Synthetic and computer-assisted analysis of the structural requirements for selective, high-affinity ligand binding to diazepam-insensitive benzodiazepine receptors

1993 ◽  
Vol 36 (13) ◽  
pp. 1820-1830 ◽  
Author(s):  
Garry Wong ◽  
Konrad F. Koehler ◽  
Phil Skolnick ◽  
Zi Qiang Gu ◽  
Subramaniam Ananthan ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Benzodiazepine Receptors ◽  
Computer Assisted ◽  
Structural Requirements ◽  
High Affinity ◽  
Affinity Ligand ◽  
Assisted Analysis ◽  
High Affinity Ligand

scholarly journals High Affinity Ligand Binding by Integrins Does Not Involve Head Separation

2003 ◽  
Vol 278 (19) ◽  
pp. 17185-17189 ◽  
Author(s):  
Bing-Hao Luo ◽  
Timothy A. Springer ◽  
Junichi Takagi
Keyword(s):  
Ligand Binding ◽  
High Affinity ◽  
Affinity Ligand ◽  
High Affinity Ligand

scholarly journals Rat and human neutrophil N-formyl-peptide chemotactic receptors. Species difference in the glycosylation of similar 35-38 kDa polypeptide cores

1991 ◽  
Vol 277 (1) ◽  
pp. 67-72 ◽  
Author(s):  
J J Remes ◽  
U E Petäjä-Repo ◽  
H J Rajaniemi
Keyword(s):  
Ligand Binding ◽  
Species Difference ◽  
Cross Linking ◽  
Complex Type ◽  
High Affinity ◽  
Affinity Ligand ◽  
Pngase F ◽  
Formyl Peptide ◽  
Membrane Bound ◽  
High Affinity Ligand

Rat and human neutrophil N-formyl-peptide chemotactic receptors were subjected to glycosidase and proteinase treatments to determine the extent and species differences of glycosylation and the carbohydrate requirement in the high-affinity ligand binding. N-Formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys was attached to rat and human neutrophils either before or after glycosidase and proteinase treatments, and the labelled receptors were solubilized after glutaraldehyde cross-linking and analysed by SDS/PAGE and autoradiography. Both the rat and human N-formyl-peptide chemotactic receptors contain only N-linked oligosaccharides, as demonstrated by their sensitivity to peptide N-glycosidase F (PNGase F) and resistance to O-glycanase treatment. The N-linked oligosaccharides seem to be of the complex type rather than the high-mannose or hybrid type and lack terminal sialic acid, as demonstrated by their resistance to endoglycosidases D and H and neuraminidase treatments. This sensitivity pattern was similar in both species, and the shift in the molecular size of the receptors to 35-38 kDa after PNGase F treatment occurred through one intermediate product, suggesting that both receptors contain a similar 35-38 kDa polypeptide core with two N-linked complex-type oligosaccharides, the heterogeneity of which is responsible for the species difference in receptor size. Papain treatment alone or followed by PNGase F produced in both species a 33-36 kDa membrane-bound fragment that was still able to bind the ligand, suggesting that the oligosaccharides are located on the approx. 2 kDa papain-cleavable polypeptide fragment of the receptors. The cleavage sites for both papain and PNGase F were hidden in occupied receptors, suggesting a conformational or topographical change in these upon ligand binding. Scatchard analyses and cross-linking experiments demonstrated that carbohydrates are not required for high-affinity ligand binding and that the 33-36 kDa membrane-bound papain fragment of both receptors contains the ligand-binding site.

Long Range Conformational Transmission Of Integrin Activation and Signaling By α1/α1’-Helix Unbending At The Junction

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 198-198
Author(s):  
Jieqing Zhu ◽  
Chuanmei Zhang ◽  
Jiafu Liu ◽  
Xiuli Jiang ◽  
Nada Haydar ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Ligand Binding ◽  
Focal Adhesion ◽  
Helical Conformation ◽  
Integrin Activation ◽  
High Affinity ◽  
Affinity Ligand ◽  
Inside Out ◽  
High Affinity Ligand ◽  
Αiibβ3 Integrin

Abstract Platelet specific integrin αIIbβ3 plays an essential role in hemostasis and thrombosis. It has been used as a prototype for understanding integrin activation and conformational regulation. Crystal structures of αIIbβ3 headpiece composed of the αIIb β-propeller and β3 βI, hybrid, and PSI domains in the absence or presence of RGD-mimetic drugs revealed the headpiece changing from a closed to an open conformation upon ligand binding. A striking change is the swing-out motion of the β3 hybrid domain away from the βI and the αIIb thigh domains. This is accompanied by the changing of the α1/α1'-helix from a bent to a merged and straightened α-helical conformation. The α1/α1'-helix is bent at the α1/α1' junction (β3 Ile-131 to Gly-135) as revealed by the crystal structures of β3, β1, β2, and β7 integrins in the closed headpiece conformation. The β3 Gly-135 at the α1/α1' junction is completely conserved among all the β integrins. We propose that the conserved glycine at the α1/α1' junction is critical for maintaining the bent α1/α1'-helix conformation, and the α1/α1'-helix unbending is required for integrin activation and bidirectional signaling. To test this hypothesis, we mutated the β3 Gly-135 to alanine and showed that the β3-G135A mutation rendered αIIbβ3 integrin constitutively binding the activation-dependent mAb PAC-1. In contrast, the β3-G135P mutation had minor effect on integrin activation. This is consistent with the idea that alanine tends to stabilize a straight α-helical structure, while glycine and proline tend to introduce a bend or kink into the α-helical conformation when present at the internal positions of an α-helix. That is, the conserved β3 Gly-135 is essential for restraining the α1/α1'-helix in the bent conformation. The β3 Gly-135 is partially exposed in the bent conformation of α1/α1'-helix and buried deeply into the hydrophobic environment upon the α1/α1'-helix unbending. We rationalized that the hydrophilic substitutions will restrain, while the hydrophobic substitutions will facilitate the burying of β3 Gly-135, and thus block and induce α1/α1'-helix unbending, respectively. As expected, the β3-G135R and G135K mutations completely blocked PAC-1 binding to αIIbβ3 integrin stimulated by Mn2+ or by the αIIb-R995D mutation that mimics integrin inside-out activation. In sharp contrast, the β3-G135L and G135M mutations constitutively induced PAC-1 binding to αIIbβ3 integrin. To further confirm the α1/α1'-helix unbending is required for integrin activation and signaling, we introduced tandem double or triple glycine substitutions into the α1/α1' junction to reinforce the bent conformation of α1/α1'-helix. Remarkably, all the double or triple glycine mutations completely abolished soluble PAC-1 binding stimulated by Mn2+ from outside or by the αIIb-R995D or αIIb-F993A mutation from inside the cell. This data provide compelling evidence that the integrin α1/α1'-helix unbending is indispensible for high affinity ligand binding. Interestingly, the β3-G135R or double glycine mutant still mediated cell adhesion to immobilized PAC-1 or fibrinogen, but at a reduced level. The cell adhesion could be blocked by eptifibatide, indicating the binding ability of the mutant integrins with the high affinity small molecule ligand. However, eptifibatide failed to induce the ectodomain extension of the mutant integrins. In addition, integrin-mediated outside-in signaling, such as cell spreading, focal adhesion and F-actin stress fiber formation, and focal adhesion kinase activation was inhibited by the β3-G135R or double glycine mutations. This data demonstrated that the conformational communication initiated by ligand binding is interrupted due to the defect of α1/α1'-helix unbending. We further showed that overexpression of talin1 head domain failed to induce PAC-1 binding to the αIIbβ3 integrin with double glycine mutations at the α1/α1' junction, but still induced integrin ectodomain extension. That is, in the inside-out integrin activation, the ectodomain extension alone does not result in high affinity ligand binding. The conformational signal has to be relayed to the ligand binding site through α1/α1'-helix unbending. In conclusion, our data established the structural role of the α1/α1' junction that allows relaxation of the α1/α1'-helix in the resting state and transmission of bidirectional conformational signals by helix unbending upon integrin activation. Disclosures: No relevant conflicts of interest to declare.

Letters to the Editor

1997 ◽  
Vol 273 (4) ◽  
pp. C1437-C1439
Author(s):  
A. W. Cuthbert
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Single Channel ◽  
Letters To The Editor ◽  
High Affinity ◽  
Channel Proteins ◽  
Affinity Ligand ◽  
Ligand Binding Sites ◽  
Kinetics Of ◽  
High Affinity Ligand

The following is the abstract of the article discussed in the subsequent letter: Blazer-Yost, Bonnie L., and Sandy I. Helman.The amiloride-sensitive epithelial Na+ channel: binding sites and channel densities. Am. J. Physiol. 272 ( Cell Physiol. 41): C761–C769, 1997.—The amiloride-sensitive Na+ channel found in many transporting epithelia plays a key role in regulating salt and water homeostasis. Both biochemical and biophysical approaches have been used to identify, characterize, and quantitate this important channel. Among biophysical methods, there is agreement as to the single-channel conductance and gating kinetics of the highly selective Na+ channel found in native epithelia. Amiloride and its analogs inhibit transport through the channel by binding to high-affinity ligand-binding sites. This characteristic of high-affinity binding has been used biochemically to quantitate channel densities and to isolate presumptive channel proteins. Although the goals of biophysical and biochemical experiments are the same in elucidating mechanisms underlying regulation of Na+transport, our review highlights a major quantitative discrepancy between methods in estimation of channel densities involved in transport. Because the density of binding sites measured biochemically is three to four orders of magnitude in excess of channel densities measured biophysically, it is unlikely that high-affinity ligand binding can be used physiologically to quantitate channel densities and characterize the channel proteins.

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