Paul J. Focke

Opposite Movement of the External Gate of a Glutamate Transporter Homolog upon Binding Cotransported Sodium Compared with Substrate

Recently, a new model for glutamate uptake by glutamate transporters was proposed based on crystal structures of the bacterial glutamate transporter homolog GltPh. It was proposed that hairpin two (HP2) functions as the extracellular gate and that Na+ and glutamate binding closes HP2, thereby allowing for the translocation of the glutamate binding pocket across the membrane. However, the conformation of HP2 in the apo state and the Na+ bound state is unknown. We here use double site-directed spin-labeling electron paramagnetic resonance spectroscopy on the bacterial transporter GltPh from Pyrococcus horikoshi to examine conformational changes in HP2. Surprisingly, the cotransported substrates Na+ and aspartate induce opposite movements of HP2. We find that in the apo state, HP2 is in a similar conformation as in the aspartate-bound closed state. Na+ binding to the apo state opens HP2, whereas the subsequent binding of aspartate closes HP2. Our findings show that Na+ binding opens and stabilizes the extracellular gate, thereby allowing for amino acid substrate binding. In contrast, in the absence of Na+ and aspartate, HP2 closes, suggesting a potential mechanism for the translocation of the empty binding pocket necessary to complete the transport cycle. The finding that physiological Na+ concentrations stabilize the open HP2 state would ensure that the outward-facing conformation of the transporter is maintained in physiological solutions and that glutamate transporters are ready to quickly bind glutamate released from glutamatergic synapses.

Neurotransmitter Transporters: Structure Meets Function

At synapses, sodium-coupled transporters remove released neurotransmitters, thereby recycling them and maintaining a low extracellular concentration of the neurotransmitter. The molecular mechanism underlying sodium-coupled neurotransmitter uptake is not completely understood. Several structures of homologs of human neurotransmitter transporters have been solved with X-ray crystallography. These crystal structures have spurred a plethora of computational and experimental work to elucidate the molecular mechanism underlying sodium-coupled transport. Here, we compare the structures of GltPh, a glutamate transporter homolog, and LeuT, a homolog of neurotransmitter transporters for the biogenic amines and inhibitory molecules GABA and glycine. We relate these structures to data obtained from experiments and computational simulations, to draw conclusions about the mechanism of uptake by sodium-coupled neurotransmitter transporters. Here, we propose how sodium and substrate binding is coupled and how binding of sodium and substrate opens and closes the gates in these transporters, thereby leading to an efficient coupled transport.

Ion-binding properties of a K+ channel selectivity filter in different conformations.

Significance The selectivity filter of K+ channels is responsible for their exquisite ion selectivity. This region is also responsible for C-type inactivation, a regulatory process in many voltage-dependent K+ channels. Although the functional properties of inactivated channels have been known for decades, the first potential glimpse of their structure emerged from crystal structures of a constricted selectivity filter in an open channel. However, recent studies challenged the suggestion that the constricted selectivity filter is the inactivated structure, leaving open the question of what the inactivated structure looks like. Here, we provide evidence that the thermodynamic properties of the selectivity filter in an inactivated channel are more similar to properties of the conductive channel rather than the constricted open channel. K+ channels are membrane proteins that selectively conduct K+ ions across lipid bilayers. Many voltage-gated K+ (KV) channels contain two gates, one at the bundle crossing on the intracellular side of the membrane and another in the selectivity filter. The gate at the bundle crossing is responsible for channel opening in response to a voltage stimulus, whereas the gate at the selectivity filter is responsible for C-type inactivation. Together, these regions determine when the channel conducts ions. The K+ channel from Streptomyces lividians (KcsA) undergoes an inactivation process that is functionally similar to KV channels, which has led to its use as a practical system to study inactivation. Crystal structures of KcsA channels with an open intracellular gate revealed a selectivity filter in a constricted conformation similar to the structure observed in closed KcsA containing only Na+ or low [K+]. However, recent work using a semisynthetic channel that is unable to adopt a constricted filter but inactivates like WT channels challenges this idea. In this study, we measured the equilibrium ion-binding properties of channels with conductive, inactivated, and constricted filters using isothermal titration calorimetry (ITC). EPR spectroscopy was used to determine the state of the intracellular gate of the channel, which we found can depend on the presence or absence of a lipid bilayer. Overall, we discovered that K+ ion binding to channels with an inactivated or conductive selectivity filter is different from K+ ion binding to channels with a constricted filter, suggesting that the structures of these channels are different.

Studies of ion channels using expressed protein ligation

Expressed protein ligation (EPL) is a semisynthetic technique for the chemoselective ligation of a synthetic peptide to a recombinant peptide that results in a native peptide bond at the ligation site. EPL therefore allows us to engineer proteins with chemically defined, site-specific modifications. While EPL has been used mainly in investigations of soluble proteins, in recent years it has been increasingly used in investigations of integral membrane proteins. These include studies on the KcsA K(+) channel, the non-selective cation channel NaK, and the porin OmpF. These studies are discussed in this review.

Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression

Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an in vitro folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein. We demonstrate the feasibility of using this approach for the K(+) channels KcsA and MVP and the amino acid transporter LeuT. We determine the crystal structure of the KcsA channel obtained by CFPS and in vitro folding to show the structural similarity to the cellular expressed KcsA channel and to establish the feasibility of using this two-step approach for membrane protein production for structural studies. Our studies show that the correct folding of these membrane proteins with complex topologies can take place in vitro without the involvement of the cellular machinery for membrane protein biogenesis. This indicates that the folding instructions for these complex membrane proteins are contained entirely within the protein sequence.

Engineering the glutamate transporter homologue GltPh using protein semisynthesis

Glutamate transporters catalyze the concentrative uptake of glutamate from synapses and are essential for normal synaptic function. Despite extensive investigations of glutamate transporters, the mechanisms underlying substrate recognition, ion selectivity, and the coupling of substrate and ion transport are not well-understood. Deciphering these mechanisms requires the ability to precisely engineer the transporter. In this study, we describe the semisynthesis of GltPh, an archaeal homologue of glutamate transporters. Semisynthesis allows the precise engineering of GltPh through the incorporation of unnatural amino acids and peptide backbone modifications. In the semisynthesis, the GltPh polypeptide is initially assembled from a recombinantly expressed thioester peptide and a chemically synthesized peptide using the native chemical ligation reaction followed by in vitro folding to the native state. We have developed a robust procedure for the in vitro folding of GltPh. Biochemical characterization of the semisynthetic GltPh indicates that it is similar to the native transporter. We used semisynthesis to substitute Arg397, a highly conserved residue in the substrate binding site, with the unnatural analogue, citrulline. Our studies demonstrate that Arg397 is required for high-affinity substrate binding, and on the basis of our results, we propose that Arg397 is involved in a Na+-dependent remodeling of the substrate binding site required for high-affinity Asp binding. We anticipate that the semisynthetic approach developed in this study will be extremely useful in investigating functional mechanisms in GltPh. Further, the approach developed in this study should also be applicable to other membrane transport proteins.

A facile approach for the in vitro assembly of multimeric membrane transport proteins

Membrane proteins such as ion channels and transporters are frequently homomeric. The homomeric nature raises important questions regarding coupling between subunits and complicates the application of techniques such as FRET or DEER spectroscopy. These challenges can be overcome if the subunits of a homomeric protein can be independently modified for functional or spectroscopic studies. Here, we describe a general approach for in vitro assembly that can be used for the generation of heteromeric variants of homomeric membrane proteins. We establish the approach using GltPh, a glutamate transporter homolog that is trimeric in the native state. We use heteromeric GltPh transporters to directly demonstrate the lack of coupling in substrate binding and demonstrate how heteromeric transporters considerably simplify the application of DEER spectroscopy. Further, we demonstrate the general applicability of this approach by carrying out the in vitro assembly of VcINDY, a Na+-coupled succinate transporter and CLC-ec1, a Cl-/H+ antiporter.