The mechanisms responsible for the trafficking of carboxylate ions across cell membranes are becoming clearer.
Small carboxylate ions such as citrate and succinate are intermediates in the citric acid cycle, which is a crucial metabolic pathway in aerobic organisms. However, small carboxylate ions also have many other roles: for example, they function as signaling molecules in processes ranging from DNA transcription and replication (Wellen et al., 2009) to heat generation (Mills et al., 2018), and they have also been linked to obesity (Birkenfeld et al., 2011) and seizures (Thevenon et al., 2014).
Cells rely on transmembrane proteins belonging to the DASS family (short for divalent anion sodium symporter) to move small carboxylate ions into and out of cells. There are two clades in the DASS family: cotransporters that import carboxylate ions from the bloodstream into cells (Prakash et al., 2003), and antiporters/exchangers that move some carboxylate ions into cells while moving others out (Pos et al., 1998).
Previously the structure of just one member of the DASS family – a cotransporter called VcINDY, which is found in the bacterium Vibrio cholerae – had been determined (Mancusso et al., 2012; Mulligan et al., 2016; Nie et al., 2017). VcINDY contains two subunits, and each of these contains two domains: (i) a scaffold domain, which is anchored in the plasma membrane of the cell and is not, therefore, free to move; (ii) a transport domain, which is more mobile.
It has been predicted that DASS proteins operate with an 'elevator mechanism' that involves the transport domain (to which the carboxylate ion is attached) sliding up and down the scaffold domain between an inward-facing state and an outward-facing state (Figure 1; Mulligan et al., 2016). However, since the structure of VcINDY has only ever been determined for the inward-facing state, evidence in support of this mechanism has remained inconclusive. Now, in eLife, Da-Neng Wang (New York University School of Medicine), Emad Tajkhorshid (University of Illinois at Urbana-Champaign) and co-workers – including David Sauer as first author – report the results of a combined experimental and computational study that helps to shed light on this mystery (Sauer et al., 2020).
The researchers used a combination of X-ray crystallography and cryo-electron microscopy to determine structures for VcINDY and also for LaINDY, an antiporter that is found in the bacterium Lactobacillus acidophilus. Remarkably, they were able to obtain structures for the previously elusive outward-facing state for both. Moreover, they determined the structures when a carboxylate ion was bound to the transport domain and also for the substrate-free case. Relative to the inward-facing state, the transport domain in the outward-facing state is rotated by an angle of 37° and is about 13 Å further away from the inside of the cell (Figure 1).
Sauer et al. then used computer simulations to model the transition from an initial state in which two succinate ions were bound to the outward-facing state in the LaINDY antiporter (based on their experimental structures) to a final state in which the succinate ions were inside the cell and the antiporter was in an inward-facing state: the researchers used an approximate structure for the final state as the actual structure for the inward-facing state in LaINDY has not been determined yet. Jointly, the experiments and simulations lend strong support to the elevator mechanism.
One may ask: how does the cotransporter or antiporter make sure that the carboxylate ion has been loaded into the elevator before the button is pressed? The experimental structures suggest that a 'passport check' is enforced via electrostatic effects. In the absence of the carboxylate ion, the binding site on the transport domain has a positive net charge, which makes it difficult for this domain to pass through the membrane, because charged particles prefer polar environments (such as aqueous solutions) to the nonpolar environments found in the membrane. Loading the negative carboxylate ion onto the binding site neutralizes the postive charge, allowing the transport domain to cruise through. This mechanism limits a form of unproductive transport called slippage (that is, the passage of substrate-free transport domains) in both cotransporters and antiporters.
The work of Sauer et al. also highlights interesting differences between the cotransporter and the antiporter. For the VcINDY cotransporter the positive charge on the transport domain is provided by sodium ions, and the binding of the carboxylate ion to the transport domain leads to large changes in conformation. It also appears that the relatively weak initial binding of sodium ions in the cotransporter is strengthened by the arrival of the carboxylate ion. In contrast, the positive charges in the LaINDY antiporter are provided by amino acid residues rather than sodium ions, and the conformational changes caused by binding appeared to be small and local.
It would be interesting to explore the effects of introducing mutations to the residues at the sites of the sodium ions in VcINDY cotransporters or the equivalent sites in LaINDY antiporters. Would it be possible to engineer a sodium-independent cotransporter? And could a cotransporter be converted into an antiporter, and vice versa? Answers to these questions will lead to a deeper understanding of the structure-function relationships for proteins belonging to the DASS family.
The bacterial dicarboxylate transporter VcINDY uses a two-domain elevator-type mechanismNature Structural & Molecular Biology 23:256–263.https://doi.org/10.1038/nsmb.3166
Mutations in SLC13A5 cause autosomal-recessive epileptic encephalopathy with seizure onset in the first days of lifeAmerican Journal of Human Genetics 95:113–120.https://doi.org/10.1016/j.ajhg.2014.06.006
Downloads (link to download the article as PDF)
Download citations (links to download the citations from this article in formats compatible with various reference manager tools)
Open citations (links to open the citations from this article in various online reference manager services)
PPP-family phosphatases such as PP1 have little intrinsic specificity. Cofactors can target PP1 to substrates or subcellular locations, but it remains unclear how they might confer sequence-specificity on PP1. The cytoskeletal regulator Phactr1 is a neuronally enriched PP1 cofactor that is controlled by G-actin. Structural analysis showed that Phactr1 binding remodels PP1's hydrophobic groove, creating a new composite surface adjacent to the catalytic site. Using phosphoproteomics, we identified mouse fibroblast and neuronal Phactr1/PP1 substrates, which include cytoskeletal components and regulators. We determined high-resolution structures of Phactr1/PP1 bound to the dephosphorylated forms of its substrates IRSp53 and spectrin αII. Inversion of the phosphate in these holoenzyme-product complexes supports the proposed PPP-family catalytic mechanism. Substrate sequences C-terminal to the dephosphorylation site make intimate contacts with the composite Phactr1/PP1 surface, which are required for efficient dephosphorylation. Sequence specificity explains why Phactr1/PP1 exhibits orders-of-magnitude enhanced reactivity towards its substrates, compared to apo-PP1 or other PP1 holoenzymes.
Sulfur-aromatic interactions occur in the majority of protein structures, yet little is known about their functional roles in ion channels. Here, we describe a novel molecular motif, the M101 gate latch, which is essential for gating of human Orai1 channels via its sulfur-aromatic interactions with the F99 hydrophobic gate. Molecular dynamics simulations of different Orai variants reveal that the gate latch is engaged in open but not in closed channels. In experimental studies, we use metal ion bridges to show that promoting an M101-F99 bond directly activates Orai1, whereas disrupting this interaction triggers channel closure. Mutational analysis demonstrates that methionine at this position has a unique length, flexibility, and chemistry to act as an effective latch for the phenylalanine gate. Because sulfur-aromatic interactions provide additional stabilization compared to purely hydrophobic interactions, we postulate that the six M101-F99 pairs in the hexameric channel represent a substantial energetic contribution to Orai1 activation.