scholarly journals High Affinity vs. Native Fibronectin in the Modulation of αvβ3 Integrin Conformational Dynamics: Insights from Computational Analyses and Implications for Molecular Design

2017 ◽  
Vol 13 (1) ◽  
pp. e1005334 ◽  
Author(s):  
Antonella Paladino ◽  
Monica Civera ◽  
Laura Belvisi ◽  
Giorgio Colombo
Keyword(s):  
Molecular Design ◽  
Conformational Dynamics ◽  
Αvβ3 Integrin ◽  
High Affinity ◽  
Computational Analyses

scholarly journals Transmembrane-truncated αvβ3 integrin retains high affinity for ligand binding: evidence for an ‘inside-out’ suppressor?

1998 ◽  
Vol 330 (2) ◽  
pp. 861-869 ◽  
Author(s):  
J. Raj MEHTA ◽  
Beate DIEFENBACH ◽  
Alex BROWN ◽  
Eilish CULLEN ◽  
Alfred JONCZYK ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Wound Repair ◽  
Molecular Mechanisms ◽  
Full Length ◽  
Αvβ3 Integrin ◽  
Cytoplasmic Domains ◽  
Bivalent Cation ◽  
High Affinity ◽  
Cellular Environment

The molecular mechanisms of αvβ3 integrin affinity regulation have important biological implications in tumour development, wound repair and angiogenesis. We expressed, purified and characterized recombinant forms of human αvβ3 (r-αvβ3) and compared the activation state of these with αvβ3 in its cellular environment. The ligand specificity and selectivity of recombinant full-length and double transmembrane truncations of r-αvβ3 cloned in BacPAK6 vectors and expressed in Sf9 and High Five insect cells were compared with those of native placental αvβ3 and the receptor in situ on the cell surface. r-αvβ3 integrins were purified by affinity chromatography from detergent extracts of cells (full-length), and from the culture medium of cells expressing double-truncated r-αvβ3. r-αvβ3 had the same epitopes, ligand-binding specificities, bivalent cation requirements and susceptibility to RGD-containing peptides as native αvβ3. On M21-L4 melanoma cells, αvβ3 mediated binding to vitronectin, but not to fibrinogen unless activated with Mn2+. Non-activated αIIbβ3 integrin as control in M21-L-IIb cells had the opposite profile, mediating binding to fibrinogen, but not to vitronectin unless activated with Mn2+. Thus these receptors had moderate to low ligand affinity. In marked contrast, purified αvβ3 receptors, with or without transmembrane and cytoplasmic domains, were constitutively of high affinity and able to bind strongly to vitronectin, fibronectin and fibrinogen under physiological conditions. Our data suggest that, in contrast with the positive regulation of αIIbβ3 in situ, intracellular controls lower the affinity of αvβ3, and the cytoplasmic domains may act as a target for negative regulators of αvβ3 activity.

scholarly journals DNA sequence is a major determinant of tetrasome dynamics

2019 ◽  
Author(s):  
O. Ordu ◽  
A. Lusser ◽  
N. H. Dekker
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
Dynamic Properties ◽  
Conformational Dynamics ◽  
Magnetic Tweezers ◽  
Torsional Stress ◽  
High Affinity ◽  
Canonical State ◽  
Left And Right ◽  
Dna Wrapping

ABSTRACTEukaryotic genomes are hierarchically organized into protein-DNA assemblies for compaction into the nucleus. Nucleosomes, with the (H3-H4)2 tetrasome as a likely intermediate, are highly dynamic in nature by way of several different mechanisms. We have recently shown that tetrasomes spontaneously change the direction of their DNA wrapping between left- and right-handed conformations, which may prevent torque build-up in chromatin during active transcription or replication. DNA sequence has been shown to strongly affect nucleosome positioning throughout chromatin. It is not known, however, whether DNA sequence also impacts the dynamic properties of tetrasomes. To address this question, we examined tetrasomes assembled on a high-affinity DNA sequence using freely orbiting magnetic tweezers. In this context, we also studied the effects of mono- and divalent salts on the flipping dynamics. We found that neither DNA sequence nor altered buffer conditions affect overall tetrasome structure. In contrast, tetrasomes bound to high-affinity DNA sequences showed significantly altered flipping kinetics, predominantly via a reduction in the lifetime of the canonical state of left-handed wrapping. Increased mono- and divalent salt concentrations counteracted this behaviour. Thus, our study indicates that high-affinity DNA sequences impact not only the positioning of the nucleosome, but that they also endow the subnucleosomal tetrasome with enhanced conformational plasticity. This may provide a means to prevent histone loss upon exposure to torsional stress, thereby contributing to the integrity of chromatin at high-affinity sites.STATEMENT OF SIGNIFICANCECanonical (H3-H4)2 tetrasomes possess high conformational flexibility, as evidenced by their spontaneous flipping between states of left- and right-handed DNA wrapping. Here, we show that these conformational dynamics of tetrasomes cannot be described by a fixed set of rates over all conditions. Instead, an accurate description of their behavior must take into account details of their loading, in particular the underlying DNA sequence. In vivo, differences in tetrasome flexibility could be regulated by modifications of the histone core or the tetrasomal DNA, and as such constitute an intriguing, potentially adjustable mechanism for chromatin to accommodate the torsional stress generated by processes such as transcription and replication.

scholarly journals A p.Arg127Gln variant in GPIbα LRR5 allosterically enhances affinity for VWF: a novel form of platelet-type VWD

2021 ◽  
Author(s):  
Loredana Bury ◽  
Emanuela Falcinelli ◽  
Haripriya Kuchi Bhotla ◽  
Anna Maria Mezzasoma ◽  
Giuseppe Guglielmini ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Conformational Dynamics ◽  
Von Willebrand Disease ◽  
High Affinity ◽  
Laboratory Test Results ◽  
Hydrogen Deuterium ◽  
Von Willebrand ◽  
The Impact ◽  
First Time ◽  
Willebrand Factor

Gain-of-function (GoF) variants in GP1BA cause platelet-type von Willebrand disease (PT-VWD), a rare inherited autosomal dominant bleeding disorder characterized by enhanced platelet GPIbα-von Willebrand factor (VWF) interaction and thrombocytopenia. To date, only 6 variants causing PT-VWD have been described, 5 in the C-terminal disulfide loop of the VWF-binding domain of GPIbα and 1 in the macroglycopeptide. GoF GP1BA variants generate a high affinity conformation of the C-terminal disulfide loop with a consequent allosteric conformational change on another region of GPIbα, the leucine-rich-repeat (LRR) domain. We identified a novel GP1BA variant (p.Arg127Gln) affecting the LRR5 domain of GPIbα in a boy with easy bruising and laboratory test results suggestive of PT-VWD. We thus aimed to investigate the impact of the p.Arg127Gln variant on GPIbα affinity for VWF and on GPIbα structure. CHO cells expressing p.Arg127Gln GPIbα showed increased binding of VWF induced by ristocetin and enhanced tethering on immobilized VWF as compared with cells expressing wild-type (WT) GPIbα. Surface plasmon resonance confirmed that p.Arg127Gln enhances the binding affinity of GPIbα for VWF. Hydrogen-deuterium exchange mass spectrometryshowed that p.Arg127Gln of LRR, while having little effect on the dynamics of the LRR locally, enhances the conformational dynamics of the GPIbα C-terminal disulfide loop structure. Our data demonstrate for the first time that GoF variants outside the GPIbα C-terminal disulfide loop may be pathogenic and that aminoacidic changes in the LRR may cause allosterically conformational changes in the C-terminal disulfide loop of GPIbα inducing a conformation with high affinity for VWF.

scholarly journals CD45 functions as a signaling gatekeeper in T cells

2019 ◽  
Vol 12 (604) ◽  
pp. eaaw8151 ◽  
Author(s):  
Adam H. Courtney ◽  
Alexey A. Shvets ◽  
Wen Lu ◽  
Gloria Griffante ◽  
Marianne Mollenauer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Tcr Signaling ◽  
Downstream Signaling ◽  
High Affinity ◽  
Signaling Events ◽  
Genetic Perturbations ◽  
Computational Analyses

T cells require the protein tyrosine phosphatase CD45 to detect and respond to antigen because it activates the Src family kinase Lck, which phosphorylates the T cell antigen receptor (TCR) complex. CD45 activates Lck by opposing the negative regulatory kinase Csk. Paradoxically, CD45 has also been implicated in suppressing TCR signaling by dephosphorylating the same signaling motifs within the TCR complex upon which Lck acts. We sought to reconcile these observations using chemical and genetic perturbations of the Csk/CD45 regulatory axis incorporated with computational analyses. Specifically, we titrated the activities of Csk and CD45 and assessed their influence on Lck activation, TCR-associated ζ-chain phosphorylation, and more downstream signaling events. Acute inhibition of Csk revealed that CD45 suppressed ζ-chain phosphorylation and was necessary for a regulatable pool of active Lck, thereby interconnecting the activating and suppressive roles of CD45 that tune antigen discrimination. CD45 suppressed signaling events that were antigen independent or induced by low-affinity antigen but not those initiated by high-affinity antigen. Together, our findings reveal that CD45 acts as a signaling “gatekeeper,” enabling graded signaling outputs while filtering weak or spurious signaling events.

scholarly journals Adhesion Molecules in Melanoma—More than Just Superglue?

1996 ◽  
Vol 89 (7) ◽  
pp. 393-395 ◽  
Author(s):  
MD Mason ◽  
R Allman ◽  
M Quibell
Keyword(s):  
Signal Transduction ◽  
Cyclic Peptide ◽  
Αvβ3 Integrin ◽  
Cell Surface Molecules ◽  
Malignant Cells ◽  
Cell Behaviour ◽  
Surface Molecules ◽  
High Affinity ◽  
Natural Ligands ◽  
Integrin Family

Malignant melanoma is increasing in incidence, and, though early lesions are readily treatable, systemic therapy for metastatic disease remains disappointing. Integrins are a family of cell-surface molecules that mediate adhesion between the cell and the extracellular matrix. One member of the integrin family, the αvβ3 integrin, is associated with progression of melanomas, in that the most malignant cells express the highest levels of αvβ3. Like many members of the integrin family, αvβ3 recognizes the sequence Arg-Gly-Asp (RGD) in its ligands, and other molecules that contain this sequence will compete with the natural ligands (such as vitronectin) for binding. There is growing evidence that integrins function as receptors for signal transduction, and that integrin-mediated signalling can affect cell behaviour and even cell survival. Under certain circumstances, loss of integrin-mediated signalling will induce apoptosis, or programmed cell death, and we have demonstrated that melanoma cells treated with a cyclic peptide with high affinity for the αvβ3 integrin will undergo apoptosis within three days. This mechanism might be exploited therapeutically.

Aβ under stress: the effects of acidosis, Cu2+-binding, and oxidation on amyloid β-peptide dimers

2018 ◽  
Vol 54 (56) ◽  
pp. 7766-7769 ◽  
Author(s):  
Qinghua Liao ◽  
Michael C. Owen ◽  
Sofia Bali ◽  
Bogdan Barz ◽  
Birgit Strodel
Keyword(s):  
Conformational Dynamics ◽  
Amyloid Β ◽  
Amyloid Β Peptide ◽  
Acidic Environment ◽  
High Affinity

In light of the high affinity of Cu2+ for Alzheimer's Aβ1–42 and its ability to subsequently catalyze the formation of radicals, we examine the effects of Cu2+ binding, Aβ oxidation, and an acidic environment on the conformational dynamics of the smallest Aβ1–42 oligomer, the Aβ1–42 dimer.

scholarly journals How the headpiece hinge angle is opened: new insights into the dynamics of integrin activation

2006 ◽  
Vol 175 (2) ◽  
pp. 349-360 ◽  
Author(s):  
Eileen Puklin-Faucher ◽  
Mu Gao ◽  
Klaus Schulten ◽  
Viola Vogel
Keyword(s):  
Crystal Structure ◽  
Peptide Ligand ◽  
Αvβ3 Integrin ◽  
Integrin Activation ◽  
Structural Variations ◽  
High Affinity ◽  
Hybrid Domain ◽  
Conformational Constraints ◽  
Allosteric Pathway ◽  
Hinge Angle

How the integrin head transitions to the high-affinity conformation is debated. Although experiments link activation with the opening of the hinge angle between the βA and hybrid domains in the ligand-binding headpiece, this hinge is closed in the liganded αvβ3 integrin crystal structure. We replaced the RGD peptide ligand of this structure with the 10th type III fibronectin module (FnIII10) and discovered through molecular dynamics (MD) equilibrations that when the conformational constraints of the leg domains are lifted, the βA/hybrid hinge opens spontaneously. Together with additional equilibrations on the same nanosecond timescale in which small structural variations impeded hinge-angle opening, these simulations allowed us to identify the allosteric pathway along which ligand-induced strain propagates via elastic distortions of the α1 helix to the βA/hybrid domain hinge. Finally, we show with steered MD how force accelerates hinge-angle opening along the same allosteric pathway. Together with available experimental data, these predictions provide a novel framework for understanding integrin activation.

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