scholarly journals Novel activating and inactivating mutations in the integrin beta1 subunit A domain

2004 ◽  
Vol 380 (2) ◽  
pp. 401-407 ◽  
Author(s):  
Stephanie J. BARTON ◽  
Mark A. TRAVIS ◽  
Janet A. ASKARI ◽  
Patrick A. BUCKLEY ◽  
Susan E. CRAIG ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Conformational Changes ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Head Region ◽  
Hybrid Domain ◽  
Factor Type ◽  
A Domains ◽  
Von Willebrand ◽  
Ligand Binding Activity

The ligand-binding activity of integrins is regulated by shape changes that convert these receptors from a resting (or inactive) state to an active state. However, the precise conformational changes that take place in head region of integrins (the site of ligand binding) during activation are not well understood. The portion of the integrin β subunit involved in ligand recognition contains a von Willebrand factor type A domain, which comprises a central β-sheet surrounded by seven α helices (α1–α7). Using site-directed mutagenesis, we show here that point mutation of hydrophobic residues in the α1 and α7 helices (which would be predicted to increase the mobility of these helices) markedly increases the ligand-binding activity of both integrins α5β1 and α4β1. In contrast, mutation of a hydrophilic residue near the base of the α1 helix decreases activity and also suppresses exposure of activation epitopes on the underlying hybrid domain. Our results provide new evidence that shifts of the α1 and α7 helices are involved in activation of the A domain. Although these changes are grossly similar to those defined in the A domains found in some integrin α subunits, movement of the α1 helix appears to play a more prominent role in βA domain activation.

scholarly journals Structural analysis of natriuretic peptide receptor-C by truncation and site-directed mutagenesis

1997 ◽  
Vol 322 (2) ◽  
pp. 585-590 ◽  
Author(s):  
Makoto ITAKURA ◽  
Hiroyuki SUZUKI ◽  
Shigehisa HIROSE
Keyword(s):  
Ligand Binding ◽  
Natriuretic Peptide ◽  
Gel Filtration ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Natriuretic Peptide Receptor ◽  
Peptide Receptor ◽  
Mammalian Expression ◽  
Ligand Binding Activity

Natriuretic peptide receptor-C (NPR-C) has a unique structure consisting of pre-existing covalent homodimers, but it is not known whether each subunit has ligand-binding activity or whether the dimeric structure is necessary for binding activity. To answer this question, a number of C-terminally truncated mutants were designed, subcloned into the mammalian expression vector pcDNA3 and expressed by transient transfection in COS-1 cells. Truncation at position 461, which eliminates the residue Cys469 that is involved in disulphide-linked dimerization, produced a soluble and monomeric form of NPR-C, as determined by gel filtration on Superose 12. Binding assays of the gel-filtration fractions clearly demonstrated that even monomeric NPR-C contains a high-affinity binding site for natriuretic peptides. Site-directed mutagenesis of the invariant residues (Asp407-Arg408 and Asp411-Phe412) in a region highly conserved among various species established that these invariant residues are essential for ligand-binding activity.

Overlap of IGF- and heparin-binding sites in rat IGF-binding protein-5

2000 ◽  
Vol 24 (1) ◽  
pp. 43-51 ◽  
Author(s):  
H Song ◽  
J Beattie ◽  
IW Campbell ◽  
GJ Allan
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Binding Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Heparin Binding ◽  
Igf I ◽  
Heparin Binding Domain ◽  
Igf Binding

Using site-directed mutagenesis, we have undertaken a study of a potential IGF-binding site in the C-terminal domain of rat IGFBP-5, lying close to or within a previously described heparin-binding domain (residues 201-218) in this protein. After analysis of binding activity using three different methods - ligand blotting, solution phase equilibrium binding and biosensor measurement of real-time on- and off-rates - we report that the mutation of two highly conserved residues within this region (glycine 203 and glutamine 209) reduces the affinity of the binding protein for both IGF-I and IGF-II, while having no effect on heparin binding. In addition, we confirm that mutation of basic residues within the heparin-binding domain (R201L, K202E, K206Q and R214A) results in a protein that has attenuated heparin binding but shows only a small reduction in affinity for IGF-I and -II. Previous findings have described the reduction in affinity of IGFBP-5 for IGFs that occurs after complexation of the binding protein with heparin or other components of the extracellular matrix (ECM) and have postulated that such an interaction may result in conformational changes in protein structure, affecting subsequent IGF interaction. Our data suggesting potential overlap of heparin- and IGF-binding domains argue for a more direct effect of ECM modulation of the affinity of IGFBP-5 for ligand by partial occlusion of the IGF-binding site after interaction with ECM.

scholarly journals Characterization of the High-Affinity Drug Ligand Binding Site of Mouse Recombinant TSPO

2019 ◽  
Vol 20 (6) ◽  
pp. 1444 ◽  
Author(s):  
Soria Iatmanen-Harbi ◽  
lucile Senicourt ◽  
Vassilios Papadopoulos ◽  
Olivier Lequin ◽  
Jean-Jacques Lacapere
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Conformational Changes ◽  
Protoporphyrin Ix ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Translocator Protein ◽  
Binding Affinities ◽  
Ligand Selectivity ◽  
High Affinity

The optimization of translocator protein (TSPO) ligands for Positron Emission Tomography as well as for the modulation of neurosteroids is a critical necessity for the development of TSPO-based diagnostics and therapeutics of neuropsychiatrics and neurodegenerative disorders. Structural hints on the interaction site and ligand binding mechanism are essential for the development of efficient TSPO ligands. Recently published atomic structures of recombinant mammalian and bacterial TSPO1, bound with either the high-affinity drug ligand PK 11195 or protoporphyrin IX, have revealed the membrane protein topology and the ligand binding pocket. The ligand is surrounded by amino acids from the five transmembrane helices as well as the cytosolic loops. However, the precise mechanism of ligand binding remains unknown. Previous biochemical studies had suggested that ligand selectivity and binding was governed by these loops. We performed site-directed mutagenesis to further test this hypothesis and measured the binding affinities. We show that aromatic residues (Y34 and F100) from the cytosolic loops contribute to PK 11195 access to its binding site. Limited proteolytic digestion, circular dichroism and solution two-dimensional (2-D) NMR using selective amino acid labelling provide information on the intramolecular flexibility and conformational changes in the TSPO structure upon PK 11195 binding. We also discuss the differences in the PK 11195 binding affinities and the primary structure between TSPO (TSPO1) and its paralogous gene product TSPO2.

Broadly distributed nucleophilic reactivity of proteins coordinated with specific ligand binding activity

2005 ◽  
Vol 18 (4) ◽  
pp. 295-306 ◽  
Author(s):  
Yasuhiro Nishiyama ◽  
Yukie Mitsuda ◽  
Hiroaki Taguchi ◽  
Stephanie Planque ◽  
Mariko Hara ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Activity ◽  
Nucleophilic Reactivity ◽  
Ligand Binding Activity ◽  
Specific Ligand
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