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author:("Laine, elodia")
1.  Hotspot Mutations in KIT Receptor Differentially Modulate Its Allosterically Coupled Conformational Dynamics: Impact on Activation and Drug Sensitivity 
PLoS Computational Biology  2014;10(7):e1003749.
Receptor tyrosine kinase KIT controls many signal transduction pathways and represents a typical allosterically regulated protein. The mutation-induced deregulation of KIT activity impairs cellular physiological functions and causes serious human diseases. The impact of hotspots mutations (D816H/Y/N/V and V560G/D) localized in crucial regulatory segments, the juxtamembrane region (JMR) and the activation (A-) loop, on KIT internal dynamics was systematically studied by molecular dynamics simulations. The mutational outcomes predicted in silico were correlated with in vitro and in vivo activation rates and drug sensitivities of KIT mutants. The allosteric regulation of KIT in the native and mutated forms is described in terms of communication between the two remote segments, JMR and A-loop. A strong correlation between the communication profile and the structural and dynamical features of KIT in the native and mutated forms was established. Our results provide new insight on the determinants of receptor KIT constitutive activation by mutations and resistance of KIT mutants to inhibitors. Depiction of an intra-molecular component of the communication network constitutes a first step towards an integrated description of vast communication pathways established by KIT in physiopathological contexts.
Author Summary
Receptor tyrosine kinase KIT plays a crucial role in the regulation of cell signaling. This allosterically controlled activity may be affected by gain-of-function mutations that promote the development of several cancers. Identification of the molecular basis of KIT constitutive activation and allosteric regulation has inspired computational study of KIT hotspot mutations. In the present contribution, we investigated the mutation-induced effects on KIT conformational dynamics and intra-protein communication conditionally on the mutation location and the nature of the substituting amino acid. Our data elucidate that all studied mutations stabilize an inactive non-autoinhibited state of KIT over the inactive auto-inhibited state prevalent for the native protein. This shift in the protein conformational landscape promotes KIT constitutive activation. Our in silico analysis established correlations between the structural and dynamical effects induced by oncogenic mutations and the mutants auto-activation rates and drug sensitivities measured in vitro and in vivo. Particularly, the A-loop mutations stabilize the drug-resistant forms, while the JMR mutations may facilitate inhibitors binding to the active site. Cross-correlations established between local and long-range structural and dynamical effects demonstrate the allosteric character of the gain-of-function mutations mode of action.
doi:10.1371/journal.pcbi.1003749
PMCID: PMC4117417  PMID: 25079768
2.  Differential Effects of CSF-1R D802V and KIT D816V Homologous Mutations on Receptor Tertiary Structure and Allosteric Communication 
PLoS ONE  2014;9(5):e97519.
The colony stimulating factor-1 receptor (CSF-1R) and the stem cell factor receptor KIT, type III receptor tyrosine kinases (RTKs), are important mediators of signal transduction. The normal functions of these receptors can be compromised by gain-of-function mutations associated with different physiopatological impacts. Whereas KIT D816V/H mutation is a well-characterized oncogenic event and principal cause of systemic mastocytosis, the homologous CSF-1R D802V has not been identified in human cancers. The KIT D816V oncogenic mutation triggers resistance to the RTK inhibitor Imatinib used as first line treatment against chronic myeloid leukemia and gastrointestinal tumors. CSF-1R is also sensitive to Imatinib and this sensitivity is altered by mutation D802V. Previous in silico characterization of the D816V mutation in KIT evidenced that the mutation caused a structure reorganization of the juxtamembrane region (JMR) and facilitated its departure from the kinase domain (KD). In this study, we showed that the equivalent CSF-1R D802V mutation does not promote such structural effects on the JMR despite of a reduction on some key H-bonds interactions controlling the JMR binding to the KD. In addition, this mutation disrupts the allosteric communication between two essential regulatory fragments of the receptors, the JMR and the A-loop. Nevertheless, the mutation-induced shift towards an active conformation observed in KIT D816V is not observed in CSF-1R D802V. The distinct impact of equivalent mutation in two homologous RTKs could be associated with the sequence difference between both receptors in the native form, particularly in the JMR region. A local mutation-induced perturbation on the A-loop structure observed in both receptors indicates the stabilization of an inactive non-inhibited form, which Imatinib cannot bind.
doi:10.1371/journal.pone.0097519
PMCID: PMC4020833  PMID: 24828813
3.  Protein-Protein Interactions in a Crowded Environment: An Analysis via Cross-Docking Simulations and Evolutionary Information 
PLoS Computational Biology  2013;9(12):e1003369.
Large-scale analyses of protein-protein interactions based on coarse-grain molecular docking simulations and binding site predictions resulting from evolutionary sequence analysis, are possible and realizable on hundreds of proteins with variate structures and interfaces. We demonstrated this on the 168 proteins of the Mintseris Benchmark 2.0. On the one hand, we evaluated the quality of the interaction signal and the contribution of docking information compared to evolutionary information showing that the combination of the two improves partner identification. On the other hand, since protein interactions usually occur in crowded environments with several competing partners, we realized a thorough analysis of the interactions of proteins with true partners but also with non-partners to evaluate whether proteins in the environment, competing with the true partner, affect its identification. We found three populations of proteins: strongly competing, never competing, and interacting with different levels of strength. Populations and levels of strength are numerically characterized and provide a signature for the behavior of a protein in the crowded environment. We showed that partner identification, to some extent, does not depend on the competing partners present in the environment, that certain biochemical classes of proteins are intrinsically easier to analyze than others, and that small proteins are not more promiscuous than large ones. Our approach brings to light that the knowledge of the binding site can be used to reduce the high computational cost of docking simulations with no consequence in the quality of the results, demonstrating the possibility to apply coarse-grain docking to datasets made of thousands of proteins. Comparison with all available large-scale analyses aimed to partner predictions is realized. We release the complete decoys set issued by coarse-grain docking simulations of both true and false interacting partners, and their evolutionary sequence analysis leading to binding site predictions. Download site: http://www.lgm.upmc.fr/CCDMintseris/
Author Summary
Protein-protein interactions (PPI) are at the heart of the molecular processes governing life and constitute an increasingly important target for drug design. Given their importance, it is vital to determine which protein interactions have functional relevance and to characterize the protein competition inherent to crowded environments, as the cytoplasm or the cellular organelles. We show that combining coarse-grain molecular cross-docking simulations and binding site predictions based on evolutionary sequence analysis is a viable route to identify true interacting partners for hundreds of proteins with a variate set of protein structures and interfaces. Also, we realize a large-scale analysis of protein binding promiscuity and provide a numerical characterization of partner competition and level of interaction strength for about 28000 false-partner interactions. Finally, we demonstrate that binding site prediction is useful to discriminate native partners, but also to scale up the approach to thousands of protein interactions. This study is based on the large computational effort made by thousands of internautes helping World Community Grid over a period of 7 months. The complete dataset issued by the computation and the analysis is released to the scientific community.
doi:10.1371/journal.pcbi.1003369
PMCID: PMC3854762  PMID: 24339765
4.  Allosteric Communication across the Native and Mutated KIT Receptor Tyrosine Kinase 
PLoS Computational Biology  2012;8(8):e1002661.
A fundamental goal in cellular signaling is to understand allosteric communication, the process by which signals originated at one site in a protein propagate dependably to affect remote functional sites. Here, we describe the allosteric regulation of the receptor tyrosine kinase KIT. Our analysis evidenced that communication routes established between the activation loop (A-loop) and the distant juxtamembrane region (JMR) in the native protein were disrupted by the oncogenic mutation D816V positioned in the A-loop. In silico mutagenesis provided a plausible way of restoring the protein communication detected in the native KIT by introducing a counter-balancing second mutation D792E. The communication patterns observed in the native and mutated KIT correlate perfectly with the structural and dynamical features of these proteins. Particularly, a long-distance effect of the D816V mutation manifested as an important structural re-organization of the JMR in the oncogenic mutant was completely vanished in the double mutant D816V/D792E. This detailed characterization of the allosteric communication in the different forms of KIT, native and mutants, was performed by using a modular network representation composed of communication pathways and independent dynamic segments. Such representation permits to enrich a purely mechanistic interaction-based model of protein communication by the introduction of concerted local atomic fluctuations. This method, validated on KIT receptor, may guide a rational modulation of the physiopathological activities of other receptor tyrosine kinases.
Author Summary
The majority of functionally important biological processes are regulated by allosteric communication within individual proteins and across protein complexes. Receptor tyrosine kinases (RTKs) control signal transduction pathways and consequently represent a typical paradigm. The mutation-induced deregulation of RTK activity impairs crucial cellular physiological functions and causes serious human diseases. The present study focuses on the allosteric communication across the three-dimensional structure of the RTK KIT cytoplasmic region. Combining a mechanistic model of information transmission with the analysis of concerted local atomic fluctuations we examined and compared the communication profiles in the native and D816V-mutated proteins. This approach permitted to localize and visualize communication routes in the native KIT and revealed that these routes were disrupted in the mutant D816V. We proposed in silico mutagenesis as a mean to restore the communication detected in the native KIT. Our work sheds light on the allosteric communication in RTKs, a phenomenon playing an essential role in signaling pathways albeit experiments do not provide the atomic details of the path followed in going from one structural element to the other. A rational understanding of the molecular determinants underlying the effects of disease-related kinase mutations may contribute to the improvement of targeted therapies.
doi:10.1371/journal.pcbi.1002661
PMCID: PMC3426562  PMID: 22927810
5.  In Silico and In Vitro Comparison of HIV-1 Subtypes B and CRF02_AG Integrases Susceptibility to Integrase Strand Transfer Inhibitors 
Advances in Virology  2012;2012:548657.
Most antiretroviral medical treatments were developed and tested principally on HIV-1 B nonrecombinant strain, which represents less than 10% of the worldwide HIV-1-infected population. HIV-1 circulating recombinant form CRF02_AG is prevalent in West Africa and is becoming more frequent in other countries. Previous studies suggested that the HIV-1 polymorphisms might be associated to variable susceptibility to antiretrovirals. This study is pointed to compare the susceptibility to integrase (IN) inhibitors of HIV-1 subtype CRF02_AG IN respectively to HIV-1 B. Structural models of B and CRF02_AG HIV-1 INs as unbound enzymes and in complex with the DNA substrate were built by homology modeling. IN inhibitors—raltegravir (RAL), elvitegravir (ELV) and L731,988—were docked onto the models, and their binding affinity for both HIV-1 B and CRF02_AG INs was compared. CRF02_AG INs were cloned and expressed from plasma of integrase strand transfer inhibitor (INSTI)-naïve infected patients. Our in silico and in vitro studies showed that the sequence variations between the INs of CRF02_AG and B strains did not lead to any notable difference in the structural features of the enzyme and did not impact the susceptibility to the IN inhibitors. The binding modes and affinities of INSTI inhibitors to B and CRF02_AG INs were found to be similar. Although previous studies suggested that several naturally occurring variations of CRF02_AG IN might alter either IN/vDNA interactions or INSTIs binding, our study demonstrate that these variations do affect neither IN activity nor its susceptibility to INSTIs.
doi:10.1155/2012/548657
PMCID: PMC3398581  PMID: 22829822
6.  Mutation D816V Alters the Internal Structure and Dynamics of c-KIT Receptor Cytoplasmic Region: Implications for Dimerization and Activation Mechanisms 
PLoS Computational Biology  2011;7(6):e1002068.
The type III receptor tyrosine kinase (RTK) KIT plays a crucial role in the transmission of cellular signals through phosphorylation events that are associated with a switching of the protein conformation between inactive and active states. D816V KIT mutation is associated with various pathologies including mastocytosis and cancers. D816V-mutated KIT is constitutively active, and resistant to treatment with the anti-cancer drug Imatinib. To elucidate the activating molecular mechanism of this mutation, we applied a multi-approach procedure combining molecular dynamics (MD) simulations, normal modes analysis (NMA) and binding site prediction. Multiple 50-ns MD simulations of wild-type KIT and its mutant D816V were recorded using the inactive auto-inhibited structure of the protein, characteristic of type III RTKs. Computed free energy differences enabled us to quantify the impact of D816V on protein stability in the inactive state. We evidenced a local structural alteration of the activation loop (A-loop) upon mutation, and a long-range structural re-organization of the juxta-membrane region (JMR) followed by a weakening of the interaction network with the kinase domain. A thorough normal mode analysis of several MD conformations led to a plausible molecular rationale to propose that JMR is able to depart its auto-inhibitory position more easily in the mutant than in wild-type KIT and is thus able to promote kinase mutant dimerization without the need for extra-cellular ligand binding. Pocket detection at the surface of NMA-displaced conformations finally revealed that detachment of JMR from the kinase domain in the mutant was sufficient to open an access to the catalytic and substrate binding sites.
Author Summary
Protein kinases are involved in a huge amount of cellular processes through phosphorylation, a crucial mechanism in cell signaling, and their misregulation often results in disease. The deactivation of protein tyrosine kinases (PTKs) or their oncogenic activation arises from mutations which affect the protein primary structure and the configuration of the enzymatic site apparently by stabilizing the activation loop (A-loop) extended conformation. Particularly, mutation D816V of receptor tyrosine kinase (RTK) KIT, found in patients with pediatric mastocytosis, acute leukemia or germ cell tumors, can be considered as the archetype of mutation inducing a displacement of the population equilibrium toward the active conformation. We present a comprehensive computational study of the activating mechanism(s) of this mutation. Our multi-approach in silico procedure evidenced a local alteration of the A-loop structure, and a long-range structural re-organization of the juxta-membrane region (JMR) followed by a weakening of the interaction network with the kinase domain. Our results provided a plausible conception of how the observed departure of JMR from kinase domain in the mutant promotes kinase mutant dimerization without requiring extra-cellular ligand binding. The pocket profiles we obtained suggested putative allosteric binding sites that could be targeted by ligands/modulators that trap the mutated enzyme.
doi:10.1371/journal.pcbi.1002068
PMCID: PMC3116893  PMID: 21698178

Results 1-6 (6)