The mechanism of activation of monomeric B-Raf V600E

Oncogenic mutations in the serine/threonine kinase B-Raf, particularly the V600E mutation, are frequent in cancer, making it a major drug target. Although much is known about B-Raf's active and inactive states, questions remain about the mechanism by which the protein changes between these two states. Here, we utilize molecular dynamics to investigate both wild-type and V600E B-Raf to gain mechanistic insights into the impact of the Val to Glu mutation. The results show that the wild-type and mutant follow similar activation pathways involving an extension of the activation loop and an inward motion of the αC-helix. The V600E mutation, however, destabilizes the inactive state by disrupting hydrophobic interactions present in the wild-type structure while the active state is stabilized through the formation of a salt bridge between Glu600 and Lys507. Additionally, when the activation loop is extended, the αC-helix is able to move between an inward and outward orientation as long as the DFG motif adopts a specific orientation. In that orientation Phe595 rotates away from the αC-helix, allowing the formation of a salt bridge between Lys483 and Glu501. These mechanistic insights have implications for the development of new Raf inhibitors.

Download Full-text

The Impact of Mutation L138F/L210F on the Orai Channel: A Molecular Dynamics Simulation Study

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.755247 ◽

2021 ◽

Vol 8 ◽

Author(s):

Xiaoqian Zhang ◽

Hua Yu ◽

Xiangdong Liu ◽

Chen Song

Keyword(s):

Molecular Dynamics ◽

Conformational Changes ◽

Calcium Release ◽

Hydrophobic Interactions ◽

Dynamics Simulation ◽

Wild Type ◽

Hydrophobic Region ◽

In The Wild ◽

Dynamics Simulations ◽

The Impact

The calcium release-activated calcium channel, composed of the Orai channel and the STIM protein, plays a crucial role in maintaining the Ca2+ concentration in cells. Previous studies showed that the L138F mutation in the human Orai1 creates a constitutively open channel independent of STIM, causing severe myopathy, but how the L138F mutation activates Orai1 is still unclear. Here, based on the crystal structure of Drosophila melanogaster Orai (dOrai), molecular dynamics simulations for the wild-type (WT) and the L210F (corresponding to L138F in the human Orai1) mutant were conducted to investigate their structural and dynamical properties. The results showed that the L210F dOrai mutant tends to have a more hydrated hydrophobic region (V174 to F171), as well as more dilated basic region (K163 to R155) and selectivity filter (E178). Sodium ions were located deeper in the mutant than in the wild-type. Further analysis revealed two local but essential conformational changes that may be the key to the activation. A rotation of F210, a previously unobserved feature, was found to result in the opening of the K163 gate through hydrophobic interactions. At the same time, a counter-clockwise rotation of F171 occurred more frequently in the mutant, resulting in a wider hydrophobic gate with more hydration. Ultimately, the opening of the two gates may facilitate the opening of the Orai channel independent of STIM.

Download Full-text

Crystal structure of microtubule affinity-regulating kinase 4 catalytic domain in complex with a pyrazolopyrimidine inhibitor

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15024747 ◽

2016 ◽

Vol 72 (2) ◽

pp. 129-134 ◽

Cited By ~ 22

Author(s):

John S. Sack ◽

Mian Gao ◽

Susan E. Kiefer ◽

Joseph E. Myers ◽

John A. Newitt ◽

...

Keyword(s):

Crystal Structure ◽

Catalytic Domain ◽

Neurofibrillary Tangle ◽

Binding Pocket ◽

Atp Binding ◽

Activation Loop ◽

Serine Threonine Kinase ◽

Atp Binding Site ◽

Threonine Kinase ◽

Catalytic Loop

Microtubule-associated protein/microtubule affinity-regulating kinase 4 (MARK4) is a serine/threonine kinase involved in the phosphorylation of MAP proteins that regulate microtubule dynamics. Abnormal activity of MARK4 has been proposed to contribute to neurofibrillary tangle formation in Alzheimer's disease. The crystal structure of the catalytic and ubiquitin-associated domains of MARK4 with a potent pyrazolopyrimidine inhibitor has been determined to 2.8 Å resolution with anRworkof 22.8%. The overall structure of MARK4 is similar to those of the other known MARK isoforms. The inhibitor is located in the ATP-binding site, with the pyrazolopyrimidine group interacting with the inter-lobe hinge region while the aminocyclohexane moiety interacts with the catalytic loop and the DFG motif, forcing the activation loop out of the ATP-binding pocket.

Download Full-text

Arabidopsis MLK3, a Plant-specific Casein Kinase 1, Negatively Regulated Flowering and Phosphorylated Histone H3 in vivo

10.21203/rs.2.16145/v1 ◽

2019 ◽

Author(s):

Zhen Wang ◽

Junmei Kang ◽

Shangang Jia ◽

Tiejun Zhang ◽

Zhihai Wu ◽

...

Keyword(s):

Kinase Activity ◽

Histone H3 ◽

Casein Kinase ◽

Kinase Assay ◽

Wild Type ◽

Serine Threonine Kinase ◽

Altered Expression ◽

Casein Kinase 1 ◽

Threonine Kinase

Abstract Background: Casein kinase 1 (CK1) family members are highly conserved serine/threonine kinase present in most eukaryotes with multiple biological functions. Arabidopsis MUT9-like kinases ( MLKs ) belong to a clade CK1 specific to the plant kingdom and have been implicated collectively in modulating flowering related processes. Three of the four MLKs ( MLK1/2/4 ) have been characterized, however, little is known about MLK3 , the most divergent MLKs. Results: We demonstrated that compared with wild type, mlk3 , a truncated MLK3 , flowered slightly early under long day conditions and ectopic expression of MLK3 rescued the morphological defects of mlk3 , indicating that MLK3 negatively regulates flowering. GA 3 application accelerated flowering of both wild type and mlk3 , suggesting that mlk3 had normal GA response. The recombinant MLK3-GFP was localized in the nucleus exclusively. In vitro kinase assay revealed that the nuclear protein MLK3 phosphorylated histone 3 at threonine 3 (H3T3ph). Mutation of a conserved catalytic residue (Lysine 175) abolished the kinase activity and resulted in failure to complement the early flowering phenotype of mlk3 . Interestingly, the global level of H3T3 phosphorylation in mlk3 did not differ significantly from wild type, suggesting the redundant roles of MLKs in flowering regulation. The transcriptomic analysis demonstrated that 425 genes significantly altered expression level in mlk3 relative to wild type. The mlk3 mlk4 double mutant generated by crossing mlk3 with mlk4 , a loss-of-function mutant of MLK4 showing late flowering, flowered between the two parental lines, suggesting that MLK3 played an antagonistic role to MLK4 in plant transition to flowering. Conclusions: A serine/threonine kinase encoding gene MLK3 is a casein kinase 1 specific to the plant species and represses flowering slightly. MLK3 located in nucleus catalyzes the phosphorylation of histone H3 at threonine 3 in vitro and an intact lysine residue (K175) is indispensible for the kinase activity. This study sheds new light on the delicate control of flowering by the plant-specific CK1 in Arabidopsis.

Download Full-text

Relevance of STK11 Mutations Regarding Immune Cell Infiltration, Drug Sensitivity, and Cellular Processes in Lung Adenocarcinoma

Frontiers in Oncology ◽

10.3389/fonc.2020.580027 ◽

2020 ◽

Vol 10 ◽

Author(s):

Zhenqing Li ◽

Bo Ding ◽

Jianxun Xu ◽

Kai Mao ◽

Pengfei Zhang ◽

...

Keyword(s):

Lung Adenocarcinoma ◽

Drug Sensitivity ◽

Immune Cell ◽

Differentially Expressed ◽

Cell Infiltration ◽

Immune Cell Infiltration ◽

Wild Type ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Cellular Processes

Serine/threonine kinase 11 (STK11) is one member of the serine/threonine kinase family, which is involved in regulating cell polarity, apoptosis, and DNA damage repair. In lung adenocarcinoma (LUAD), it can play as one tumor suppressor and always be mutated. In this study, we aimed to assess the relevance of STK11 mutations in LUAD, in which we also studied the correlation among immune cell infiltration, drug sensitivity, and cellular processes. By performing the bioinformatics analysis of the Cancer Genome Atlas (TCGA) about LUAD patients, we found that the mutation efficiency of STK11 mutations is about 19%. Additionally, the differentially expressed gene analysis showed that there were 746 differentially expressed genes (DEGs) between LUAD patients with and without STK11 mutations. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis showed that the DEGs were enriched in various tumorigenesis signaling pathways and metabolic processes. Among these DEGs, the top ranking 21 genes were found that they were more frequently mutated in the STK11 mutation group than in the wild-type group (p-value<0.01). Finally, the LUAD patients with STK11 mutations suffered the worse immune cell infiltration levels than the LUAD patients with wild-type. The STK11 gene copy number was correlated with immune cell infiltration. Aiming to develop the therapeutic drugs, we performed Genomics of Drug Sensitivity in Cancer (GDSC) data to identify the potential therapeutic candidate and the results showed that Nutlin-3a(-) may be a sensitive drug for LUAD cases harboring STK11 mutations. The specific genes and pathways shown to be associated with LUAD cases involving STK11 mutations may serve as targets for individualized LUAD treatment.

Download Full-text

Mutations in Four Regulatory Genes Have Interrelated Effects on Heterocyst Maturation in Anabaena sp. Strain PCC 7120

Journal of Bacteriology ◽

10.1128/jb.00974-06 ◽

2006 ◽

Vol 188 (21) ◽

pp. 7387-7395 ◽

Cited By ~ 16

Author(s):

Sigal Lechno-Yossef ◽

Qing Fan ◽

Shigeki Ehira ◽

Naoki Sato ◽

C. Peter Wolk

Keyword(s):

Type Strain ◽

Wild Type Strain ◽

Response Regulator ◽

Regulatory System ◽

Transcript Abundance ◽

Regulatory Genes ◽

Wild Type ◽

Serine Threonine Kinase ◽

In The Wild ◽

Pcc 7120

ABSTRACT Regulatory genes hepK, hepN, henR, and hepS are required for heterocyst maturation in Anabaena sp. strain PCC 7120. They presumptively encode two histidine kinases, a response regulator, and a serine/threonine kinase, respectively. To identify relationships between those genes, we compared global patterns of gene expression, at 14 h after nitrogen step-down, in corresponding mutants and in the wild-type strain. Heterocyst envelopes of mutants affected in any of those genes lack a homogeneous, polysaccharide layer. Those of a henR mutant also lack a glycolipid layer. patA, which encodes a positive effector of heterocyst differentiation, was up-regulated in all mutants except the hepK mutant, suggesting that patA expression may be inhibited by products related to heterocyst development. hepS and hepK were up-regulated if mutated and so appear to be negatively autoregulated. HepS and HenR regulated a common set of genes and so appear to belong to one regulatory system. Some nontranscriptional mechanism may account for the observation that henR mutants lack, and hepS mutants possess, a glycolipid layer, even though both mutations down-regulated genes involved in formation of the glycolipid layer. HepK and HepN also affected transcription of a common set of genes and therefore appear to share a regulatory pathway. However, the transcript abundance of other genes differed very significantly from expression in the wild-type strain in either the hepK or hepN mutant while differing very little from wild-type expression in the other of those two mutants. Therefore, hepK and hepN appear to participate also in separate pathways.

Download Full-text

PRKD2 Serine-Threonine Kinase, a Downstream Effector Of GABP, Plays Essential Roles For The Maintenance Of HSCs

Blood ◽

10.1182/blood.v122.21.2430.2430 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2430-2430

Author(s):

Zhong-Fa Yang ◽

Wang Junling ◽

Alan G. Rosmarin

Keyword(s):

Bone Marrow ◽

Transcription Factor ◽

Protein Kinase ◽

Bone Marrow Cells ◽

Specific Cell ◽

Wild Type ◽

Serine Threonine Kinase ◽

Hematopoietic Stem ◽

Threonine Kinase ◽

Marrow Cells

Abstract Hematopoietic stem cells (HSCs) are the source of all blood lineages, and HSCs must balance quiescence, self-renewal, and differentiation to meet lifelong needs for blood cell development. GABP is an ets-related transcription factor that controls critical genes in myeloid and lymphoid development, and has been implicated in control of HSC growth. GABP is an obligate multimeric transcription factor that includes the DNA-binding ets component, GABPa, along with various GABPb partner proteins. We conditionally deleted Gabpa in mouse bone marrow and found that Gabpa cells have a profound growth disadvantage due to cell cycle arrest in HSCs. We identified Protein Kinase D2 (PRKD2) as a candidate effector of GABP. PRKD2 is a diacyl glycerol- and Protein Kinase C-activated serine-threonine kinase, because deletion of Gabpa markedly reduced PRKD2 expression in normal HSCs and progenitor cells. In a Prkd2ki/ki mouse model, in which two functionally essential phosphorylation serines were inactivated genetically, their bone marrow long term HSCs reduced dramatically and the short term HSCs increased accordingly. Mice transplanted with a 1:1 mixture of Prkd2ki/ki and wild type bone marrow cells demonstrated the decreased proportion of the Prkd2ki/ki bone marrow cells with the corresponding increase of the wild type cells. Although ectopic expression of the human Chronic Myeloid Leukemia (CML) fusion oncogene BCR-ABL in wild type bone marrow cells induced rapid CML development, expression of BCR-ABL in Prkd2ki/ki bone marrow cells failed to develop CML in transplanted recipient mice. Analysis of the peripheral blood, bone marrow and spleen of these mice revealed that the BCR-ABL+, Prkd2ki/ki cells did not express myeloid or lymphoid specific cell surface antigens CD11b, Gr1, B220, or CD3e. They demonstrated an immature blast-like microscopic morphology, and recipient mice transplanted with these cells died before the onset of CML development. We conclude that the phosphorylation activated Prkd2 is required for the maintenance of HSC pool and the development of mature hematopoietic lineages from HSCs. These findings suggest that PRKD2 kinase mediate key downstream events of both PKC and transcription factor GABP, and that PRKD2 may serve as a novel therapeutic target in leukemia. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

TheMycobacterium tuberculosis serine/threonine kinase PknL phosphorylates Rv2175c: Mass spectrometric profiling of the activation loop phosphorylation sites and their role in the recruitment of Rv2175c

PROTEOMICS ◽

10.1002/pmic.200700442 ◽

2008 ◽

Vol 8 (3) ◽

pp. 521-533 ◽

Cited By ~ 46

Author(s):

Marc J. Canova ◽

Romain Veyron-Churlet ◽

Isabelle Zanella-Cleon ◽

Martin Cohen-Gonsaud ◽

Alain J. Cozzone ◽

...

Keyword(s):

Mass Spectrometric ◽

Activation Loop ◽

Phosphorylation Sites ◽

Serine Threonine Kinase ◽

Threonine Kinase

Download Full-text

Molecular Dynamics Approach in the Comparison of Wild-Type and Mutant Paraoxonase-1 Apoenzyme Form

Bioinformatics and Biology Insights ◽

10.4137/bbi.s25626 ◽

2015 ◽

Vol 9 ◽

pp. BBI.S25626 ◽

Cited By ~ 3

Author(s):

Khadija Amine ◽

Lamia Miri ◽

Adil Naimi ◽

Rachid Saile ◽

Abderrahmane El Kharrim ◽

...

Keyword(s):

Molecular Dynamics ◽

Active Site ◽

Paraoxonase 1 ◽

Wild Type ◽

Repetitive Movement ◽

Catalytic Core ◽

In The Wild ◽

Dynamics Simulations ◽

L1 And L2 ◽

The Impact

There is some evidence linking the mammalian paraoxonase-1 (PON1) loops (L1 and L2) to an increased flexibility and reactivity of its active site with potential substrates. The aim of this work is to study the structural, dynamical, and functional effects of the most flexible regions close to the active site and to determine the impact of mutations on the protein. For both models, wild-type (PON1wild) and PON1 mutant (PON1mut) models, the L1 loop and Q/R and L/M mutations were constructed using MODELLER software. Molecular dynamics simulations of 20 ns at 300 K on fully modeled PON1wild and PON1mut apoenzyme have been done. Detailed analyses of the root-mean-square deviation and fluctuations, H-bonding pattern, and torsion angles have been performed. The PON1wild results were then compared with those obtained for the PON1mut. Our results show that the active site in the wild-type structure is characterized by two distinct movements of opened and closed conformations of the L1 and L2 loops. The alternating and repetitive movement of loops at specific times is consistent with the presence of 11 defined hydrogen bonds. In the PON1mut, these open-closed movements are therefore totally influenced and repressed by the Q/R and L/M mutations. In fact, these mutations seem to impact the PON1mut active site by directly reducing the catalytic core flexibility, while maintaining a significant mobility of the switch regions delineated by the loops surrounding the active site. The impact of the studied mutations on structure and dynamics proprieties of the protein may subsequently contribute to the loss of both flexibility and activity of the PON1 enzyme.

Download Full-text

Origin of Conformational Dynamics in a Globular Protein

10.1101/724286 ◽

2019 ◽

Author(s):

Adam M. Damry ◽

Marc M. Mayer ◽

Aron Broom ◽

Natalie K. Goto ◽

Roberto A. Chica

Keyword(s):

Conformational Dynamics ◽

Protein Structures ◽

Vital Role ◽

Globular Protein ◽

Conformational Exchange ◽

Solution Nmr ◽

Wild Type ◽

In The Wild ◽

Dynamics Simulations ◽

The Impact

AbstractProtein structures are dynamic, undergoing specific motions that can play a vital role in function. However, the link between primary sequence and conformational dynamics remains poorly understood. Here, we studied how conformational dynamics can arise in a globular protein by evaluating the impact of individual substitutions of core residues in DANCER-3, a streptococcal protein G domain β1 (Gβ1) variant that we previously designed to undergo a specific mode of conformational exchange that has never been observed in the wild-type protein. Using a combination of solution NMR experiments and molecular dynamics simulations, we demonstrate that only two mutations are necessary to create this conformational exchange, and that these mutations work synergistically, with one destabilizing the native Gβ1 structure and the other allowing two new conformational states to be accessed on the energy landscape. Overall, our results show how conformational dynamics can appear in a stable globular fold, a critical step in the molecular evolution of new dynamics-linked functions.

Download Full-text

Autophagy Deficiency Promotes β-Lactam Production inPenicillium chrysogenum

Applied and Environmental Microbiology ◽

10.1128/aem.01531-10 ◽

2010 ◽

Vol 77 (4) ◽

pp. 1413-1422 ◽

Cited By ~ 39

Author(s):

Magdalena Bartoszewska ◽

Jan A. K. W. Kiel ◽

Roel A. L. Bovenberg ◽

Marten Veenhuis ◽

Ida J. van der Klei

Keyword(s):

Penicillium Chrysogenum ◽

Biosynthetic Pathway ◽

Morphological Analysis ◽

Wild Type ◽

Serine Threonine Kinase ◽

Cell Degeneration ◽

Threonine Kinase ◽

Enhanced Production ◽

Electron And Fluorescence Microscopy ◽

Lactam Antibiotic

ABSTRACTWe have investigated the significance of autophagy in the production of the β-lactam antibiotic penicillin (PEN) by the filamentous fungusPenicillium chrysogenum. In this fungus PEN production is compartmentalized in the cytosol and in peroxisomes. We demonstrate that under PEN-producing conditions significant amounts of cytosolic and peroxisomal proteins are degraded via autophagy. Morphological analysis, based on electron and fluorescence microscopy, revealed that this phenomenon might contribute to progressive deterioration of late subapical cells. We show that deletion of theP. chrysogenumortholog ofSaccharomyces cerevisiaeserine-threonine kinaseatg1results in impairment of autophagy. InP. chrysogenum atg1cells, a distinct delay in cell degeneration is observed relative to wild-type cells. This phenomenon is associated with an increase in the enzyme levels of the PEN biosynthetic pathway and enhanced production levels of this antibacterial compound.

Download Full-text