On the kinetics and conformational dynamics of elevator transporters

In this thesis are collected information about the transport kinetics and dynamics of GltTk and CitS, two secondary active membrane transporters which translocate aspartate and citrate respectively along the sodium gradient. In proteins that translocate substrates following steady state kinetics, such as GltTk, the analysis of substrate transport rates in a wide range of substrate and co-transported ions allow the calculation of elusive kinetic parameters. In proteins that follow rapid equilibrium kinetics, such as CitS, the rates can be analyzed to extrapolate the order of binding of substrates and co-ions. In addition, an HS-AFM study carried out on CitS revealed the presence of three meta-stable protein conformations that occur during transport. In chapter 5, the crystal structure of GltTk in complex with the non-canonical amino acid D-aspartate reveals the binding mode of the substrate's stereoisomer. Finally the role of a conserved methionine residue which is putatively linked to coordinating a sodium ion in GltTk has been investigated by measuring the transport stoichiometry on several mutants

Discovery and characterization of an F420-dependent glucose-6-phosphate dehydrogenase (Rh-FGD1) from Rhodococcus jostii RHA1

Cofactor F420, a 5-deazaflavin involved in obligatory hydride transfer, is widely distributed among archaeal methanogens and actinomycetes. Owing to the low redox potential of the cofactor, F420-dependent enzymes play a pivotal role in central catabolic pathways and xenobiotic degradation processes in these organisms. A physiologically essential deazaflavoenzyme is the F420-dependent glucose-6-phosphate dehydrogenase (FGD), which catalyzes the reaction F420 + glucose-6-phosphate → F420H2 + 6-phospho-gluconolactone. Thereby, FGDs generate the reduced F420 cofactor required for numerous F420H2-dependent reductases, involved e.g., in the bioreductive activation of the antitubercular prodrugs pretomanid and delamanid. We report here the identification, production, and characterization of three FGDs from Rhodococcus jostii RHA1 (Rh-FGDs), being the first experimental evidence of F420-dependent enzymes in this bacterium. The crystal structure of Rh-FGD1 has also been determined at 1.5 Å resolution, showing a high similarity with FGD from Mycobacterium tuberculosis (Mtb) (Mtb-FGD1). The cofactor-binding pocket and active-site catalytic residues are largely conserved in Rh-FGD1 compared with Mtb-FGD1, except for an extremely flexible insertion region capping the active site at the C-terminal end of the TIM-barrel, which also markedly differs from other structurally related proteins. The role of the three positively charged residues (Lys197, Lys258, and Arg282) constituting the binding site of the substrate phosphate moiety was experimentally corroborated by means of mutagenesis study. The biochemical and structural data presented here provide the first step towards tailoring Rh-FGD1 into a more economical biocatalyst, e.g., an F420-dependent glucose dehydrogenase that requires a cheaper cosubstrate and can better match the demands for the growing applications of F420H2-dependent reductases in industry and bioremediation

Correction to:On the Role of a Conserved Methionine in the Na+-Coupling Mechanism of a Neurotransmitter Transporter Homolog (Neurochemical Research, (2022), 47, 1, (163-175), 10.1007/s11064-021-03253-w)

After publication, the authors realized that the version of the supplementary information that was originally submitted was incomplete in that it omitted results examining alternative NBFIX corrections to the force field. Those data have now been added as Supplementary Fig. S3 and they reaffirm the conclusions of the manuscript. In addition, the legend to Fig. 3b should read: “Using the NBFIX correction of Na+- methionine interactions, all Na+ ions and the substrate remain stably bound throughout the trajectory. See also Fig. S3.

Kinetic mechanism of Na+-coupled aspartate transport catalyzed by GltTk

Trinco et al. measure aspartate uptake rates in proteoliposomes containing purified prokaryotic Na+-coupled aspartate transporter GltTk. To overcome limitation of protein orientation, they use synthetic nanobody that blocks transporters from outside and reveal mechanistic features of Na+-aspartate symport that cannot be observed in detergent solution

On the Role of a Conserved Methionine in the Na+-Coupling Mechanism of a Neurotransmitter Transporter Homolog

Excitatory amino acid transporters (EAAT) play a key role in glutamatergic synaptic communication. Driven by transmembrane cation gradients, these transporters catalyze the reuptake of glutamate from the synaptic cleft once this neurotransmitter has been utilized for signaling. Two decades ago, pioneering studies in the Kanner lab identified a conserved methionine within the transmembrane domain as key for substrate turnover rate and specificity; later structural work, particularly for the prokaryotic homologs Glt(Ph) and Glt(Tk), revealed that this methionine is involved in the coordination of one of the three Na(+) ions that are co-transported with the substrate. Albeit extremely atypical, the existence of this interaction is consistent with biophysical analyses of Glt(Ph) showing that mutations of this methionine diminish the binding cooperativity between substrates and Na(+). It has been unclear, however, whether this intriguing methionine influences the thermodynamics of the transport reaction, i.e., its substrate:ion stoichiometry, or whether it simply fosters a specific kinetics in the binding reaction, which, while influential for the turnover rate, do not fundamentally explain the ion-coupling mechanism of this class of transporters. Here, studies of Glt(Tk) using experimental and computational methods independently arrive at the conclusion that the latter hypothesis is the most plausible, and lay the groundwork for future efforts to uncover the underlying mechanism. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s11064-021-03253-w) contains supplementary material, which is available to authorized users

Binding and transport of D-aspartate by the glutamate transporter homologue GltTk

Mammalian glutamate transporters are crucial players in neuronal communication as they perform neurotransmitter reuptake from the synaptic cleft. Besides L-glutamate and L-aspartate, they also recognize D-aspartate, which might participate in mammalian neurotransmission and/or neuromodulation. Much of the mechanistic insight in glutamate transport comes from studies of the archaeal homologues GltPh from Pyrococcus horikoshii and GltTk from Thermococcus kodakarensis. Here, we show that GltTk transports D-aspartate with identical Na+ : substrate coupling stoichiometry as L-aspartate, and that the affinities (Kd and Km) for the two substrates are similar. We determined a crystal structure of GltTk with bound D-aspartate at 2.8 Å resolution. Comparison of the L- and D-aspartate bound GltTk structures revealed that D-aspartate is accommodated with only minor rearrangements in the structure of the binding site. The structure explains how the geometrically different molecules L- and D-aspartate are recognized and transported by the protein in the same way

Structural Aspects of Photopharmacology: Insight into the Binding of Photoswitchable and Photocaged Inhibitors to the Glutamate Transporter Homologue

Photopharmacology addresses the challenge of drug selectivity and side effects through creation of photoresponsive molecules activated with light with high spatiotemporal precision. This is achieved through incorporation of molecular photoswitches and photocages into the pharmacophore. However, the structural basis for the light-induced modulation of inhibitory potency in general is still missing, which poses a major design challenge for this emerging field of research. Here we solved crystal structures of the glutamate transporter homologue GltTk in complex with photoresponsive transport inhibitors—azobenzene derivative of TBOA (both in trans and cis configuration) and with the photocaged compound ONB-hydroxyaspartate. The essential role of glutamate transporters in the functioning of the central nervous system renders them potential therapeutic targets in the treatment of neurodegenerative diseases. The obtained structures provide a clear structural insight into the origins of photocontrol in photopharmacology and lay the foundation for application of photocontrolled ligands to study the transporter dynamics by using time-resolved X-ray crystallography

High-speed atomic force microscopy reveals a three-state elevator mechanism in the citrate transporter CitS

The secondary active transporter CitS shuttles citrate across the cytoplasmic membrane of gram-negative bacteria by coupling substrate translocation to the transport of two Na(+) ions. Static crystal structures suggest an elevator type of transport mechanism with two states: up and down. However, no dynamic measurements have been performed to substantiate this assumption. Here, we use high-speed atomic force microscopy for real-time visualization of the transport cycle at the level of single transporters. Unexpectedly, instead of a bimodal height distribution for the up and down states, the experiments reveal movements between three distinguishable states, with protrusions of ∼0.5 nm, ∼1.0 nm, and ∼1.6 nm above the membrane, respectively. Furthermore, the real-time measurements show that the individual protomers of the CitS dimer move up and down independently. A three-state elevator model of independently operating protomers resembles the mechanism proposed for the aspartate transporter Glt(Ph). Since CitS and Glt(Ph) are structurally unrelated, we conclude that the three-state elevators have evolved independently