Conformational spread and dynamics in allostery of NMDA receptors.

Allostery can be manifested as a combination of repression and activation in multidomain proteins allowing for fine tuning of regulatory mechanisms. Here we have used single molecule fluorescence resonance energy transfer (smFRET) and molecular dynamics simulations to study the mechanism of allostery underlying negative cooperativity between the two agonists glutamate and glycine in the NMDA receptor. These data show that binding of one agonist leads to conformational flexibility and an increase in conformational spread at the second agonist site. Mutational and cross-linking studies show that the dimer-dimer interface at the agonist-binding domain mediates the allostery underlying the negative cooperativity. smFRET on the transmembrane segments shows that they are tightly coupled in the unliganded and single agonist-bound form and only upon binding both agonists the transmembrane domain explores looser packing which would facilitate activation.

The structure-energy landscape of NMDA receptor gating.

N-Methyl-D-aspartate (NMDA) receptors are the main calcium-permeable excitatory receptors in the mammalian central nervous system. The NMDA receptor gating is complex, exhibiting multiple closed, open, and desensitized states; however, central questions regarding the conformations and energetics of the transmembrane domains as they relate to the gating states are still unanswered. Here, using single-molecule Förster resonance energy transfer (smFRET), we map the energy landscape of the first transmembrane segment of the Rattus norvegicus NMDA receptor under resting and various liganded conditions. These results show kinetically and structurally distinct changes associated with apo, agonist-bound, and inhibited receptors linked by a linear mechanism of gating at this site. Furthermore, the smFRET data suggest that allosteric inhibition by zinc occurs by an uncoupling of the agonist-induced changes at the extracellular domains from the gating motions leading to an apo-like state, while dizocilpine, a pore blocker, stabilizes multiple closely packed transmembrane states.

Computational and biochemical characterization of two partially overlapping interfaces and multiple weak-affinity K-Ras dimers.

Recent studies found that membrane-bound K-Ras dimers are important for biological function. However, the structure and thermodynamic stability of these complexes remained unknown because they are hard to probe by conventional approaches. Combining data from a wide range of computational and experimental approaches, here we describe the structure, dynamics, energetics and mechanism of assembly of multiple K-Ras dimers. Utilizing a range of techniques for the detection of reactive surfaces, protein-protein docking and molecular simulations, we found that two largely polar and partially overlapping surfaces underlie the formation of multiple K-Ras dimers. For validation we used mutagenesis, electron microscopy and biochemical assays under non-denaturing conditions. We show that partial disruption of a predicted interface through charge reversal mutation of apposed residues reduces oligomerization while introduction of cysteines at these positions enhanced dimerization likely through the formation of an intermolecular disulfide bond. Free energy calculations indicated that K-Ras dimerization involves direct but weak protein-protein interactions in solution, consistent with the notion that dimerization is facilitated by membrane binding. Taken together, our atomically detailed analyses provide unique mechanistic insights into K-Ras dimer formation and membrane organization as well as the conformational fluctuations and equilibrium thermodynamics underlying these processes.

Stargazin Modulation of AMPA Receptors.

Fast excitatory synaptic signaling in the mammalian brain is mediated by AMPA-type ionotropic glutamate receptors. In neurons, AMPA receptors co-assemble with auxiliary proteins, such as stargazin, which can markedly alter receptor trafficking and gating. Here, we used luminescence resonance energy transfer measurements to map distances between the full-length, functional AMPA receptor and stargazin expressed in HEK293 cells and to determine the ensemble structural changes in the receptor due to stargazin. In addition, we used single-molecule fluorescence resonance energy transfer to study the structural and conformational distribution of the receptor and how this distribution is affected by stargazin. Our nanopositioning data place stargazin below the AMPA receptor ligand-binding domain, where it is well poised to act as a scaffold to facilitate the long-range conformational selection observations seen in single-molecule experiments. These data support a model of stargazin acting to stabilize or select conformational states that favor activation.

Conformational Selection and Submillisecond Dynamics of the Ligand-binding Domain of the N-Methyl-d-aspartate Receptor.

The N-methyl-d-aspartate (NMDA) receptors are heteromeric non-selective cation channels that require the binding of glycine and glutamate for gating. Based on crystal structures, the mechanism of partial agonism at the glycine-binding site is thought to be mediated by a shift in the conformational equilibrium between an open clamshell and a closed clamshell-like structure of the bilobed ligand-binding domain (LBD). Using single-molecule Förster resonance energy transfer (smFRET) and multiparameter fluorescence detection, which allows us to study the conformational states and dynamics in the submillisecond time scale, we show that there are at least three conformational states explored by the LBD: the low FRET, medium FRET, and high FRET states. The distance of the medium and low FRET states corresponds to what has been observed in crystallography structures. We show that the high FRET state, which would represent a more closed clamshell conformation than that observed in the crystal structure, is most likely the state initiating activation, as evidenced by the fact that the fraction of the protein in this state correlates well with the extent of activation. Furthermore, full agonist bound LBDs show faster dynamic motions between the medium and high FRET states, whereas they show slower dynamics when bound to weaker agonists or to antagonists.

Synaptic connections of amacrine cells containing vesicular glutamate transporter 3 in baboon retinas.

The goals of these experiments were to describe the morphology and synaptic connections of amacrine cells in the baboon retina that contain immunoreactive vesicular glutamate transporter 3 (vGluT3). These amacrine cells had the morphology characteristic of knotty bistratified type 1 cells, and their dendrites formed two plexuses on either side of the center of the inner plexiform layer. The primary dendrites received large synapses from amacrine cells, and the higher-order dendrites were both pre- and postsynaptic to other amacrine cells. Based on light microscopic immunolabeling results, these include AII cells and starburst cells, but not the polyaxonal amacrine cells tracer-coupled to ON parasol ganglion cells. The vGluT3 cells received input from ON bipolar cells at ribbon synapses and made synapses onto OFF bipolar cells, including the diffuse DB3a type. Many synapses from vGluT3 cells onto retinal ganglion cells were observed in both plexuses. At synapses where vGluT3 cells were presynaptic, two types of postsynaptic densities were observed; there were relatively thin ones characteristic of inhibitory synapses and relatively thick ones characteristic of excitatory synapses. In the light microscopic experiments with Neurobiotin-injected ganglion cells, vGluT3 cells made contacts with midget and parasol ganglion cells, including both ON and OFF types. Puncta containing immunoreactive gephyrin, an inhibitory synapse marker, were found at appositions between vGluT3 cells and each of the four types of labeled ganglion cells. The vGluT3 cells did not have detectable levels of immunoreactive γ-aminobutyric acid (GABA) or immunoreactive glycine transporter 1. Thus, the vGluT3 cells would be expected to have ON responses to light and make synapses onto neurons in both the ON and the OFF pathways. Taken with previous results, these findings suggest that vGluT3 cells release glycine at some of their output synapses and glutamate at others.

Conformational transitions in the glycine-bound GluN1 NMDA receptor LBD via single-molecule FRET.

The N-methyl-D-aspartate receptor (NMDAR) is a member of the glutamate receptor family of proteins and is responsible for excitatory transmission. Activation of the receptor is thought to be controlled by conformational changes in the ligand binding domain (LBD); however, glutamate receptor LBDs can occupy multiple conformations even in the activated form. This work probes equilibrium transitions among NMDAR LBD conformations by monitoring the distance across the glycine-bound LBD cleft using single-molecule Förster resonance energy transfer (smFRET). Recent improvements in photoprotection solutions allowed us to monitor transitions among the multiple conformations. Also, we applied a recently developed model-free algorithm called "step transition and state identification" to identify the number of states, their smFRET efficiencies, and their interstate kinetics. Reversible interstate conversions, corresponding to transitions among a wide range of cleft widths, were identified in the glycine-bound LBD, on much longer timescales compared to channel opening. These transitions were confirmed to be equilibrium in nature by shifting the distribution reversibly via denaturant. We found that the NMDAR LBD proceeds primarily from one adjacent smFRET state to the next under equilibrium conditions, consistent with a cleft-opening/closing mechanism. Overall, by analyzing the state-to-state transition dynamics and distributions, we achieve insight into specifics of long-lived LBD equilibrium structural dynamics, as well as obtain a more general description of equilibrium folding/unfolding in a conformationally dynamic protein. The relationship between such long-lived LBD dynamics and channel function in the full receptor remains an open and interesting question.

Structural dynamics of the glycine-binding domain of the N-methyl-D-aspartate receptor.

N-Methyl-D-aspartate receptors mediate the slow component of excitatory neurotransmission in the central nervous system. These receptors are obligate heteromers containing glycine- and glutamate-binding subunits. The ligands bind to a bilobed agonist-binding domain of the receptor. Previous x-ray structures of the glycine-binding domain of NMDA receptors showed no significant changes between the partial and full agonist-bound structures. Here we have used single molecule fluorescence resonance energy transfer (smFRET) to investigate the cleft closure conformational states that the glycine-binding domain of the receptor adopts in the presence of the antagonist 5,7-dichlorokynurenic acid (DCKA), the partial agonists 1-amino-1-cyclobutanecarboxylic acid (ACBC) and L-alanine, and full agonists glycine and D-serine. For these studies, we have incorporated the unnatural amino acid p-acetyl-L-phenylalanine for specific labeling of the protein with hydrazide derivatives of fluorophores. The single molecule fluorescence resonance energy transfer data show that the agonist-binding domain can adopt a wide range of cleft closure states with significant overlap in the states occupied by ligands of varying efficacy. The difference lies in the fraction of the protein in a more closed-cleft form, with full agonists having a larger fraction in the closed-cleft form, suggesting that the ability of ligands to select for these states could dictate the extent of activation.

Luminescence resonance energy transfer to study conformational changes in membrane proteins expressed in mammalian cells.

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 Å. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.