Smriti Mishra

Conformational dynamics of the nucleotide binding domains and the power stroke of a heterodimeric ABC transporter

Multidrug ATP binding cassette (ABC) exporters are ubiquitous ABC transporters that extrude cytotoxic molecules across cell membranes. Despite recent progress in structure determination of these transporters, the conformational motion that transduces the energy of ATP hydrolysis to the work of substrate translocation remains undefined. Here, we have investigated the conformational cycle of BmrCD, a representative of the heterodimer family of ABC exporters that have an intrinsically impaired nucleotide binding site. We measured distances between pairs of spin labels monitoring the movement of the nucleotide binding (NBD) and transmembrane domains (TMD). The results expose previously unobserved structural intermediates of the NBDs arising from asymmetric configuration of catalytically inequivalent nucleotide binding sites. The two-state transition of the TMD, from an inward- to an outward-facing conformation, is driven exclusively by ATP hydrolysis. These findings provide direct evidence of divergence in the mechanism of ABC exporters. DOI: http://dx.doi.org/10.7554/eLife.02740.001

Protonation drives the conformational switch in the multidrug transporter LmrP

Multidrug antiporters of the major facilitator superfamily couple proton translocation to the extrusion of cytotoxic molecules. The conformational changes that underlie the transport cycle and the structural basis of coupling of these transporters have not been elucidated. Here we used extensive double electron-electron resonance measurements to uncover the conformational equilibrium of LmrP, a multidrug transporter from Lactococcus lactis, and to investigate how protons and ligands shift this equilibrium to enable transport. We find that the transporter switches between outward-open and outward-closed conformations, depending on the protonation states of specific acidic residues forming a transmembrane protonation relay. Our data can be framed in a model of transport wherein substrate binding initiates the transport cycle by opening the extracellular side. Subsequent protonation of membrane-embedded acidic residues induces substrate release to the extracellular side and triggers a cascade of conformational changes that concludes in proton release to the intracellular side.

Lipids modulate the conformational dynamics of a secondary multidrug transporter.

Direct interactions with lipids have emerged as key determinants of the folding, structure and function of membrane proteins, but an understanding of how lipids modulate protein dynamics is still lacking. Here, we systematically explored the effects of lipids on the conformational dynamics of the proton-powered multidrug transporter LmrP from Lactococcus lactis, using the pattern of distances between spin-label pairs previously shown to report on alternating access of the protein. We uncovered, at the molecular level, how the lipid headgroups shape the conformational-energy landscape of the transporter. The model emerging from our data suggests a direct interaction between lipid headgroups and a conserved motif of charged residues that control the conformational equilibrium through an interplay of electrostatic interactions within the protein. Together, our data lay the foundation for a comprehensive model of secondary multidrug transport in lipid bilayers.

Unfolding of a Temperature-Sensitive Domain Controls Voltage-Gated Channel Activation

Voltage-gated ion channels (VGICs) are outfitted with diverse cytoplasmic domains that impact function. To examine how such elements may affect VGIC behavior, we addressed how the bacterial voltage-gated sodium channel (BacNa(V)) C-terminal cytoplasmic domain (CTD) affects function. Our studies show that the BacNa(V) CTD exerts a profound influence on gating through a temperature-dependent unfolding transition in a discrete cytoplasmic domain, the neck domain, proximal to the pore. Structural and functional studies establish that the BacNa(V) CTD comprises a bi-partite four-helix bundle that bears an unusual hydrophilic core whose integrity is central to the unfolding mechanism and that couples directly to the channel activation gate. Together, our findings define a general principle for how the widespread four-helix bundle cytoplasmic domain architecture can control VGIC responses, uncover a mechanism underlying the diverse BacNa(V) voltage dependencies, and demonstrate that a discrete domain can encode the temperature-dependent response of a channel.

Protonation-dependent conformational dynamics of the multidrug transporter EmrE

Significance Small multidrug resistance (SMR) transporters play an important role in the protection of prokaryotes from cytotoxic molecules. They exploit the proton electrochemical gradient to drive the transport of these molecules out of the cell against their concentration gradient. This work investigated how binding of protons power the conformational changes that enable substrate binding and subsequent alternating access of the Escherichia coli SMR transporter EmrE. The results show that protonation induces large-scale reconfiguration of the structure, including helical rotation and tilt and repacking of loops. A highly conserved charged residue primarily accounts for proton sensing, but other acidic residues control local structures. Our findings stimulate a structural model of transport, offering a novel perspective on proton-coupled multidrug transport. The small multidrug transporter from Escherichia coli, EmrE, couples the energetically uphill extrusion of hydrophobic cations out of the cell to the transport of two protons down their electrochemical gradient. Although principal mechanistic elements of proton/substrate antiport have been described, the structural record is limited to the conformation of the substrate-bound state, which has been shown to undergo isoenergetic alternating access. A central but missing link in the structure/mechanism relationship is a description of the proton-bound state, which is an obligatory intermediate in the transport cycle. Here we report a systematic spin labeling and double electron electron resonance (DEER) study that uncovers the conformational changes of EmrE subsequent to protonation of critical acidic residues in the context of a global description of ligand-induced structural rearrangements. We find that protonation of E14 leads to extensive rotation and tilt of transmembrane helices 1–3 in conjunction with repacking of loops, conformational changes that alter the coordination of the bound substrate and modulate its access to the binding site from the lipid bilayer. The transport model that emerges from our data posits a proton-bound, but occluded, resting state. Substrate binding from the inner leaflet of the bilayer releases the protons and triggers alternating access between inward- and outward-facing conformations of the substrate-loaded transporter, thus enabling antiport without dissipation of the proton gradient.

Navigating Membrane Protein Structure, Dynamics, and Energy Landscapes Using Spin Labeling and EPR Spectroscopy.

A detailed understanding of the functional mechanism of a protein entails the characterization of its energy landscape. Achieving this ambitious goal requires the integration of multiple approaches including determination of high-resolution crystal structures, uncovering conformational sampling under distinct biochemical conditions, characterizing the kinetics and thermodynamics of transitions between functional intermediates using spectroscopic techniques, and interpreting and harmonizing the data into novel computational models. With increasing sophistication in solution-based and ensemble-oriented biophysical approaches such as electron paramagnetic resonance (EPR) spectroscopy, atomic resolution structural information can be directly linked to conformational sampling in solution. Here, we detail how recent methodological and technological advances in EPR spectroscopy have contributed to the elucidation of membrane protein mechanisms. Furthermore, we aim to assist investigators interested in pursuing EPR studies by providing an introduction to the technique, a primer on experimental design, and a description of the practical considerations of the method toward generating high quality data.

Na⁺-substrate coupling in the multidrug antiporter norm probed with a spin-labeled substrate.

NorM of the multidrug and toxic compound extrusion (MATE) family of transporters couples the efflux of a broad range of hydrophobic molecules to an inward Na⁺ gradient across the cell membrane. Several crystal structures of MATE transporters revealed distinct substrate binding sites leading to differing models of the mechanism of ion-coupled substrate extrusion. In the experiments reported here, we observed that a spin-labeled derivative of daunorubicin, Ruboxyl, is transported by NorM from Vibrio cholerae. It is therefore ideal for characterizing mechanistically relevant binding interactions with NorM and directly addressing the coupling of ion and drug binding. Fluorescence and electron paramagnetic resonance experiments revealed that Ruboxyl binds to NorM with micromolar affinity and becomes immobilized upon binding, even in the presence of Na⁺. Using double electron-electron resonance spectroscopy, we determined that Ruboxyl binds to a single site on the periplasmic side of the protein. The presence of Na⁺ did not translocate the substrate to a second site as previously proposed. These experiments surprisingly show that Na⁺ does not affect the affinity or location of the substrate binding site on detergent-solubilized NorM, thus suggesting that additional factors beyond simple mutual exclusivity of binding, such as the presence of a Na⁺ gradient across the native membrane, govern Na⁺-drug coupling during antiport.

Direct Spectroscopic Detection of ATP Turnover Reveals Mechanistic Divergence of ABC Exporters

We have applied high-field (W-band) pulse electron-nuclear double resonance (ENDOR) and electron-electron double resonance (ELDOR)-detected nuclear magnetic resonance (EDNMR) to characterize the coordination sphere of the Mn2+ co-factor in the nucleotide binding sites (NBSs) of ABC transporters. MsbA and BmrCD are two efflux transporters hypothesized to represent divergent catalytic mechanisms. Our results reveal distinct coordination of Mn2+ to ATP and transporter residues in the consensus and degenerate NBSs of BmrCD. In contrast, the coordination of Mn2+ at the two NBSs of MsbA is similar, which provides a mechanistic rationale for its higher rate constant of ATP hydrolysis relative to BmrCD. Direct detection of vanadate ion, trapped in a high-energy post-hydrolysis intermediate, further supports the notion of asymmetric hydrolysis by the two NBSs of BmrCD. The integrated spectroscopic approach presented here, which link energy input to conformational dynamics, can be applied to a variety of systems powered by ATP turnover.