Functional anatomy of an allosteric protein

Purohit, Prasad; Gupta, Shaweta; Jadey, Snehal; Auerbach, Anthony

doi:10.1038/ncomms3984

Cited by 66 publications

(102 citation statements)

References 45 publications

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“…Further, alanine mutations of the aromatic residues at each site have approximately independent energetic consequences and, hence, do not create a newfound interdependence. These results, and others reported elsewhere (24), are consistent with the idea that ΔG B1 is generated mainly by local interactions between the agonist and a few structural elements at each binding site. With regard to binding free energy, each agonist site can be considered a small and independent working part of the larger AChR complex.…”

Section: Discussionsupporting

confidence: 81%

Functional differences between neurotransmitter binding sites of muscle acetylcholine receptors

Nayak

Bruhova

Chakraborty

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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A muscle acetylcholine receptor (AChR) has two neurotransmitter binding sites located in the extracellular domain, at αδ and either αe (adult) or αγ (fetal) subunit interfaces. We used single-channel electrophysiology to measure the effects of mutations of five conserved aromatic residues at each site with regard to their contribution to the difference in free energy of agonist binding to active versus resting receptors (ΔG B1 ). The two binding sites behave independently in both adult and fetal AChRs. For four different agonists, including ACh and choline, ΔG B1 is ∼−2 kcal/mol more favorable at αγ compared with at αe and αδ. Only three of the aromatics contribute significantly to ΔG B1 at the adult sites (αY190, αY198, and αW149), but all five do so at αγ (as well as αY93 and γW55). γW55 makes a particularly large contribution only at αγ that is coupled energetically to those contributions of some of the α-subunit aromatics. The hydroxyl and benzene groups of loop C residues αY190 and αY198 behave similarly with regard to ΔG B1 at all three kinds of site. ACh binding energies estimated from molecular dynamics simulations are consistent with experimental values from electrophysiology and suggest that the αγ site is more compact, better organized, and less dynamic than αe and αδ. We speculate that the different sensitivities of the fetal αγ site versus the adult αe and αδ sites to choline and ACh are important for the proper maturation and function of the neuromuscular synapse.allosteric protein | ion channel | ligand binding sites | single-channel electrophysiology | synaptic maturation R eceptors at synapses respond to specific chemical signals in the extracellular environment because the active conformation of the protein has a higher affinity for the ligand compared with the resting conformation (1, 2). The active vs. resting difference in binding free energy increases the relative stability of the active state and, hence, the probability of a cellular response. In this report, we describe and distinguish sources of ligandbinding free energy in three kinds of agonist site present in mouse muscle nicotinic acetylcholine receptors (AChRs). Our goal was to use single-channel electrophysiology to assess the relative contribution of significant functional groups to the overall free energy generated by the affinity change at each type of site.At cholinergic synapses, the main chemical signals are ACh released from nerve terminals and choline, which is an ACh precursor, hydrolysis product, and stable component of serum (3). The muscle AChR has central pore surrounded by five subunits of composition α 2 βδe in adult-type and α 2 βδγ in fetal-type (Fig. 1A) (4). The fetal, γ, subunit is essential for proper synapse maturation, and the adult, e, subunit is necessary for proper function of mature synapses (5-7). Each AChR pentamer has two agonist binding sites in the extracellular domain, at αδ and either αe (adult) or αγ (fetal) subunit interfaces.The change in agonist affinity occurs within the global, resting↔active...

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Section: Discussionsupporting

confidence: 81%

Functional differences between neurotransmitter binding sites of muscle acetylcholine receptors

Nayak

Bruhova

Chakraborty

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…In the ivermectin-bound conformation, Val 45 is lodged against Pro 268, thus providing a steric block on the M2/M3 loop and, in turn, stabilizing the M2 and M3 helices and the entire transmembrane domain in an open pore conformation 27 . Furthermore, Pro 268 is strictly conserved throughout the family of Cys-loop receptors and mutations of this residue, as well as others nearby, have profound effects on the channel gating and desensitization behavior 1, 28 .…”

mentioning

confidence: 99%

X-ray structures of GluCl in apo states reveal a gating mechanism of Cys-loop receptors

Althoff¹,

Hibbs²,

Banerjee

et al. 2014

Nature

237

294

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Summary Cys-loop receptors are neurotransmitter-gated ion channels that are essential mediators of fast chemical neurotransmission and are associated with a large number of neurological diseases and disorders, as well as parasitic infections1–4. Members of this ion channel superfamily mediate excitatory or inhibitory neurotransmission depending on their ligand and ion selectivity. Structural information for Cys-loop receptors comes from several sources including electron microscopic studies of the nicotinic acetylcholine receptor 5, high resolution x-ray structures of extracellular domains6 and x-ray structures of bacterial orthologs 7–10. In 2011 our group published structures of the Caenorhabditis elegans glutamate-gated chloride channel (GluCl) in complex with the allosteric partial agonist, ivermectin, which provided insights into the structure of a possibly open state of a eukaryotic Cys-loop receptor, the basis for anion selectivity and channel block, and the mechanism by which ivermectin and related molecules stabilize the open state and potentiate neurotransmitter binding11. However, there remain unanswered questions about the mechanism of channel opening and closing, the location and nature of the shut ion channel gate, the transitions between the closed/resting, open/activated and closed/desensitized states, and the mechanism by which conformational changes are coupled between the extracellular, orthosteric agonist binding domain and the transmembrane, ion channel domain. Here we present two conformationally distinct structures of GluCl in the absence of ivermectin. Structural comparisons reveal a quaternary activation mechanism arising from rigid body movements between the extracellular and transmembrane domains and a mechanism for modulation of the receptor by phospholipids.

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“…1A, inset). Channel activation proceeds as a wave-like progression of structural rearrangements, initiated by the ligand binding reaction (1,2) and conveyed to the gate via interactions across the TZ (3)(4)(5).…”

mentioning

confidence: 99%

“…The glycine receptor channel (GlyR) 2 is an anion-selective member of the pentameric ligand-gated ion-channel (pLGIC) family. pLGICs are comprised of modular domains (Fig.…”

mentioning

confidence: 99%

Correlating Structural and Energetic Changes in Glycine Receptor Activation

Scott

Lynch

Keramidas

2015

Journal of Biological Chemistry

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Background: Interactions between GlnϪ26Ј (in M1 domain), Arg 19Ј (in M2 domain), and Lys 24Ј (in M2-M3 linker) may reveal molecular mechanisms of glycine receptor activation. Results: ␣1QϪ26ЈE -containing receptors have longer active periods and lower conductances. Conclusion:The energy for activation is distributed broadly at the transduction zone. Significance: These energetic interactions are likely present in multiple pentameric ligand-gated ion channels.

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Functional anatomy of an allosteric protein

Cited by 66 publications

References 45 publications

Functional differences between neurotransmitter binding sites of muscle acetylcholine receptors

Functional differences between neurotransmitter binding sites of muscle acetylcholine receptors

X-ray structures of GluCl in apo states reveal a gating mechanism of Cys-loop receptors

Correlating Structural and Energetic Changes in Glycine Receptor Activation

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