Ekaterina Zaitseva

Tracking G-protein-coupled receptor activation using genetically encoded infrared probes

Rhodopsin is a prototypical heptahelical family A G-protein-coupled receptor (GPCR) responsible for dim-light vision. Light isomerizes rhodopsins retinal chromophore and triggers concerted movements of transmembrane helices, including an outward tilting of helix 6 (H6) and a smaller movement of H5, to create a site for G-protein binding and activation. However, the precise temporal sequence and mechanism underlying these helix rearrangements is unclear. We used site-directed non-natural amino acid mutagenesis to engineer rhodopsin with p-azido-l-phenylalanine residues incorporated at selected sites, and monitored the azido vibrational signatures using infrared spectroscopy as rhodopsin proceeded along its activation pathway. Here we report significant changes in electrostatic environments of the azido probes even in the inactive photoproduct Meta I, well before the active receptor state was formed. These early changes suggest a significant rotation of H6 and movement of the cytoplasmic part of H5 away from H3. Subsequently, a large outward tilt of H6 leads to opening of the cytoplasmic surface to form the active receptor photoproduct Meta II. Thus, our results reveal early conformational changes that precede larger rigid-body helix movements, and provide a basis to interpret recent GPCR crystal structures and to understand conformational sub-states observed during the activation of other GPCRs.

Highly conserved tyrosine stabilizes the active state of rhodopsin

Light-induced isomerization of the 11-cis-retinal chromophore in the visual pigment rhodopsin triggers displacement of the second extracellular loop (EL2) and motion of transmembrane helices H5, H6, and H7 leading to the active intermediate metarhodopsin II (Meta II). We describe solid-state NMR measurements of rhodopsin and Meta II that target the molecular contacts in the region of the ionic lock involving these three helices. We show that a contact between Arg1353.50 and Met2576.40 forms in Meta II, consistent with the outward rotation of H6 and breaking of the dark-state Glu1343.49-Arg1353.50-Glu2476.30 ionic lock. We also show that Tyr2235.58 and Tyr3067.53 form molecular contacts with Met2576.40. Together these results reveal that the crystal structure of opsin in the region of the ionic lock reflects the active state of the receptor. We further demonstrate that Tyr2235.58 and Ala1323.47 in Meta II stabilize helix H5 in an active orientation. Mutation of Tyr2235.58 to phenylalanine or mutation of Ala1323.47 to leucine decreases the lifetime of the Meta II intermediate. Furthermore, the Y223F mutation is coupled to structural changes in EL2. In contrast, mutation of Tyr3067.53 to phenylalanine shows only a moderate influence on the Meta II lifetime and is not coupled to EL2.

Sequential Rearrangement of Interhelical Networks Upon Rhodopsin Activation in Membranes: the Meta IIa Conformational Substate

Photon absorption by rhodopsin is proposed to lead to an activation pathway that is described by the extended reaction scheme Meta I <==>Meta II(a) <==> Meta II(b) <==> Meta II(b)H(+), where Meta II(b)H(+) is thought to be the conformational substate that activates the G protein transducin. Here we test this extended scheme for rhodopsin in a membrane bilayer environment by investigating lipid perturbation of the activation mechanism. We found that symmetric membrane lipids having two unsaturated acyl chains, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), selectively stabilize the Meta II(a) substate in the above mechanism. By combining FTIR and UV-visible difference spectroscopy, we characterized the structural and functional changes involved in the transition to the Meta II(a) intermediate, which links the inactive Meta I intermediate with the Meta II(b) states formed by helix rearrangement. Besides the opening of the Schiff base ionic lock, the Meta II(a) substate is characterized by an activation switch in a conserved water-mediated hydrogen-bonded network involving transmembrane helices H1/H2/H7, which is sensed by its key residue Asp83. On the other hand, movement of retinal toward H5 and its interaction with another interhelical H3/H5 network mediated by His211 and Glu122 is absent in Meta II(a). The latter rearrangement takes place only in the subsequent transition to Meta II(b), which has been previously associated with movement of H6. Our results imply that activating structural changes in the H1/H2/H7 network are triggered by disruption of the Schiff base salt bridge and occur prior to other chromophore-induced changes in the H3/H5 network and the outward tilt of H6 in the activation process.

Automated Formation of Lipid Membrane Microarrays for Ionic Single-Molecule Sensing with Protein Nanopores

Efficient use of membrane protein nanopores in ionic single-molecule sensing requires technology for the reliable formation of suspended molecular membranes densely arrayed in formats that allow high-resolution electrical recording. Here, automated formation of bimolecular lipid layers is shown using a simple process where a poly(tetrafluoroethylene)-coated magnetic bar is remotely actuated to perform a turning motion, thereby spreading phospholipid in organic solvent on a nonpolar surface containing a <1 mm(2) 4 × 4 array of apertures with embedded microelectrodes (microelectrode cavity array). Parallel and high-resolution single-molecule detection by single nanopores is demonstrated on the resulting bilayer arrays, which are shown to form by a classical but very rapid self-assembly process. The technique provides a robust and scalable solution for the problem of reliable, automated formation of multiple independent lipid bilayers in a dense microarray format, while preserving the favorable electrical properties of the microelectrode cavity array.

Structural Impact of the E113Q Counterion Mutation on the Activation and Deactivation Pathways of the G Protein-coupled Receptor Rhodopsin

Disruption of an interhelical salt bridge between the retinal protonated Schiff base linked to H7 and Glu113 on H3 is one of the decisive steps during activation of rhodopsin. Using previously established stabilization strategies, we engineered a stabilized E113Q counterion mutant that converted rhodopsin to a UV-absorbing photoreceptor with deprotonated Schiff base and allowed reconstitution into native-like lipid membranes. Fourier-transform infrared difference spectroscopy reveals a deprotonated Schiff base in the photoproducts of the mutant up to the active state Meta II, the absence of the classical pH-dependent Meta I/Meta II conformational equilibrium in favor of Meta II, and an anticipation of active state features under conditions that stabilize inactive photoproduct states in wildtype rhodopsin. Glu181 on extracellular loop 2, is found to be unable to maintain a counterion function to the Schiff base on the activation pathway of rhodopsin in the absence of the primary counterion, Glu113. The Schiff base becomes protonated in the transition to Meta III. This protonation is, however, not associated with a deactivation of the receptor, in contrast to wildtype rhodopsin. Glu181 is suggested to be the counterion in the Meta III state of the mutant and appears to be capable of stabilizing a protonated Schiff base in Meta III, but not of constraining the receptor in an inactive conformation.

Retinal orientation and interactions in rhodopsin reveal a two-stage trigger mechanism for activation

The 11-cis retinal chromophore is tightly packed within the interior of the visual receptor rhodopsin and isomerizes to the all-trans configuration following absorption of light. The mechanism by which this isomerization event drives the outward rotation of transmembrane helix H6, a hallmark of activated G protein-coupled receptors, is not well established. To address this question, we use solid-state NMR and FTIR spectroscopy to define the orientation and interactions of the retinal chromophore in the active metarhodopsin II intermediate. Here we show that isomerization of the 11-cis retinal chromophore generates strong steric interactions between its β-ionone ring and transmembrane helices H5 and H6, while deprotonation of its protonated Schiffs base triggers the rearrangement of the hydrogen-bonding network involving residues on H6 and within the second extracellular loop. We integrate these observations with previous structural and functional studies to propose a two-stage mechanism for rhodopsin activation.

SEIRA spectroscopy on a membrane receptor monolayer using lipoprotein particles as carriers.

Surface-enhanced infrared absorption (SEIRA) difference spectroscopy can probe reactions in a protein monolayer tethered to a nanostructured gold surface. SEIRA studies of membrane proteins, however, remain challenging due to sample stability, effects of the metal surface on function, and the need for a membrane-mimicking environment. Here we demonstrate and characterize a model system for membrane receptor investigations using SEIRA spectroscopy. The system employs nanoscale apolipoprotein bound bilayer (NABB) particles, similar to discoidal high-density lipoprotein particles, as soluble carriers for the G-protein-coupled receptor rhodopsin. The His-tag of the engineered apolipoprotein allows for selective binding of the NABBs to a Ni-NTA modified surface, while the lipid environment of the particle ensures stability and protection of the embedded receptor. Using SEIRA spectroscopy, we followed specific binding of rhodopsin-loaded NABB particles to the surface and formation of a membrane protein monolayer. Functionality of the photoreceptor in the immobilized NABBs was probed by SEIRA difference spectroscopy confirming protein conformational changes associated with photoactivation. Orientation of the immobilized NABB particles was assessed by comparing SEIRA data with polarized attenuated total reflection-Fourier-transform infrared spectroscopy. Thus, SEIRA difference spectroscopy supported by the NABB technology provides a promising approach for further functional studies of transmembrane receptors.

Validation of ADAM10 metalloprotease as a Bacillus thuringiensis Cry3Aa toxin functional receptor in Colorado potato beetle (Leptinotarsa decemlineata).

Bacillus thuringiensis parasporal crystal proteins (Cry proteins) are insecticidal pore‐forming toxins that bind to specific receptor molecules on the brush border membrane of susceptible insect midgut cells to exert their toxic action. In the Colorado potato beetle (CPB), a coleopteran pest, we previously proposed that interaction of Cry3Aa toxin with a CPB ADAM10 metalloprotease is an essential part of the mode of action of this toxin. Here, we annotated the gene sequence encoding an ADAM10 metalloprotease protein (CPB‐ADAM10) in the CPB genome sequencing project, and using RNA interference gene silencing we demonstrated that CPB‐ADAM10 is a Cry3Aa toxin functional receptor in CPB. Cry3Aa toxicity was significantly lower in CPB‐ADAM10 silenced larvae and in vitro toxin pore‐forming ability was greatly diminished in lipid planar bilayers fused with CPB brush border membrane vesicles (BBMVs) prepared from CPB‐ADAM10 silenced larvae. In accordance with our previous data that indicated this toxin was a substrate of ADAM10 in CPB, Cry3Aa toxin membrane‐associated proteolysis was altered when CPB BBMVs lacked ADAM10. The functional validation of CPB‐ADAM10 as a Cry3Aa toxin receptor in CPB expands the already recognized role of ADAM10 as a pathogenicity determinant of pore‐forming toxins in humans to an invertebrate species.