway (DNA inter-crosslink repair pathway) proteins mainly FANCI, FANCD2 and BRCA2 were expressed to repair the covalent crosslink between the dsDNA [2]. We have performed the multimodal approach to evaluate the structure and protein-protein interactions (PPI) between the different regions present in FANCI, FANCD2 and BRCA2 proteins. It has been observed that FANCI ARM repeat interacts with FANCD2 Cue domain and BRCA2 central region. Interestingly, FANCD2 Cue domain forms strong interaction with BRCA2 central region. We also tested the interaction between BRCA2 and functionally relevant mutations present in FANCD2 Cue domain, Ser222Ala (cell cycle checkpoint mutant) and Leu231Arg (DNA ICL repair mutant), and observed that mutations abrogates the binding ability. These results suggest that (1) domain and regions present in FANCI, FANCD2 and BRCA2 play important role in PPI, (2) mutations cause the failure in the PPI of these proteins, that affect the cell cycle and DNA repair processes. [1]Alter, B. P. (2003). Cancer 97, 425-440. [2]Cohen, M. M., Simpson, S. J., Honig, G. R., Maurer, H. S., Nicklas, J. W. & Martin, A. O. (1982). American journal of human genetics 34, 794-810.

Structural characterization of retinoic acid receptor-α (RARA)

Acute promyelocytic leukemia (APL) is a hematological malignancy characterized by the presence of balanced reciprocal translocation between promyelocytic leukemia (PML) gene on chromosome 15 and the retinoic acid receptor-alpha (RARA) gene on chromosome 17. This translocation leads to a fusion transcript known as PML-RARA which is present in 98% cases of APL [1]. It is well established that PML-RARA associated APL is sensitive to both all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) [2]. It promotes the degradation of chimeric oncogenic protein through a series of molecular events leading to differentiation of promyelocytes. The mutations identified in RARA moiety such as Val218Asp, Arg72Gln, Thr278Ala, Thr291Ile, Asn299Asp, Arg294Trp, Ala300Gly and Gly391Glu affect the binding of ATRA which in turn result the non-degradation of chimeric oncoprotein [3]. Considering the importance of RARA, the RARA region comprising 60-462 amino acids was cloned, expressed and purified in bacterial system. Further purified by size exclusion chromatography using FPLC to get active protein in native conditions. Secondary structure and folding pattern of protein was characterised by Circular-Dicroism (CD) and Fluorimetry. It has been concluded that purified protein has correctly folded secondary and tertiary structures. We have observed the melting temperature (Tm) using Thermal denaturation by CD spectroscopy.

Structureomics in Systems-Based Drug Discovery

Selection of the appropriate drug target is crucial and furthermore its association with small molecule binding partners is the subject of extensive studies. Drug discovery has evolved from the traditional serendipity to system based approaches of using the three dimensional structures of biological molecules. This chapter provides a brief description about the contribution of large number of proteins structures in drug discovery. This further elucidates the target selection and its role in reducing the time and efforts required for drug development.