Daniel S. Terry

Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue

Neurotransmitter/Na+ symporters (NSSs) terminate neuronal signalling by recapturing neurotransmitter released into the synapse in a co-transport (symport) mechanism driven by the Na+ electrochemical gradient. NSSs for dopamine, noradrenaline and serotonin are targeted by the psychostimulants cocaine and amphetamine, as well as by antidepressants. The crystal structure of LeuT, a prokaryotic NSS homologue, revealed an occluded conformation in which a leucine (Leu) and two Na+ are bound deep within the protein. This structure has been the basis for extensive structural and computational exploration of the functional mechanisms of proteins with a LeuT-like fold. Subsequently, an ‘outward-open’ conformation was determined in the presence of the inhibitor tryptophan, and the Na+-dependent formation of a dynamic outward-facing intermediate was identified using electron paramagnetic resonance spectroscopy. In addition, single-molecule fluorescence resonance energy transfer imaging has been used to reveal reversible transitions to an inward-open LeuT conformation, which involve the movement of transmembrane helix TM1a away from the transmembrane helical bundle. We investigated how substrate binding is coupled to structural transitions in LeuT during Na+-coupled transport. Here we report a process whereby substrate binding from the extracellular side of LeuT facilitates intracellular gate opening and substrate release at the intracellular face of the protein. In the presence of alanine, a substrate that is transported ∼10-fold faster than leucine, we observed alanine-induced dynamics in the intracellular gate region of LeuT that directly correlate with transport efficiency. Collectively, our data reveal functionally relevant and previously hidden aspects of the NSS transport mechanism that emphasize the functional importance of a second substrate (S2) binding site within the extracellular vestibule. Substrate binding in this S2 site appears to act cooperatively with the primary substrate (S1) binding site to control intracellular gating more than 30 Å away, in a manner that allows the Na+ gradient to power the transport mechanism.

Single-molecule dynamics of gating in a neurotransmitter transporter homologue

Neurotransmitter:Na+ symporters (NSS) remove neurotransmitters from the synapse in a reuptake process that is driven by the Na+ gradient. Drugs that interfere with this reuptake mechanism, such as cocaine and antidepressants, profoundly influence behaviour and mood. To probe the nature of the conformational changes that are associated with substrate binding and transport, we have developed a single-molecule fluorescence imaging assay and combined it with functional and computational studies of the prokaryotic NSS homologue LeuT. Here we show molecular details of the modulation of intracellular gating of LeuT by substrates and inhibitors, as well as by mutations that alter binding, transport or both. Our direct observations of single-molecule transitions, reflecting structural dynamics of the intracellular region of the transporter that might be masked by ensemble averaging or suppressed under crystallographic conditions, are interpreted in the context of an allosteric mechanism that couples ion and substrate binding to transport.

Crystal structure, conformational fixation and entry-related interactions of mature ligand-free HIV-1 Env

As the sole viral antigen on the HIV-1–virion surface, trimeric Env is a focus of vaccine efforts. Here we present the structure of the ligand-free HIV-1–Env trimer, fix its conformation and determine its receptor interactions. Epitope analyses revealed trimeric ligand-free Env to be structurally compatible with broadly neutralizing antibodies but not poorly neutralizing ones. We coupled these compatibility considerations with binding antigenicity to engineer conformationally fixed Envs, including a 201C 433C (DS) variant specifically recognized by broadly neutralizing antibodies. DS-Env retained nanomolar affinity for the CD4 receptor, with which it formed an asymmetric intermediate: a closed trimer bound by a single CD4 without the typical antigenic hallmarks of CD4 induction. Antigenicity-guided structural design can thus be used both to delineate mechanism and to fix conformation, with DS-Env trimers in virus-like-particle and soluble formats providing a new generation of vaccine antigens.

Cyanine fluorophore derivatives with enhanced photostability

Fluorescence applications requiring high photostability often depend on the use of solution additives to enhance fluorophore performance. Here we demonstrate that the direct or proximal conjugation of cyclooctatetraene (COT), 4-nitrobenzyl alcohol (NBA) or Trolox to the cyanine fluorophore Cy5 dramatically enhanced fluorophore photostability without otherwise affecting its native spectral characteristics. Such conjugation is a powerful means of improving the robustness of fluorescence-based applications demanding long-lived, nonblinking fluorescence emission.

Mitigating unwanted photophysical processes for improved single-molecule fluorescence imaging.

Organic fluorophores common to fluorescence-based investigations suffer from unwanted photophysical properties, including blinking and photobleaching, which limit their overall experimental performance. Methods to control such processes are particularly important for single-molecule fluorescence and fluorescence resonance energy transfer imaging where uninterrupted, stable fluorescence is paramount. Fluorescence and FRET-based assays have been carried out on dye-labeled DNA and RNA-based systems to quantify the effect of including small-molecule solution additives on the fluorescence and FRET behaviors of both cyanine and Alexa fluorophores. A detailed dwell time analysis of the fluorescence and FRET trajectories of more than 200,000 individual molecules showed that two compounds identified previously as triplet state quenchers, cyclooctatetraene, and Trolox, as well as 4-nitrobenzyl alcohol, act to favorably attenuate blinking, photobleaching, and influence the rate of photoresurrection in a concentration-dependent and context-dependent manner. In both biochemical systems examined, a unique cocktail of compounds was shown to be optimal for imaging performance. By simultaneously providing the most rapid and direct access to multiple photophysical kinetic parameters, smFRET imaging provides a powerful avenue for future investigations aimed at discovering new compounds, and effective combinations thereof. These efforts may ultimately facilitate tuning organic dye molecule performance according to each specific experimental demand.

Conformational sampling of aminoacyl-tRNA during selection on the bacterial ribosome.

Aminoacyl-tRNA (aa-tRNA), in a ternary complex with elongation factor-Tu and GTP, enters the aminoacyl (A) site of the ribosome via a multi-step, mRNA codon-dependent mechanism. This process gives rise to the preferential selection of cognate aa-tRNAs for each mRNA codon and, consequently, the fidelity of gene expression. The ribosome actively facilitates this process by recognizing structural features of the correct substrate, initiated in its decoding site, to accelerate the rates of elongation factor-Tu-catalyzed GTP hydrolysis and ribosome-catalyzed peptide bond formation. Here, the order and timing of conformational events underpinning the aa-tRNA selection process were investigated from multiple structural perspectives using single-molecule fluorescence resonance energy transfer. The time resolution of these measurements was extended to 2.5 and 10 ms, a 10- to 50-fold improvement over previous studies. The data obtained reveal that aa-tRNA undergoes fast conformational sampling within the A site, both before and after GTP hydrolysis. This suggests that the alignment of aa-tRNA with respect to structural elements required for irreversible GTP hydrolysis and peptide bond formation plays a key role in the fidelity mechanism. These observations provide direct evidence that the selection process is governed by motions of aa-tRNA within the A site, adding new insights into the physical framework that helps explain how the rates of GTP hydrolysis and peptide bond formation are controlled by the mRNA codon and other fidelity determinants within the system.

High-resolution structure of the Escherichia coli ribosome

Protein synthesis by the ribosome is highly dependent on the ionic conditions in the cellular environment, but the roles of ribosome solvation have remained poorly understood. Moreover, the functions of modifications to ribosomal RNA and ribosomal proteins have also been unclear. Here we present the structure of the Escherichia coli 70S ribosome at 2.4-Å resolution. The structure reveals details of the ribosomal subunit interface that are conserved in all domains of life, and it suggests how solvation contributes to ribosome integrity and function as well as how the conformation of ribosomal protein uS12 aids in mRNA decoding. This structure helps to explain the phylogenetic conservation of key elements of the ribosome, including post-transcriptional and post-translational modifications, and should serve as a basis for future antibiotic development.

Aminoglycoside activity observed on single pre-translocation ribosome complexes

Aminoglycoside-class antibiotics bind directly to ribosomal RNA, imparting pleiotropic effects on ribosome function. Despite in-depth structural investigations of aminoglycoside-RNA oligonucleotide and aminoglycoside-ribosome interactions, mechanisms explaining the unique ribosome inhibition profiles of chemically similar aminoglycosides remain elusive. Here, using single-molecule fluorescence resonance energy transfer (smFRET) methods, we show that high-affinity aminoglycoside binding to the conserved decoding site region of the functional pre-translocation ribosome complex specifically remodels the nature of intrinsic dynamic processes within the particle. The extents of these effects, which are distinct for each member of the aminoglycoside class, strongly correlate with their inhibition of EF-G-catalyzed translocation. Neomycin, a 4,5-linked aminoglycoside, binds with lower affinity to one or more secondary binding sites, mediating distinct structural and dynamic perturbations that further enhance translocation inhibition. These new insights help explain why closely related aminoglycosides elicit pleiotropic translation activities and demonstrate the potential utility of smFRET as a tool for dissecting the mechanisms of antibiotic action.

Enhanced photostability of cyanine fluorophores across the visible spectrum

protective agents. Notably, these positive effects varied substantially for each fluorophore type. For instance, each of the COT-linked fluorophores showed dramatic enhancements in overall performance. For Cy2-COT, we observed an ~25-fold increase in the number of photons emitted before a dark-state transition. For Cy3-COT and Cy3.5-COT, this parameter changed very little, but the SNR of fluorescence for both molecules increased approximately fourto fivefold. Cy5.5-COT and Cy7-COT showed 50and 70-fold increases, respectively, in the number of photons emitted before entering a dark state. Consequently, the average Cy5.5-COT and Cy7-COT molecule could be continuously imaged for ~3 min at an SNR >7:1 (Fig. 1c). Consistent with our previous work1, we observed only modest improvements for most NBA-linked fluorophores. However, we observed an almost 70-fold enhancement of photon count for Cy7-NBA. Trolox, which shows the most favorable impact on the performance of the Cy5 fluorophore1, also had output before blinking or photobleaching. However, a quantitative understanding of the distinct mechanisms for enhancing fluorophore performance can only be delineated with confidence when married with bulk electrochemical and spectroscopic investigations. These combined approaches, although requiring large quantities of material, could provide grounded insights into the impact and relative weighting of ‘self-healing’ and ‘self-protecting’ mechanisms for distinct fluorophore types. Progress on this front will enable both additional improvements in the performances of known protective agents and the search for new compounds with similar or improved properties. Developments of this kind may ultimately facilitate the design and synthesis of new classes of fluorophores spanning the visible spectrum with enhancements in performance even greater than those observed for the cyanine class4 and tailored properties for distinct experimental demands.

Transport domain unlocking sets the uptake rate of an aspartate transporter

Glutamate transporters terminate neurotransmission by clearing synaptically released glutamate from the extracellular space, allowing repeated rounds of signalling and preventing glutamate-mediated excitotoxicity. Crystallographic studies of a glutamate transporter homologue from the archaeon Pyrococcus horikoshii, GltPh, showed that distinct transport domains translocate substrates into the cytoplasm by moving across the membrane within a central trimerization scaffold. Here we report direct observations of these ‘elevator-like’ transport domain motions in the context of reconstituted proteoliposomes and physiological ion gradients using single-molecule fluorescence resonance energy transfer (smFRET) imaging. We show that GltPh bearing two mutations introduced to impart characteristics of the human transporter exhibits markedly increased transport domain dynamics, which parallels an increased rate of substrate transport, thereby establishing a direct temporal relationship between transport domain motion and substrate uptake. Crystallographic and computational investigations corroborated these findings by revealing that the ‘humanizing’ mutations favour structurally ‘unlocked’ intermediate states in the transport cycle exhibiting increased solvent occupancy at the interface between the transport domain and the trimeric scaffold.