Maria E. Zoghbi

Permeation of Calcium through Purified Connexin 26 Hemichannels

Background: Indirect evidence suggests that connexin hemichannels are permeable to Ca2+, but direct demonstration is lacking. Results: Calcium moves into liposomes containing purified Cx26 in response to a concentration gradient. Conclusion: Cx26 hemichannels are permeable to Ca2+. Significance: Cx26 hemichannels may play a role in Ca2+ influx into cells under conditions that lead to hemichannel activation, such as ischemic damage. Gap junction channels communicate the cytoplasms of two cells and are formed by head to head association of two hemichannels, one from each of the cells. Gap junction channels and hemichannels are permeable to ions and hydrophilic molecules of up to Mr 1,000, including second messengers and metabolites. Intercellular Ca2+ signaling can occur by movement of a number of second messengers, including Ca2+, through gap junction channels, or by a paracrine pathway that involves activation of purinergic receptors in neighboring cells following ATP release through hemichannels. Understanding Ca2+ permeation through Cx26 hemichannels is important to assess the role of gap junction channels and hemichannels in health and disease. In this context, it is possible that increased Ca2+ influx through hemichannels under ischemic conditions contributes to cell damage. Previous studies suggest Ca2+ permeation through hemichannels, based on indirect arguments. Here, we demonstrate for the first time hemichannel permeability to Ca2+ by measuring Ca2+ transport through purified Cx26 hemichannels reconstituted in liposomes. We trapped the low affinity Ca2+-sensitive fluorescent probe Fluo-5N into the liposomes and followed the increases in intraliposomal [Ca2+] in response to an imposed [Ca2+] gradient. We show that Ca2+ does move through Cx26 hemichannels and that the permeability of the hemichannels to Ca2+ is high, similar to that for Na+. We suggest that hemichannels can be a significant pathway for Ca2+ influx into cells under conditions such as ischemia.

The Lipid Bilayer Modulates the Structure and Function of an ATP-binding Cassette Exporter.

ATP-binding cassette exporters use the energy of ATP hydrolysis to transport substrates across membranes by switching between inward- and outward-facing conformations. Essentially all structural studies of these proteins have been performed with the proteins in detergent micelles, locked in specific conformations and/or at low temperature. Here, we used luminescence resonance energy transfer spectroscopy to study the prototypical ATP-binding cassette exporter MsbA reconstituted in nanodiscs at 37 °C while it performs ATP hydrolysis. We found major differences when comparing MsbA in these native-like conditions with double electron-electron resonance data and the crystal structure of MsbA in the open inward-facing conformation. The most striking differences include a significantly smaller separation between the nucleotide-binding domains and a larger fraction of molecules with associated nucleotide-binding domains in the nucleotide-free apo state. These studies stress the importance of studying membrane proteins in an environment that approaches physiological conditions.

Kinetics of the Association/Dissociation Cycle of an ATP-binding Cassette Nucleotide-binding Domain

Background: ATP induces dimerization of the nucleotide-binding domains (NBD) of ATP-binding cassette proteins, followed by ATP hydrolysis and dimer dissociation. Results: The rate of dimerization induced by MgATP is faster than previously thought. Conclusion: During the hydrolysis cycle, there is a dynamic equilibrium where neither monomers nor dimers are favored. Significance: Knowledge of the mechanism of hydrolysis by NBDs will help us understand the function of ATP-binding cassette proteins. Most ATP binding cassette (ABC) proteins are pumps that transport substrates across biological membranes using the energy of ATP hydrolysis. Functional ABC proteins have two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP, but the molecular mechanism of nucleotide hydrolysis is unresolved. This is due in part to the limited kinetic information on NBD association and dissociation. Here, we show dimerization of a catalytically active NBD and follow in real time the association and dissociation of NBDs from the changes in fluorescence emission of a tryptophan strategically located at the center of the dimer interface. Spectroscopic and structural studies demonstrated that the tryptophan can be used as dimerization probe, and we showed that under hydrolysis conditions (millimolar MgATP), not only the dimer dissociation rate increases, but also the dimerization rate. Neither dimer formation or dissociation are clearly favored, and the end result is a dynamic equilibrium where the concentrations of monomer and dimer are very similar. We proposed that based on their variable rates of hydrolysis, the rate-limiting step of the hydrolysis cycle may differ among full-length ABC proteins.

Hydrolysis at One of the Two Nucleotide-binding Sites Drives the Dissociation of ATP-binding Cassette Nucleotide-binding Domain Dimers

Background: Nucleotide-binding domains (NBDs) of ABC proteins bind two ATPs, but it is unknown whether dissociation follows hydrolysis of one or both ATPs. Results: NBD dimers with one or two sites capable of ATP hydrolysis dissociate at the same rate. Conclusion: NBD dimers dissociate following a single ATP hydrolysis event. Significance: Understanding ABC proteins will help develop strategies to modify their function in disease. The functional unit of ATP-binding cassette (ABC) transporters consists of two transmembrane domains and two nucleotide-binding domains (NBDs). ATP binding elicits association of the two NBDs, forming a dimer in a head-to-tail arrangement, with two nucleotides “sandwiched” at the dimer interface. Each of the two nucleotide-binding sites is formed by residues from the two NBDs. We recently found that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii dimerizes in response to ATP binding and dissociates completely following ATP hydrolysis. However, it is still unknown whether dissociation of NBD dimers follows ATP hydrolysis at one or both nucleotide-binding sites. Here, we used luminescence resonance energy transfer to study heterodimers formed by one active (donor-labeled) and one catalytically defective (acceptor-labeled) NBD. Rapid mixing experiments in a stop-flow chamber showed that NBD heterodimers with one functional and one inactive site dissociated at a rate indistinguishable from that of dimers with two hydrolysis-competent sites. Comparison of the rates of NBD dimer dissociation and ATP hydrolysis indicated that dissociation followed hydrolysis of one ATP. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dimer dissociation.

ATP binding to two sites is necessary for dimerization of nucleotide-binding domains of ABC proteins

ATP binding cassette (ABC) transporters have a functional unit formed by two transmembrane domains and two nucleotide binding domains (NBDs). ATP-bound NBDs dimerize in a head-to-tail arrangement, with two nucleotides sandwiched at the dimer interface. Both NBDs contribute residues to each of the two nucleotide-binding sites (NBSs) in the dimer. In previous studies, we showed that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii forms ATP-bound dimers that dissociate completely following hydrolysis of one of the two bound ATP molecules. Since hydrolysis of ATP at one NBS is sufficient to drive dimer dissociation, it is unclear why all ABC proteins contain two NBSs. Here, we used luminescence resonance energy transfer (LRET) to study ATP-induced formation of NBD homodimers containing two NBSs competent for ATP binding, and NBD heterodimers with one active NBS and one binding-defective NBS. The results showed that binding of two ATP molecules is necessary for NBD dimerization. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dissociation, but two binding sites are required to form the ATP-sandwich NBD dimer necessary for hydrolysis.

Dissociation of ATP-binding cassette nucleotide-binding domain dimers into monomers during the hydrolysis cycle

Background: In ATP-binding cassette proteins, ATP binding induces formation of nucleotide-binding domain (NBD) dimers, but the mechanism of nucleotide hydrolysis is unknown. Results: ATP hydrolysis leads to complete separation of NBD dimers, as opposed to dimer opening. Conclusion: NBD dimers dissociate during the hydrolysis cycle. Significance: Elucidation of the molecular mechanism of hydrolysis will help us understand the function of ATP-binding cassette proteins. ATP-binding cassette (ABC) proteins have two nucleotide-binding domains (NBDs) that work as dimers to bind and hydrolyze ATP, but the molecular mechanism of nucleotide hydrolysis is controversial. In particular, it is still unresolved whether hydrolysis leads to dissociation of the ATP-induced dimers or opening of the dimers, with the NBDs remaining in contact during the hydrolysis cycle. We studied a prototypical ABC NBD, the Methanococcus jannaschii MJ0796, using spectroscopic techniques. We show that fluorescence from a tryptophan positioned at the dimer interface and luminescence resonance energy transfer between probes reacted with single-cysteine mutants can be used to follow NBD association/dissociation in real time. The intermonomer distances calculated from luminescence resonance energy transfer data indicate that the NBDs separate completely following ATP hydrolysis, instead of opening. The results support ABC protein NBD association/dissociation, as opposed to constant-contact models.

Substrate-induced conformational changes in the nucleotide-binding domains of lipid bilayer-associated P-glycoprotein during ATP hydrolysis

P-glycoprotein (Pgp) is an efflux pump important in multidrug resistance of cancer cells and in determining drug pharmacokinetics. Pgp is a prototype ATP-binding cassette transporter with two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. Conformational changes at the NBDs (the Pgp engines) lead to changes across Pgp transmembrane domains that result in substrate translocation. According to current alternating access models (substrate-binding pocket accessible only to one side of the membrane at a time), binding of ATP promotes NBD dimerization, resulting in external accessibility of the drug-binding site (outward-facing, closed NBD conformation), and ATP hydrolysis leads to dissociation of the NBDs with the subsequent return of the accessibility of the binding site to the cytoplasmic side (inward-facing, open NBD conformation). However, previous work has not investigated these events under near-physiological conditions in a lipid bilayer and in the presence of transport substrate. Here, we used luminescence resonance energy transfer (LRET) to measure the distances between the two Pgp NBDs. Pgp was labeled with LRET probes, reconstituted in lipid nanodiscs, and the distance between the NBDs was measured at 37 °C. In the presence of verapamil, a substrate that activates ATP hydrolysis, the NBDs of Pgp reconstituted in nanodiscs were never far apart during the hydrolysis cycle, and we never observed the NBD–NBD distances of tens of Å that have previously been reported. However, we found two main conformations that coexist in a dynamic equilibrium under all conditions studied. Our observations highlight the importance of performing studies of efflux pumps under near-physiological conditions, in a lipid bilayer, at 37 °C, and during substrate-stimulated hydrolysis.

Membrane protein reconstitution in nanodiscs for luminescence spectroscopy studies

Abstract ATP-binding cassette (ABC) exporters transport substrates across biological membranes using ATP hydrolysis by a process that involves switching between inward- and outward-facing conformations. Most of the structural studies of ABC proteins have been performed with proteins in detergent micelles, locked in specific conformations and/or at low temperature. In this article, we present recent data from our laboratories where we studied the prototypical ABC exporter MsbA during ATP hydrolysis, at 37°C, reconstituted in a lipid bilayer. These studies were possible through the use of luminescence resonance energy transfer spectroscopy in MsbA reconstituted in nanodiscs. We found major differences between MsbA in these native-like conditions and in previous studies. These include a separation between the nucleotide-binding domains that was much smaller than previously thought, and a large fraction of molecules with associated nucleotide-binding domains in the nucleotide-free apo state. These studies stress the importance of studying membrane proteins in an environment that approaches physiological conditions.

Luminescence resonance energy transfer spectroscopy of ATP-binding cassette proteins

The ATP-binding cassette (ABC) superfamily includes regulatory and transport proteins. Most human ABC exporters pump substrates out of cells using energy from ATP hydrolysis. Although major advances have been made toward understanding the molecular mechanism of ABC exporters, there are still many issues unresolved. During the last few years, luminescence resonance energy transfer has been used to detect conformational changes in real time, with atomic resolution, in isolated ABC nucleotide binding domains (NBDs) and full-length ABC exporters. NBDs are particularly interesting because they provide the power stroke for substrate transport. Luminescence resonance energy transfer (LRET) is a spectroscopic technique that can provide dynamic information with atomic-resolution of protein conformational changes under physiological conditions. Using LRET, it has been shown that NBD dimerization, a critical step in ABC proteins catalytic cycle, requires binding of ATP to two nucleotide binding sites. However, hydrolysis at just one of the sites can drive dissociation of the NBD dimer. It was also found that the NBDs of the bacterial ABC exporter MsbA reconstituted in a lipid bilayer membrane and studied at 37°C never separate as much as suggested by crystal structures. This observation stresses the importance of performing structural/functional studies of ABC exporters under physiologic conditions. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

The Lipid Bilayer Modulates the Structure and Function of an Atp-Binding Cassette Exporter

ATP-binding cassette (ABC) exporters use the energy of ATP hydrolysis to transport substrates across membranes. ABC proteins contain two transmembrane domains that form the translocation pathway, and two conserved nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. ATP binding promotes NBD dimerization, which is essential for ATP hydrolysis, whereas NBDs dissociation occurs following hydrolysis. This NBD dimerization/dissociation process is coupled to rearrangements of the transmembrane helices, switching the transporters from an inward-facing conformation (dissociated NBDs) to an outward-facing conformation (dimeric NBDs), with the concomitant translocation of substrate. Structural studies of these proteins have been performed with the proteins in detergent micelles, locked in specific conformations and/or at low temperature. As part of our efforts to study ABC proteins under more natural experimental conditions, we used luminescence resonance energy transfer (LRET) on the prototypical ABC exporter MsbA reconstituted in nanodiscs, at 37oC, and while it performs ATP hydrolysis. We have found that, in these native-like conditions, MsbA adopts two main conformations: NBDs in closed proximity (36-A distance) and NBDs partially separated (∼46-A distance), with changes in the percentage of molecules adopting each conformation during the ATP hydrolysis cycle. Essentially, our results show major differences with the published crystal structure in the open inward-facing conformation, as well as with double electron-electron resonance (DEER) experiments and LRET experiments performed with MsbA in detergent micelles. The most striking differences include a significantly smaller separation between the NBDs, and a larger fraction of molecules with associated NBDs in the apo state. These studies stress the importance of studying membrane proteins under native-like conditions that include the insertion into lipid bilayers and normal temperatures.