scholarly journals VEGFR-2 conformational switch in response to ligand binding

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Sarvenaz Sarabipour ◽  
Kurt Ballmer-Hofer ◽  
Kalina Hristova
Keyword(s):  
Ligand Binding ◽  
Blood Vessels ◽  
Biochemical Analysis ◽  
Infantile Hemangioma ◽  
Kinase Domain ◽  
Active Conformation ◽  
Wild Type ◽  
Conformational Switch ◽  
Dimer Structure ◽  
Tm Domain

VEGFR-2 is the primary regulator of angiogenesis, the development of new blood vessels from pre-existing ones. VEGFR-2 has been hypothesized to be monomeric in the absence of bound ligand, and to undergo dimerization and activation only upon ligand binding. Using quantitative FRET and biochemical analysis, we show that VEGFR-2 forms dimers also in the absence of ligand when expressed at physiological levels, and that these dimers are phosphorylated. Ligand binding leads to a change in the TM domain conformation, resulting in increased kinase domain phosphorylation. Inter-receptor contacts within the extracellular and TM domains are critical for the establishment of the unliganded dimer structure, and for the transition to the ligand-bound active conformation. We further show that the pathogenic C482R VEGFR-2 mutant, linked to infantile hemangioma, promotes ligand-independent signaling by mimicking the structure of the ligand-bound wild-type VEGFR-2 dimer.

scholarly journals The dynamic switch mechanism that leads to activation of LRRK2 is embedded in the DFGψ motif in the kinase domain

2019 ◽  
Vol 116 (30) ◽  
pp. 14979-14988 ◽  
Author(s):  
Sven H. Schmidt ◽  
Matthias J. Knape ◽  
Daniela Boassa ◽  
Natascha Mumdey ◽  
Alexandr P. Kornev ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Regulatory Mechanism ◽  
Kinase Domain ◽  
Leucine Rich Repeat ◽  
Wild Type ◽  
Conformational Switch ◽  
Multidomain Protein ◽  
Switch Mechanism ◽  
And Function

Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein, and LRRK2 mutants are recognized risk factors for Parkinson’s disease (PD). Although the precise mechanisms that control LRRK2 regulation and function are unclear, the importance of the kinase domain is strongly implicated, since 2 of the 5 most common familial LRRK2 mutations (G2019S and I2020T) are localized to the conserved DFGψ motif in the kinase core, and kinase inhibitors are under development. Combining the concept of regulatory (R) and catalytic (C) spines with kinetic and cell-based assays, we discovered a major regulatory mechanism embedded within the kinase domain and show that the DFG motif serves as a conformational switch that drives LRRK2 activation. LRRK2 is quite unusual in that the highly conserved Phe in the DFGψ motif, which is 1 of the 4 R-spine residues, is replaced with tyrosine (DY2018GI). A Y2018F mutation creates a hyperactive phenotype similar to the familial mutation G2019S. The hydroxyl moiety of Y2018 thus serves as a “brake” that stabilizes an inactive conformation; simply removing it destroys a key hydrogen-bonding node. Y2018F, like the pathogenic mutant I2020T, spontaneously forms LRRK2-decorated microtubules in cells, while the wild type and G2019S require kinase inhibitors to form filaments. We also explored 3 different mechanisms that create kinase-dead pseudokinases, including D2017A, which further emphasizes the highly synergistic role of key hydrophobic and hydrophilic/charged residues in the assembly of active LRRK2. We thus hypothesize that LRRK2 harbors a classical protein kinase switch mechanism that drives the dynamic activation of full-length LRRK2.

scholarly journals Effects of I Domain Deletion on the Function of the β2 Integrin Lymphocyte Function-associated Antigen-1

2000 ◽  
Vol 11 (2) ◽  
pp. 677-690 ◽  
Author(s):  
Birgit Leitinger ◽  
Nancy Hogg
Keyword(s):  
Ligand Binding ◽  
Intracellular Signaling ◽  
Lymphocyte Function ◽  
Active Conformation ◽  
Wild Type ◽  
Β2 Integrin ◽  
Β1 Integrins ◽  
Enhanced Expression ◽  
Α Subunits

A subset of integrin α subunits contain an I domain, which is important for ligand binding. We have deleted the I domain from the β2 integrin lymphocyte function-asssociated antigen-1 (LFA-1) and expressed the resulting non–I domain-containing integrin (ΔI-LFA-1) in an LFA-1-deficient T cell line. ΔI-LFA-1 showed no recognition of LFA-1 ligands, confirming the essential role of the I domain in ligand binding. Except for I domain monoclonal antibodies (mAbs), ΔI-LFA-1 was recognized by a panel of anti-LFA-1 mAbs similarly to wild-type LFA-1. However, ΔI-LFA-1 had enhanced expression of seven mAb epitopes that are associated with β2 integrin activation, suggesting that it exhibited an “active” conformation. In keeping with this characteristic, ΔI-LFA-1 induced constitutive activation of α4β1 and α5β1, suggesting intracellular signaling to these integrins. This “cross-talk” was not due to an effect on β1 integrin affinity. However, the enhanced activity was susceptible to inhibition by cytochalasin D, indicating a role for the cytoskeleton, and also correlated with clustering of β1 integrins. Thus, removal of the I domain from LFA-1 created an integrin with the hallmarks of a constitutively active receptor mediating signals into the cell. These findings suggest a key role for the I domain in controlling integrin activity.

Distinct Modulation of Wild-Type and Selective Gene Mutated Vitamin D Receptor by Essential Poly Unsaturated Fatty Acids

2021 ◽  
Vol 21 ◽  
Author(s):  
Hari Balaji ◽  
Selvaraj Ayyamperuma ◽  
Niladri Saha ◽  
Shyam Sundar Pottabathula ◽  
Jubie Selvaraj ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Vitamin D ◽  
Ligand Binding ◽  
Vitamin D Deficiency ◽  
Binding Affinity ◽  
Unsaturated Fatty Acids ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Binding Energies ◽  
Wild Type

: Vitamin-D deficiency is a global concern. Gene mutations in the vitamin D receptor’s (VDR) ligand binding domain (LBD) variously alter the ligand binding affinity, heterodimerization with retinoid X receptor (RXR) and inhibit coactivator interactions. These LBD mutations may result in partial or total hormone unresponsiveness. A plethora of evidence report that selective long chain polyunsaturated fatty acids (PUFAs) including eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) bind to the ligand-binding domain of VDR and lead to transcriptional activation. We therefore hypothesize that selective PUFAs would modulate the dynamics and kinetics of VDRs, irrespective bioactive of vitamin-D binding. The spatial arrangements of the selected PUFAs in VDR active site were examined by in-silico docking studies. The docking results revealed that PUFAs have fatty acid structure-specific binding affinity towards VDR. The calculated EPA, DHA & AA binding energies (Cdocker energy) were lesser compared to vitamin-D in wild type of VDR (PDB id: 2ZLC). Of note, the DHA has higher binding interactions to the mutated VDR (PDB id: 3VT7) when compared to the standard Vitamin-D. Molecular dynamic simulation was utilized to confirm the stability of potential compound binding of DHA with mutated VDR complex. These findings suggest the unique roles of PUFAs in VDR activation and may offer alternate strategy to circumvent vitamin-D deficiency.

scholarly journals Novel Myosin Heavy Chain Kinase Involved in Disassembly of Myosin II Filaments and Efficient Cleavage in MitoticDictyostelium Cells

2002 ◽  
Vol 13 (12) ◽  
pp. 4333-4342 ◽  
Author(s):  
Akira Nagasaki ◽  
Go Itoh ◽  
Shigehiko Yumura ◽  
Taro Q.P. Uyeda
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Myosin Ii ◽  
Kinase Domain ◽  
Cleavage Furrow ◽  
Wild Type ◽  
Daughter Cells ◽  
Cleavage Process ◽  
Interphase Cells ◽  
Myosin Heavy Chain Kinase

We have cloned a full-length cDNA encoding a novel myosin II heavy chain kinase (mhckC) from Dictyostelium. Like other members of the myosin heavy chain kinase family, themhckC gene product, MHCK C, has a kinase domain in its N-terminal half and six WD repeats in the C-terminal half. GFP-MHCK C fusion protein localized to the cortex of interphase cells, to the cleavage furrow of mitotic cells, and to the posterior of migrating cells. These distributions of GFP-MHCK C always corresponded with that of myosin II filaments and were not observed in myosin II-null cells, where GFP-MHCK C was diffusely distributed in the cytoplasm. Thus, localization of MHCK C seems to be myosin II-dependent. Cells lacking the mhckC gene exhibited excessive aggregation of myosin II filaments in the cleavage furrows and in the posteriors of the daughter cells once cleavage was complete. The cleavage process of these cells took longer than that of wild-type cells. Taken together, these findings suggest MHCK C drives the disassembly of myosin II filaments for efficient cytokinesis and recycling of myosin II that occurs during cytokinesis.

Structure of a ligand-binding intermediate in wild-type carbonmonoxy myoglobin

Nature ◽  
2000 ◽  
Vol 403 (6772) ◽  
pp. 921-923 ◽  
Author(s):  
Kelvin Chu ◽  
Jaroslav Vojtchovský ◽  
Benjamin H. McMahon ◽  
Robert M. Sweet ◽  
Joel Berendzen ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Wild Type ◽  
Carbonmonoxy Myoglobin

Expression of SMRTβ promotes ligand-induced activation of mutated and wild-type retinoid receptors

Blood ◽  
2004 ◽  
Vol 104 (13) ◽  
pp. 4226-4235 ◽  
Author(s):  
Sylvie Côté ◽  
Suzan McNamara ◽  
Daria Brambilla ◽  
Andrea Bianchini ◽  
Giovanni Rizzo ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Nuclear Receptors ◽  
Transcriptional Activation ◽  
Leukemic Cell ◽  
Wild Type ◽  
Specific Expression ◽  
Retinoid Receptors ◽  
Leukemic Cell Lines ◽  
All Trans Retinoic Acid ◽  
New Evidence

Abstract Nuclear receptors are ligand-modulated transcription factors regulated by interactions with corepressors and coactivators, whose functions are not fully understood. Acute promyelocytic leukemia (APL) is characterized by a translocation, t(15;17), that produces a PML/RARα fusion oncoprotein, whose abnormal transcriptional function is successfully targeted by pharmacologic levels of all-trans-retinoic acid (ATRA). Mutations in the ligand-binding domain of PML/RARα that confer resistance to ATRA have been studied by expression in nonhematopoietic cells, such as Cos-1. Here, we show that ATRA binding and transcriptional activation by the same PML/RARα mutant differ markedly between nonhematopoietic and leukemic cell lines. Differential expression of the corepressor isoform silencing mediator for retinoid and thyroid receptors β (SMRTβ) correlates with increased ligand binding and transcription by the mutant PML/RARα. Transient and stable overexpression of SMRTβ in hematopoietic cells that only express SMRTα increased ATRA binding, ligand-induced transcription, and ATRA-induced cell differentiation. This effect may not be limited to abnormal nuclear receptors, because overexpression of SMRTβ increased ATRA-induced binding and transcriptional activation of wild-type receptors PML/RARα and RARα. Our results suggest a novel role for the SMRTβ isoform whereby its cell-specific expression may influence the binding and transcriptional capacities of nuclear receptors, thus providing new evidence of distinct functions of corepressor isoforms and adding complexity to transcriptional regulation.

scholarly journals Mutational activation of c-raf-1 and definition of the minimal transforming sequence.

1990 ◽  
Vol 10 (6) ◽  
pp. 2503-2512 ◽  
Author(s):  
G Heidecker ◽  
M Huleihel ◽  
J L Cleveland ◽  
W Kolch ◽  
T W Beck ◽  
...  
Keyword(s):  
Structural Features ◽  
Kinase Domain ◽  
Full Length ◽  
Wild Type ◽  
Amino Terminal ◽  
Raf Kinase ◽  
Rich Domain ◽  
Binding Residue ◽  
Transforming Activity ◽  
Mutational Activation

A series of wild-type and mutant raf genes was transfected into NIH 3T3 cells and analyzed for transforming activity. Full-length wild-type c-raf did not show transforming activity. Two types of mutations resulted in oncogenic activity similar to that of v-raf: truncation of the amino-terminal half of the protein and fusion of the full-length molecule to gag sequences. A lower level of activation was observed for a mutant with a tetrapeptide insertion mapping to conserved region 2 (CR2), a serine- and threonine-rich domain located 100 residues amino-terminal of the kinase domain. To determine essential structural features of the transforming region of raf, we analyzed point and deletion mutants of v-raf. Substitutions of Lys-56 modulated the transforming activity, whereas mutation of Lys-53, a putative ATP binding residue, abolished it. Deletion analysis established that the minimal transforming sequence coincided precisely with CR3, the conserved Raf kinase domain. Thus, oncogenic activation of the Raf kinase can be achieved by removal of CR1 and CR2 or by steric distortion and requires retention of an active kinase domain. These findings are consistent with a protein structure model for the nonstimulated enzyme in which the active site is buried within the protein.

scholarly journals Mutation in Abl kinase with altered drug-binding kinetics indicates a novel mechanism of imatinib resistance

2021 ◽  
Vol 118 (46) ◽  
pp. e2111451118
Author(s):  
Agatha Lyczek ◽  
Benedict-Tilman Berger ◽  
Aziz M. Rangwala ◽  
YiTing Paung ◽  
Jessica Tom ◽  
...  
Keyword(s):  
Binding Site ◽  
Kinase Domain ◽  
Protein Kinase Inhibitors ◽  
Drug Binding ◽  
The Body ◽  
Binding Kinetics ◽  
Wild Type ◽  
Resistance Mutations ◽  
Imatinib Resistance ◽  
Abl Kinase

Protein kinase inhibitors are potent anticancer therapeutics. For example, the Bcr-Abl kinase inhibitor imatinib decreases mortality for chronic myeloid leukemia by 80%, but 22 to 41% of patients acquire resistance to imatinib. About 70% of relapsed patients harbor mutations in the Bcr-Abl kinase domain, where more than a hundred different mutations have been identified. Some mutations are located near the imatinib-binding site and cause resistance through altered interactions with the drug. However, many resistance mutations are located far from the drug-binding site, and it remains unclear how these mutations confer resistance. Additionally, earlier studies on small sets of patient-derived imatinib resistance mutations indicated that some of these mutant proteins were in fact sensitive to imatinib in cellular and biochemical studies. Here, we surveyed the resistance of 94 patient-derived Abl kinase domain mutations annotated as disease relevant or resistance causing using an engagement assay in live cells. We found that only two-thirds of mutations weaken imatinib affinity by more than twofold compared to Abl wild type. Surprisingly, one-third of mutations in the Abl kinase domain still remain sensitive to imatinib and bind with similar or higher affinity than wild type. Intriguingly, we identified three clinical Abl mutations that bind imatinib with wild type–like affinity but dissociate from imatinib considerably faster. Given the relevance of residence time for drug efficacy, mutations that alter binding kinetics could cause resistance in the nonequilibrium environment of the body where drug export and clearance play critical roles.

scholarly journals Conformational clamping by a membrane ligand activates the EphA2 receptor

2021 ◽  
Author(s):  
Justin M Westerfield ◽  
Amita Sahoo ◽  
Daiane S Alves ◽  
Brayan Grau ◽  
Alayna Cameron ◽  
...  
Keyword(s):  
Drug Target ◽  
Transmembrane Domain ◽  
Binding Mode ◽  
Membrane Receptors ◽  
Binding Motif ◽  
Active Conformation ◽  
Dynamic Simulations ◽  
Membrane Peptide ◽  
Migration Assays ◽  
Tm Domain

The EphA2 receptor is a promising drug target for cancer treatment, since EphA2 activation can inhibit metastasis and tumor progression. It has been recently described that the TYPE7 peptide activates EphA2 using a novel mechanism that involves binding to the single transmembrane domain of the receptor. TYPE7 is a conditional transmembrane (TM) ligand, which only inserts into membranes at neutral pH in the presence of the TM region of EphA2. However, how membrane interactions can activate EphA2 is not known. We systematically altered the sequence of TYPE7 to identify the binding motif used to activate EphA2. With the resulting six peptides, we performed biophysical and cell migration assays that identified a new potent peptide variant. We also performed a mutational screen that determined the helical interface that mediates dimerization of the TM domain of EphA2 in cells. These results, together with molecular dynamic simulations, allowed to elucidate the molecular mechanism that TYPE7 uses to activate EphA2, where the membrane peptide acts as a molecular clamp that wraps around the TM dimer of the receptor. We propose that this binding mode stabilizes the active conformation of EphA2. Our data, additionally, provide clues into the properties that TM ligands need to have in order to achieve activation of membrane receptors.

scholarly journals In-cell SHAPE reveals that free 30S ribosome subunits are in the inactive state

2015 ◽  
Vol 112 (8) ◽  
pp. 2425-2430 ◽  
Author(s):  
Jennifer L. McGinnis ◽  
Qi Liu ◽  
Christopher A. Lavender ◽  
Aishwarya Devaraj ◽  
Sean P. McClory ◽  
...  
Keyword(s):  
Energy Barrier ◽  
Cell Shape ◽  
Large Scale ◽  
Neck Region ◽  
Active Conformation ◽  
Conformational Switch ◽  
Biologically Relevant ◽  
Inactive State ◽  
Active Subunits

It was shown decades ago that purified 30S ribosome subunits readily interconvert between “active” and “inactive” conformations in a switch that involves changes in the functionally important neck and decoding regions. However, the physiological significance of this conformational change had remained unknown. In exponentially growing Escherichia coli cells, RNA SHAPE probing revealed that 16S rRNA largely adopts the inactive conformation in stably assembled, mature 30S subunits and the active conformation in translating (70S) ribosomes. Inactive 30S subunits bind mRNA as efficiently as active subunits but initiate translation more slowly. Mutations that inhibited interconversion between states compromised translation in vivo. Binding by the small antibiotic paromomycin induced the inactive-to-active conversion, consistent with a low-energy barrier between the two states. Despite the small energetic barrier between states, but consistent with slow translation initiation and a functional role in vivo, interconversion involved large-scale changes in structure in the neck region that likely propagate across the 30S body via helix 44. These findings suggest the inactive state is a biologically relevant alternate conformation that regulates ribosome function as a conformational switch.

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