Characterizing Structural Transitions Using Localized Free Energy Landscape Analysis

Structural changes in molecules are frequently observed during biological processes like replication, transcription and translation. These structural changes can usually be traced to specific distortions in the backbones of the macromolecules involved. Quantitative energetic characterization of such distortions can greatly advance the atomic-level understanding of the dynamic character of these biological processes.Molecular dynamics simulations combined with a variation of the Weighted Histogram Analysis Method for potential of mean force determination are applied to characterize localized structural changes for the test case of cytosine (underlined) base flipping in a GTCAGCGCATGG DNA duplex. Free energy landscapes for backbone torsion and sugar pucker degrees of freedom in the DNA are used to understand their behavior in response to the base flipping perturbation. By simplifying the base flipping structural change into a two-state model, a free energy difference of upto 14 kcal/mol can be attributed to the flipped state relative to the stacked Watson-Crick base paired state. This two-state classification allows precise evaluation of the effect of base flipping on local backbone degrees of freedom.The calculated free energy landscapes of individual backbone and sugar degrees of freedom expectedly show the greatest change in the vicinity of the flipping base itself, but specific delocalized effects can be discerned upto four nucleotide positions away in both 5' and 3' directions. Free energy landscape analysis thus provides a quantitative method to pinpoint the determinants of structural change on the atomic scale and also delineate the extent of propagation of the perturbation along the molecule. In addition to nucleic acids, this methodology is anticipated to be useful for studying conformational changes in all macromolecules, including carbohydrates, lipids, and proteins

Hierarchical Modeling of Activation Mechanisms in the ABL and EGFR Kinase Domains: Thermodynamic and Mechanistic Catalysts of Kinase Activation by Cancer Mutations

Structural and functional studies of the ABL and EGFR kinase domains have recently suggested a common mechanism of activation by cancer-causing mutations. However, dynamics and mechanistic aspects of kinase activation by cancer mutations that stimulate conformational transitions and thermodynamic stabilization of the constitutively active kinase form remain elusive. We present a large-scale computational investigation of activation mechanisms in the ABL and EGFR kinase domains by a panel of clinically important cancer mutants ABL-T315I, ABL-L387M, EGFR-T790M, and EGFR-L858R. We have also simulated the activating effect of the gatekeeper mutation on conformational dynamics and allosteric interactions in functional states of the ABL-SH2-SH3 regulatory complexes. A comprehensive analysis was conducted using a hierarchy of computational approaches that included homology modeling, molecular dynamics simulations, protein stability analysis, targeted molecular dynamics, and molecular docking. Collectively, the results of this study have revealed thermodynamic and mechanistic catalysts of kinase activation by major cancer-causing mutations in the ABL and EGFR kinase domains. By using multiple crystallographic states of ABL and EGFR, computer simulations have allowed one to map dynamics of conformational fluctuations and transitions in the normal (wild-type) and oncogenic kinase forms. A proposed multi-stage mechanistic model of activation involves a series of cooperative transitions between different conformational states, including assembly of the hydrophobic spine, the formation of the Src-like intermediate structure, and a cooperative breakage and formation of characteristic salt bridges, which signify transition to the active kinase form. We suggest that molecular mechanisms of activation by cancer mutations could mimic the activation process of the normal kinase, yet exploiting conserved structural catalysts to accelerate a conformational transition and the enhanced stabilization of the active kinase form. The results of this study reconcile current experimental data with insights from theoretical approaches, pointing to general mechanistic aspects of activating transitions in protein kinases

Sequence and Structure Signatures of Cancer Mutation Hotspots in Protein Kinases

Protein kinases are the most common protein domains implicated in cancer, where somatically acquired mutations are known to be functionally linked to a variety of cancers. Resequencing studies of protein kinase coding regions have emphasized the importance of sequence and structure determinants of cancer-causing kinase mutations in understanding of the mutation-dependent activation process. We have developed an integrated bioinformatics resource, which consolidated and mapped all currently available information on genetic modifications in protein kinase genes with sequence, structure and functional data. The integration of diverse data types provided a convenient framework for kinome-wide study of sequence-based and structure-based signatures of cancer mutations. The database-driven analysis has revealed a differential enrichment of SNPs categories in functional regions of the kinase domain, demonstrating that a significant number of cancer mutations could fall at structurally equivalent positions (mutational hotspots) within the catalytic core. We have also found that structurally conserved mutational hotspots can be shared by multiple kinase genes and are often enriched by cancer driver mutations with high oncogenic activity. Structural modeling and energetic analysis of the mutational hotspots have suggested a common molecular mechanism of kinase activation by cancer mutations, and have allowed to reconcile the experimental data. According to a proposed mechanism, structural effect of kinase mutations with a high oncogenic potential may manifest in a significant destabilization of the autoinhibited kinase form, which is likely to drive tumorigenesis at some level. Structure-based functional annotation and prediction of cancer mutation effects in protein kinases can facilitate an understanding of the mutation-dependent activation process and inform experimental studies exploring molecular pathology of tumorigenesis

The Energy Landscape Analysis of Cancer Mutations in Protein Kinases

The growing interest in quantifying the molecular basis of protein kinase activation and allosteric regulation by cancer mutations has fueled computational studies of allosteric signaling in protein kinases. In the present study, we combined computer simulations and the energy landscape analysis of protein kinases to characterize the interplay between oncogenic mutations and locally frustrated sites as important catalysts of allostetric kinase activation. While structurally rigid kinase core constitutes a minimally frustrated hub of the catalytic domain, locally frustrated residue clusters, whose interaction networks are not energetically optimized, are prone to dynamic modulation and could enable allosteric conformational transitions. The results of this study have shown that the energy landscape effect of oncogenic mutations may be allosteric eliciting global changes in the spatial distribution of highly frustrated residues. We have found that mutation-induced allosteric signaling may involve a dynamic coupling between structurally rigid (minimally frustrated) and plastic (locally frustrated) clusters of residues. The presented study has demonstrated that activation cancer mutations may affect the thermodynamic equilibrium between kinase states by allosterically altering the distribution of locally frustrated sites and increasing the local frustration in the inactive form, while eliminating locally frustrated sites and restoring structural rigidity of the active form. The energy landsape analysis of protein kinases and the proposed role of locally frustrated sites in activation mechanisms may have useful implications for bioinformatics-based screening and detection of functional sites critical for allosteric regulation in complex biomolecular systems

Report of an International Network of Cancer Treatment and Research workshop on non-Hodgkin\u27s lymphoma in developing countries

The International Network of Cancer Treatment and Research (INCTR) recently organized a workshop on non-Hodgkin lymphomas (NHLs) in selected developing countries with the purpose of examining existing information relating to the pathology and management of these neoplasms, and identifying potential areas for research. This report provides a summary of the information presented and is focused primarily on the pathology of NHLs in children and adults. In most countries, the WHO classification of lymphomas was used and most participating centers included immunohistochemistry using a wide array of lymphoid antibodies as part of routine diagnosis. Some of the series had been reviewed by an external panel of experts. B-cell lymphomas accounted for 82-88% of all NHLs. The proportions of chronic lymphatic leukemia (4-6%), mantle cell lymphoma (MCL, 3-5%), and plasmacytoma (2-4%) were similar in the series presented. However, there was a significant variation in the proportion of follicular lymphoma (FL), which accounted for 15% and 11% in India and Kuwait, but less than 5% in Pakistan and Egypt. All of these frequencies are significantly lower than those reported in Western series. Diffuse large B-cell lymphoma accounted for about 35% of cases in India but for more 50% in other countries, but this difference was not accounted for by an increased incidence in a single lymphoma subtype in India, but rather an apparent paucity of several subtypes (such as mantle cell and marginal zone lymphomas (MZL)) in other series. There were relatively high frequencies of Burkitt lymphoma in Egypt (7%) and precursor T-cell lymphoblastic lymphoma in India (6-7%). Peripheral T-cell lymphomas (PTCLs) (not otherwise specified and angioimmunoblastic subtypes) accounted for 3-5% of NHLs, and extranodal lymphoma of T/NK cell type was rare (\u3c1%). These differences in the relative proportions of NHL subtypes among developing countries and between developing countries and the rest of the world presumably arise from differences in environmental and genetic factors that influence lymphomagenesis and strongly suggest that more research in developing countries would provide valuable insights into the pathogenesis of lymphoid neoplasms

Mapping the conformational transition in Src activation by cumulating the information from multiple molecular dynamics trajectories

The Src-family kinases are allosteric enzymes that play a key role in the regulation of cell growth and proliferation. In response to cellular signals, they undergo large conformational changes to switch between distinct inactive and active states. A computational strategy for characterizing the conformational transition pathway is presented to bridge the inactive and active states of the catalytic domain of Hck. The information from a large number (78) of independent all-atom molecular dynamics trajectories with explicit solvent is combined together to assemble a connectivity map of the conformational transition. Two intermediate states along the activation pathways are identified, and their structural features are characterized. A coarse free-energy landscape is built in terms of the collective motions corresponding to the opening of the activation loop (A-loop) and the rotation of the αC helix. This landscape shows that the protein can adopt a multitude of conformations in which the A-loop is partially open, while the αC helix remains in the orientation characteristic of the inactive conformation. The complete transition leading to the active conformation requires a concerted movement involving further opening of the A-loop, the relative alignment of N-lobe and C-lobe, and the rotation of the αC helix needed to recruit the residues necessary for catalysis in the active site. The analysis leads to a dynamic view of the full-length kinase activation, whereby transitions of the catalytic domain to intermediate configurations with a partially open A-loop are permitted, even while the SH2-SH3 clamp remains fully engaged. These transitions would render Y416 available for the transphosphorylation event that ultimately locks down the active state. The results provide a broad framework for picturing the conformational transitions leading to kinase activation

Outcome of operable oral cavity cancer and impact of maintenance metronomic chemotherapy: A retrospective study from rural India

Background: Oral cavity cancer is the most common cancer among rural India. There is a paucity of data for outcomes of operable oral cavity cancer from rural India. Use of maintenance metronomic may delay or avoid relapse. Aim: To evaluate outcomes of operable oral cavity carcinoma and evaluate impact of maintenance metronomic chemotherapy. Objectives: To evaluate disease-free survival (DFS), overall survival (OS), and factors affecting the outcome in operable oral cavity cancer. Materials and Methods: Data of patients diagnosed with oral cavity cancer registered between May 2008 and May 2014 were retrieved. Only those patients with operable oral cavity cancer and upfront definitive surgery were included in the study. Demographic profile, stage, tobacco consumption, adjuvant therapy, and pattern of failure were collected. Kaplan-Meir survival analysis was used to determine DFS and OS. Log-rank test was used to evaluate factors affecting outcome. Results: Median follow-up is 24 months. Out of 335 patients, 225 (67%) had advanced operable cancer with 42/225 (18%) and 183/225 (82%) as Stages III and IVA, respectively. Buccal mucosa was the most common subsite (178/335, 53%) followed by tongue (63/335, 19%). Ninety-two percent patients were addicted to smokeless tobacco, whereas 27% were smokers. Median DFS is 13 months with 2 years relative DFS 32%. Median OS is 30 months, with 2 years OS of 54%. Metronomic adjuvant oral chemotherapy was given in 130/225 (58%); Stage III and IVA patients with median of 14 months (3-18 months). Use of metronomic chemotherapy improved DFS (8 vs. 14 months, P = 0.22) and OS (14 vs. 26 months, P = 0.04). Conclusion: Oral cavity cancer is a major health care problem in rural India. Presentation at advanced stage leads to suboptimal outcomes. Benefit of metronomic maintenance chemotherapy in locally advanced oral cavity needs to be further evaluated prospectively