Allosteric control of an ionotropic glutamate receptor with an optical switch

Abstract

The precise regulation of protein activity is fundamental to life. The allosteric control of an active site by a remote regulatory binding site is a mechanism of regulation found across protein classes, from enzymes to motors to signaling proteins. We describe a general approach for manipulating allosteric control using synthetic optical switches. Our strategy is exemplified by a ligand-gated ion channel of central importance in neuroscience, the ionotropic glutamate receptor (iGluR). Using structure-based design, we have modified its ubiquitous clamshell-type ligand-binding domain to develop a light-activated channel, which we call LiGluR. An agonist is covalently tethered to the protein through an azobenzene moiety, which functions as the optical switch. The agonist is reversibly presented to the binding site upon photoisomerization, initiating clamshell domain closure and concomitant channel gating. Photoswitching occurs on a millisecond timescale, with channel conductances that reflect the photostationary state of the azobenzene at a given wavelength. Our device has potential uses not only in biology but also in bioelectronics and nanotechnology.Many proteins function like molecular machines that undergo mechanical movements in response to input signals. These signals can consist of changes in voltage, membrane tension, temperature or, most commonly, ligand concentration. Ligands provide information about events in the external world or about the energetic or biosynthetic state of the cell. They can be as small as a proton or as large as a whole protein. In allostery, ligand binding induces a structural change of a sensor domain, which propagates to a functional domain of the protein and alters its behavior. Such conformational control can operate over long distances, crossing a membrane or passing from one protein to another in a complex.Reengineering of nanoscopic protein machines to contain artificial control elements would be a major benefit for biology and technology. Optical switches would be especially powerful, as they could be activated remotely with precise temporal and spatial control 1,2 . A simple design Correspondence should be addressed to E.I. (ehud@berkeley.edu) and D.T. (trauner@berkeley.edu).. 5 These authors contributed equally to this work.Note: Supplementary information is available on the Nature Chemical Biology website. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript strategy would be to modify a protein by attaching a synthetic ligand whose binding ability could be altered by light. These ligands tethered via an optical switch could function in two ways. They could conditionally block the active site of an enzyme or the pore of a channel without inducing major conformational changes in the protein, or they could reversibly present an agonist to an allosteric binding site and conditionally trigger the normal conformational changes of ac...

Genetically encoding a light switch in an ionotropic glutamate receptor reveals subunit-specific interfaces

“…The genetic encoding technique is also well-suited for biological applications as it allows for both receptor-subtype selectivity and cell-type specificity. It thus complements the existing posttranslational labeling approach, which relies on photoswitchable ligands tethered to specific cysteines introduced in the receptor (29,30). Although this latter approach is limited to extracellular sites, it has the essential advantage of being reversible (the receptor can be turned on and off in a reversible manner) and has proven powerful in controlling neuronal firing and synaptic circuits in behaving animals (2,31).…”

Long-Range Conformational Transition of a Photoswitchable Allosteric Protein: Molecular Dynamics Simulation Study

“…[30][31][32][33][34][35][36][37][38] To facilitate a dynamic perspective of the allosteric mechanism, recently Buchli et al 45 presented a time-resolved study of the transition from the free to the bound state of PDZ2 triggered by a molecular photoswitch. [46][47][48][49][50][51] By covalently linking an azobenzene photoswitch across the binding groove and using a femtosecond laser pulse that affects the cis → trans photoisomerization of azobenzene, they were able to initiate a conformational change similar to the free-bound transition. The photoswitch was attached at residues 21 and 76 of PDZ2 ( Figure Figure 1), because the C α distances between these anchor residues in the free and bound state closely match the linear extension of the photoswitch in the cis and the trans state, respectively.…”

Synthesis of 3,3′-bis(sulfonato)-4,4′-bis(chloroacetamido)azobenzene and cysteine cross-linking for photo-control of protein conformation and activity

“…Examples of biomolecular systems into which azobenzene has been incorporated for photo-regulation are peptides [1][2][3] , enzymes [4][5][6][7] , oligonucleotides 8,9 and ion channels 10,11 . Methods for incorporating azobenzene chromophores into biomolecules include solid-phase peptide or oligonucleotide synthesis 7,12 , nonsense suppression via azobenzenecharged suppressor tRNAs 4 , and both non-selective 13 and targeted chemical modification of protein side chains 14 .…”