Exploring conformational equilibria of a heterodimeric ABC transporter

ABC exporters pump substrates across the membrane by coupling ATP-driven movements of nucleotide binding domains (NBDs) to the transmembrane domains (TMDs), which switch between inward- and outward-facing (IF, OF) orientations. DEER measurements on the heterodimeric ABC exporter TM287/288 from Thermotoga maritima, which contains a non-canonical ATP binding site, revealed that in the presence of nucleotides the transporter exists in an IF/OF equilibrium. While ATP binding was sufficient to partially populate the OF state, nucleotide trapping in the pre- or post-hydrolytic state was required for a pronounced conformational shift. At physiologically high temperatures and in the absence of nucleotides, the NBDs disengage asymmetrically while the conformation of the TMDs remains unchanged. Nucleotide binding at the degenerate ATP site prevents complete NBD separation, a molecular feature differentiating heterodimeric from homodimeric ABC exporters. Our data suggest hydrolysis-independent closure of the NBD dimer, which is further stabilized as the consensus site nucleotide is committed to hydrolysis

Exploring conformational dynamics and equilibria of ABC transporters

ATP-Bindungskassetten-Transporter bilden eine bedeutende Superfamilie von Membranproteinen, die Energie aus der Hydrolyse von ATP nutzen, um Substratmolekle über die Zellmembran zu transportieren. Ein detailliertes Verständnis der Transportmechanismen, sowie ihrer Inhibition ist notwendig, um Krankheiten zu bekämpfen, die durch Fehlfunktionen, Mutationen oder Überexpression von ABC-Transportern verursacht werden. Verschiedene biochemische Werkzeuge wie Elektronenmikroskopie oder Röntgenkristallographie haben tiefe Einblicke in die Struktur und Funktion von verschiedenen ABC-Transportern gegeben, jedoch liefern sie nur statische Einsichten in den Transportzyklus. Dazu sind zusätzliche Methoden nötig, die die Dynamik und die Zwischenzustände des Systems beschreiben können. Elektronenspinresonanz (ESR/EPR)-Spektroskopie in Kombination mit ortsspezifischer Spinmarkierung bietet leistungsstarke Werkzeuge um genau diese Dynamiken zu untersuchen.ATP binding cassette (ABC) transporters form a significant super family of membrane proteins coupling the energy of ATP hydrolysis to substrate transport across the cell membrane. Unraveling transport and inhibition mechanisms is crucial to address several diseases caused by malfunction, over expression and mutation of ABC transporters. Several biochemical tools including electron microscopy and X-ray crystallography have aided the mechanistic characterization of many ABC transporters in static states of the transport cycle. While providing unprecedented insight into the mechanism, static states cannot explain the complete machinery. To achieve a full understanding, all the intermediate states as well as the collective behavior of transporters throughout the cycle need to be studied. Site-directed spin labeling and electron paramagnetic resonance (EPR) techniques provide invaluable information on dynamics, structure and function of ABC transporters under physiological conditions

Spin-labeled nanobodies as protein conformational reporters for electron paramagnetic resonance in cellular membranes

Nanobodies are emerging tools in a variety of fields such as structural biology, cell imaging, and drug discovery. Here we pioneer the use of their spin-labeled variants as reporters of conformational dynamics of membrane proteins using DEER spectroscopy. At the example of the bacterial ABC transporter TM287/288, we show that two gadolinium-labeled nanobodies allow us to quantify, via analysis of the modulation depth of DEER traces, the fraction of transporters adopting the outward-facing state under different experimental conditions. Additionally, we quantitatively follow the interconversion from the outward- to the inward-facing state in the conformational ensemble under ATP turnover conditions. We finally show that the specificity of the nanobodies for the target protein allows the direct attainment of structural information on the wild-type TM287/288 expressed in cellular membranes without the need to purify or label the investigated membrane protein

Spin-labeled nanobodies as protein conformational reporters for electron paramagnetic resonance in cellular membranes

Nanobodies are emerging tools in a variety of fields such as structural biology, cell imaging, and drug discovery. Here we pioneer the use of their spin-labeled variants as reporters of conformational dynamics of membrane proteins using DEER spectroscopy. At the example of the bacterial ABC transporter TM287/288, we show that two gadolinium-labeled nanobodies allow us to quantify, via analysis of the modulation depth of DEER traces, the fraction of transporters adopting the outward-facing state under different experimental conditions. Additionally, we quantitatively follow the interconversion from the outward- to the inward-facing state in the conformational ensemble under ATP turnover conditions. We finally show that the specificity of the nanobodies for the target protein allows the direct attainment of structural information on the wild-type TM287/288 expressed in cellular membranes without the need to purify or label the investigated membrane protein.</p

Mode of Interaction of the Signal-Transducing Protein EIIA^Glc with the Maltose ABC Transporter in the Process of Inducer Exclusion

Enzyme IIA^Glc (EIIA^Glc) of the phosphoenolpyruvate phosphotransferase system for the uptake of glucose in <i>Escherichia coli</i> and <i>Salmonella</i> inhibits the maltose ATP-binding cassette transporter (MalE-FGK₂) by interaction with the nucleotide-binding and -hydrolyzing subunit MalK, a process termed inducer exclusion. We have investigated binding of EIIA^Glc to the MalK dimer by cysteine cross-linking in proteoliposomes. The results prove that the binding site I of EIIA^Glc is contacting the N-terminal subdomain of MalK while the binding site II is relatively close to the C-terminal (regulatory) subdomain, in agreement with a crystal structure [Chen, S., Oldham, M. L., Davidson, A. L., and Chen, J. (2013) Nature 499, 364−368]. Moreover, EIIA^Glc was found to bind to the MalK dimer regardless of its conformational state. Deletion of the amphipathic N-terminal peptide of EIIA^Glc, which is required for inhibition, reduced formation of cross-linked products. Using a spin-labeled transporter variant and EPR spectroscopy, we demonstrate that EIIA^Glc arrests the transport cycle by inhibiting the ATP-dependent closure of the MalK dimer

The extracellular gate shapes the energy profile of an ABC exporter

ABC exporters harness the energy of ATP to pump substrates across membranes. Extracellular gate opening and closure are key steps of the transport cycle, but the underlying mechanism is poorly understood. Here, we generated a synthetic single domain antibody (sybody) that recognizes the heterodimeric ABC exporter TM287/288 exclusively in the presence of ATP, which was essential to solve a 3.2 Å crystal structure of the outward-facing transporter. The sybody binds to an extracellular wing and strongly inhibits ATPase activity by shifting the transporter's conformational equilibrium towards the outward-facing state, as shown by double electron-electron resonance (DEER). Mutations that facilitate extracellular gate opening result in a comparable equilibrium shift and strongly reduce ATPase activity and drug transport. Using the sybody as conformational probe, we demonstrate that efficient extracellular gate closure is required to dissociate the NBD dimer after ATP hydrolysis to reset the transporter back to its inward-facing state

Atomistic Mechanism of Large-Scale Conformational Transition in a Heterodimeric ABC Exporter

ATP-binding cassette (ABC) transporters are ATP-driven molecular machines, in which ATP binding and hydrolysis in the nucleotide-binding domains (NBDs) is chemomechanically coupled to large-scale, alternating access conformational changes in the transmembrane domains (TMDs), ultimately leading to the translocation of substrates across biological membranes. The precise nature of the structural dynamics behind the large-scale conformational transition as well as the coupling of NBD and TMD motions is still unresolved. In this work, we combine all-atom molecular dynamics (MD) simulations with electron paramagnetic resonance (EPR) spectroscopy to unravel the atomic-level mechanism of the dynamic conformational transitions underlying the functional working cycle of the heterodimeric ABC exporter TM287/288. Extensive multimicrosecond simulations in an explicit membrane/water environment show how in response to ATP binding, TM287/288 undergoes spontaneous conformational transitions from the inward-facing (IF) state via an occluded (Occ) intermediate to an outward-facing (OF) state. The latter two states have thus far not been characterized at atomic level. ATP-induced tightening of the NBD dimer involves closing and reorientation of the two NBD monomers concomitant with a closure of the intracellular TMD gate, which leads to the occluded state. Subsequently, opening at the extracellular TMD gate yields the OF conformer. The obtained mechanism imposes NBD-TMD coupling via a tight orchestration of conformational transitions, between both the two domains and also within the TMDs, ensuring that the cytoplasmic and periplasmic gate regions are never open simultaneously

The extracellular gate shapes the energy profile of an ABC exporter

ABC exporters harness the energy of ATP to pump substrates across membranes. Extracellular gate opening and closure are key steps of the transport cycle, but the underlying mechanism is poorly understood. Here, we generated a synthetic single domain antibody (sybody) that recognizes the heterodimeric ABC exporter TM287/288 exclusively in the presence of ATP, which was essential to solve a 3.2 Å crystal structure of the outward-facing transporter. The sybody binds to an extracellular wing and strongly inhibits ATPase activity by shifting the transporter’s conformational equilibrium towards the outward-facing state, as shown by double electron-electron resonance (DEER). Mutations that facilitate extracellular gate opening result in a comparable equilibrium shift and strongly reduce ATPase activity and drug transport. Using the sybody as conformational probe, we demonstrate that efficient extracellular gate closure is required to dissociate the NBD dimer after ATP hydrolysis to reset the transporter back to its inward-facing state.</p

Atomistic Mechanism of Large-Scale Conformational Transition in a Heterodimeric ABC Exporter

ATP-binding cassette (ABC) transporters are ATP-driven molecular machines, in which ATP binding and hydrolysis in the nucleotide-binding domains (NBDs) is chemomechanically coupled to large-scale, alternating access conformational changes in the transmembrane domains (TMDs), ultimately leading to the translocation of substrates across biological membranes. The precise nature of the structural dynamics behind the large-scale conformational transition as well as the coupling of NBD and TMD motions is still unresolved. In this work, we combine all-atom molecular dynamics (MD) simulations with electron paramagnetic resonance (EPR) spectroscopy to unravel the atomic-level mechanism of the dynamic conformational transitions underlying the functional working cycle of the heterodimeric ABC exporter TM287/288. Extensive multimicrosecond simulations in an explicit membrane/water environment show how in response to ATP binding, TM287/288 undergoes spontaneous conformational transitions from the inward-facing (IF) state via an occluded (Occ) intermediate to an outward-facing (OF) state. The latter two states have thus far not been characterized at atomic level. ATP-induced tightening of the NBD dimer involves closing and reorientation of the two NBD monomers concomitant with a closure of the intracellular TMD gate, which leads to the occluded state. Subsequently, opening at the extracellular TMD gate yields the OF conformer. The obtained mechanism imposes NBD-TMD coupling via a tight orchestration of conformational transitions, between both the two domains and also within the TMDs, ensuring that the cytoplasmic and periplasmic gate regions are never open simultaneously

Atomistic Mechanism of Large-Scale Conformational Transition in a Heterodimeric ABC Exporter

ATP-binding cassette (ABC) transporters are ATP-driven molecular machines, in which ATP binding and hydrolysis in the nucleotide-binding domains (NBDs) is chemomechanically coupled to large-scale, alternating access conformational changes in the transmembrane domains (TMDs), ultimately leading to the translocation of substrates across biological membranes. The precise nature of the structural dynamics behind the large-scale conformational transition as well as the coupling of NBD and TMD motions is still unresolved. In this work, we combine all-atom molecular dynamics (MD) simulations with electron paramagnetic resonance (EPR) spectroscopy to unravel the atomic-level mechanism of the dynamic conformational transitions underlying the functional working cycle of the heterodimeric ABC exporter TM287/288. Extensive multimicrosecond simulations in an explicit membrane/water environment show how in response to ATP binding, TM287/288 undergoes spontaneous conformational transitions from the inward-facing (IF) state via an occluded (Occ) intermediate to an outward-facing (OF) state. The latter two states have thus far not been characterized at atomic level. ATP-induced tightening of the NBD dimer involves closing and reorientation of the two NBD monomers concomitant with a closure of the intracellular TMD gate, which leads to the occluded state. Subsequently, opening at the extracellular TMD gate yields the OF conformer. The obtained mechanism imposes NBD-TMD coupling via a tight orchestration of conformational transitions, between both the two domains and also within the TMDs, ensuring that the cytoplasmic and periplasmic gate regions are never open simultaneously