EVIDENCE FOR MULTIPLE BINDING SITES OF HIRUDIN IN THROMBIN

1987 ◽  
Author(s):  
German B Villanueva ◽  
Konno Sensuke ◽  
John Fenton
Keyword(s):  
Dissociation Constant ◽  
Active Site ◽  
Binding Sites ◽  
Specific Activity ◽  
Multiple Binding Sites ◽  
Straight Line ◽  
Multiple Binding ◽  
Hyperbolic Curve ◽  
Sigmoidal Curve ◽  
A Site

A highly purified hirudin with a specific activity of 13, 950 AT units/mg was used in these studies. Investigation of the circular dichroism of hirudin and thrombin showed that the CD spectrum of the thrombin-hirudin complex deviates significantly from additivity towards a less organized structure (i.e. loss of a-helix).A sigmoidal curve, rather than a hyperbolic curve, is generated when the deviation from additivity is plotted against hirudin concentration. This suggests cooperativity of the binding process. At low concentation, aScatchard plot of the data fits intoa straight line clearly indicating one binding site per mole of thrombin.This site binds hirudin with a dissociation constant of 500 nM. However, the data cannot be fitted to a straight line at higher concentration ofhirudin suggesting that hirudin binds also to another site with a different affinity. These results agree with the findings of Stone and Hofsteenge (Biochemistry 25, 4622-4628, 1986) and support the idea that initially hirudin binds at a site distinct from the active site, which then rearranges through a conformational change (detected by CD) to form a tigher complex in which hirudin is also bound to the active site.

scholarly journals Multiple binding sites involved in the effect of choline esters on decarbamoylation of monomethylcarbamoyl- or dimethylcarbamoly-acetylcholinesterase

1994 ◽  
Vol 301 (3) ◽  
pp. 713-720 ◽  
Author(s):  
D E Sok ◽  
Y B Kim ◽  
S J Choi ◽  
C H Jung ◽  
S H Cha
Keyword(s):  
Active Site ◽  
Binding Sites ◽  
Inhibitory Action ◽  
Competitive Inhibition ◽  
Acyl Chain ◽  
Active Centre ◽  
Multiple Binding Sites ◽  
Wide Range ◽  
Choline Esters ◽  
Multiple Binding

Multiple binding sites for inhibitory choline esters in spontaneous decarbamoylation of dimethylcarbamoyl-acetylcholinesterase (AChE) were suggested from a wide range of IC50 values, in contrast with a limited range of AC50 values (concentration giving 50% of maximal activation) at a peripheral activatory site. Association of choline esters containing a long acyl chain (C7-C12) with the hydrophobic zone in the active site could be deduced from a linear relationship between the size of the acyl group and the inhibitory potency in either spontaneous decarbamoylation or acetylthiocholine hydrolysis. Direct support for laurylcholine binding to the active site might come from the competitive inhibition (Ki 33 microM) of choline-catalysed decarbamoylation by laurylcholine. Moreover, its inhibitory action was greater for monomethylcarbamoyl-AChE than for dimethylcarbamoyl-AChE, where there is a greater steric hindrance at the active centre. In further support, the inhibition of pentanoylthiocholine-induced decarbamoylation by laurylcholine was suggested to be due to laurylcholine binding to a central site rather than a peripheral site, similar to the inhibition of spontaneous decarbamoylation by laurylcholine. Supportive data for acetylcholine binding to the active site are provided by the results that acetylcholine is a competitive inhibitor (Ki 7.6 mM) of choline-catalysed decarbamoylation, and its inhibitory action was greater for monomethylcarbamoyl-AChE than for dimethylcarbamoyl-AChE. Meanwhile, choline esters with an acyl group of an intermediate size (C4-C6), more subject to steric exclusion at the active centre, and less associable with the hydrophobic zone, appear to bind preferentially to a peripheral activity site. Thus the multiple effects of choline esters may be governed by hydrophobicity and/or a steric effect exerted by the acyl moiety at the binding sites.

scholarly journals Multiple binding sites for resin-acid derivatives on the voltage-sensor domain of the Shaker potassium channel

2020 ◽  
Author(s):  
Malin Silverå Ejneby ◽  
Arina Gromova ◽  
Nina E Ottosson ◽  
Stina Borg ◽  
Argel Estrada-Mondragón ◽  
...  
Keyword(s):  
Binding Sites ◽  
Resin Acid ◽  
Voltage Sensor ◽  
Resin Acids ◽  
Voltage Gated ◽  
Multiple Binding Sites ◽  
Gating Charge ◽  
Multiple Binding ◽  
A Site ◽  
Positively Charged

ABSTRACTNegatively charged resin acids and their derivatives open voltage-gated potassium (KV) channels by attracting the positively charged voltage-sensor helix of the channel (S4) towards the extracellular leaflet of the cellular membrane and thereby favoring gate opening. The resin acids have been proposed to primarily bind in a pocket in the periphery of the channel, located between the lipid-facing extracellular ends of the transmembrane segments S3 and S4. However, in apparent contrast to the suggested electrostatic mechanism, neutralization of the top gating charge of the Shaker KV channel did not reduce the resin-acid induced opening, but unexpectedly increased it, suggesting other mechanisms and other sites of action. Here we explored the binding of two resin-acid derivatives, Wu50 and Wu161, to the activated open state Shaker KV channel by a combination of in-silico docking, molecular dynamics simulations, and electrophysiology of mutated channels. We identified three potential resinacid binding sites around the voltage sensor helix S4: (1) The S3/S4 site suggested previously. Positively charged residues introduced at the top of S4 are critical to keep the compound bound in this site by electrostatic force. (2) A site located in the cleft between S4 and the pore domain (the S4/pore site). A tryptophan at the top of S6 and the top gating charge of S4 keeps the compound bound. (3) A site located at the extracellular side of the voltage-sensor domain in a cleft formed by S1-S4 (the top-VSD site). The presence of multiple binding sites around S4 and the anticipated helical-screw motion of the helix during activation makes the effect of resin-acid derivatives on channel function intricate. The propensity of a specific resin acid to activate and open a voltage-gated channel likely depends on its exact binding pose and the types of interactions it can form with the protein in a state-specific manner.

NMDAR PAMs: Multiple Chemotypes for Multiple Binding Sites

2019 ◽  
Vol 19 (24) ◽  
pp. 2239-2253 ◽  
Author(s):  
Paul J. Goldsmith
Keyword(s):  
Binding Sites ◽  
Receptor Activation ◽  
Research Area ◽  
Allosteric Modulators ◽  
Nonselective Cation Channels ◽  
Multiple Binding Sites ◽  
Multiple Binding ◽  
Excitatory Neurotransmission ◽  
Psychiatric Conditions ◽  
High Level

The N-methyl-D-aspartate receptor (NMDAR) is a member of the ionotropic glutamate receptor (iGluR) family that plays a crucial role in brain signalling and development. NMDARs are nonselective cation channels that are involved with the propagation of excitatory neurotransmission signals with important effects on synaptic plasticity. NMDARs are functionally and structurally complex receptors, they exist as a family of subtypes each with its own unique pharmacological properties. Their implication in a variety of neurological and psychiatric conditions means they have been a focus of research for many decades. Disruption of NMDAR-related signalling is known to adversely affect higherorder cognitive functions (e.g. learning and memory) and the search for molecules that can recover (or even enhance) receptor output is a current strategy for CNS drug discovery. A number of positive allosteric modulators (PAMs) that specifically attempt to overcome NMDAR hypofunction have been discovered. They include various chemotypes that have been found to bind to several different binding sites within the receptor. The heterogeneity of chemotype, binding site and NMDAR subtype provide a broad landscape of ongoing opportunities to uncover new features of NMDAR pharmacology. Research on NMDARs continues to provide novel mechanistic insights into receptor activation and this review will provide a high-level overview of the research area and discuss the various chemical classes of PAMs discovered so far.

Effects of Multiple Binding Sites on Studies of Hydrogen Bonding between Nitroxide Radicals and Solvent Molecules

1993 ◽  
Vol 58 (1) ◽  
pp. 47-52 ◽  
Author(s):  
Imad Al-Bala'a ◽  
Richard D. Bates
Keyword(s):  
Hydrogen Bonding ◽  
Magnetic Resonance ◽  
Binding Site ◽  
Binding Sites ◽  
Hyperfine Coupling Constant ◽  
Hydrogen Donor ◽  
Complex Formation Constants ◽  
Multiple Binding Sites ◽  
Multiple Binding ◽  
Solvent Molecules

The role of more than one binding site on a nitroxide free radical in magnetic resonance determinations of the properties of the complex formed with a hydrogen donor is examined. The expression that relates observed hyperfine couplings in EPR spectra to complex formation constants and concentrations of each species in solution becomes much more complex when multiple binding sites are present, but reduces to a simpler form when binding at the two sites occurs independently and the binding at the non-nitroxide site does not produce significant differences in the hyperfine coupling constant in the complexed radical. Effects on studies of hydrogen bonding between multiple binding site nitroxides and hydrogen donor solvent molecules by other magnetic resonance methods are potentially more extreme.

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