Crystal structure of the Na+/H+ antiporter NhaA at active pH reveals the mechanistic basis for pH sensing.

The strict exchange of protons for sodium ions across cell membranes by Na+/H+ exchangers is a fundamental mechanism for cell homeostasis. At active pH, Na+/H+ exchange can be modelled as competition between H+ and Na+ to an ion-binding site, harbouring either one or two aspartic-acid residues. Nevertheless, extensive analysis on the model Na+/H+ antiporter NhaA from Escherichia coli, has shown that residues on the cytoplasmic surface, termed the pH sensor, shifts the pH at which NhaA becomes active. It was unclear how to incorporate the pH senor model into an alternating-access mechanism based on the NhaA structure at inactive pH 4. Here, we report the crystal structure of NhaA at active pH 6.5, and to an improved resolution of 2.2 Å. We show that at pH 6.5, residues in the pH sensor rearrange to form new salt-bridge interactions involving key histidine residues that widen the inward-facing cavity. What we now refer to as a pH gate, triggers a conformational change that enables water and Na+ to access the ion-binding site, as supported by molecular dynamics (MD) simulations. Our work highlights a unique, channel-like switch prior to substrate translocation in a secondary-active transporter.

Correction to "Substrate Dynamics Contribute to Enzymatic Specificity in Human and Bacterial Methionine Adenosyltransferases".

[This corrects the article DOI: 10.1021/jacsau.1c00464.].

Silicon nitride polarization beam splitter based on polarization-independent MMIs and apodized Bragg gratings.

We present the design and experimental results of a novel polarization beam splitter (PBS) with a high polarization extinction ratio (PER) made on a Si3N4 platform. The PBS is composed of two identical polarization-independent multi-mode interferometers and two identical apodized Bragg gratings. The operating principle of this device is based on the fact that the TE and TM stopbands of the grating are centered at different wavelengths. The reflected and transmitted light from the gratings are routed to separate output ports by the two-MMI configuration. The experimental results show that a PER of > 30 dB is achieved over a bandwidth of 22 nm, with an insertion loss of ∼ 1.1 dB. The total length of the device is ∼ 820 µm.

Substrate Dynamics Contribute to Enzymatic Specificity in Human and Bacterial Methionine Adenosyltransferases.

Protein conformational changes can facilitate the binding of noncognate substrates and underlying promiscuous activities. However, the contribution of substrate conformational dynamics to this process is comparatively poorly understood. Here, we analyze human (hMAT2A) and Escherichia coli (eMAT) methionine adenosyltransferases that have identical active sites but different substrate specificity. In the promiscuous hMAT2A, noncognate substrates bind in a stable conformation to allow catalysis. In contrast, noncognate substrates sample stable productive binding modes less frequently in eMAT owing to altered mobility in the enzyme active site. Different cellular concentrations of substrates likely drove the evolutionary divergence of substrate specificity in these orthologues. The observation of catalytic promiscuity in hMAT2A led to the detection of a new human metabolite, methyl thioguanosine, that is produced at elevated levels in a cancer cell line. This work establishes that identical active sites can result in different substrate specificity owing to the effects of substrate and enzyme dynamics.

The molecular basis for sugar import in malaria parasites.

Elucidating the mechanism of sugar import requires a molecular understanding of how transporters couple sugar binding and gating events. Whereas mammalian glucose transporters (GLUTs) are specialists1, the hexose transporter from the malaria parasite Plasmodium falciparum PfHT12,3 has acquired the ability to transport both glucose and fructose sugars as efficiently as the dedicated glucose (GLUT3) and fructose (GLUT5) transporters. Here, to establish the molecular basis of sugar promiscuity in malaria parasites, we determined the crystal structure of PfHT1 in complex with D-glucose at a resolution of 3.6 Å. We found that the sugar-binding site in PfHT1 is very similar to those of the distantly related GLUT3 and GLUT5 structures4,5. Nevertheless, engineered PfHT1 mutations made to match GLUT sugar-binding sites did not shift sugar preferences. The extracellular substrate-gating helix TM7b in PfHT1 was positioned in a fully occluded conformation, providing a unique glimpse into how sugar binding and gating are coupled. We determined that polar contacts between TM7b and TM1 (located about 15 Å from D-glucose) are just as critical for transport as the residues that directly coordinate D-glucose, which demonstrates a strong allosteric coupling between sugar binding and gating. We conclude that PfHT1 has achieved substrate promiscuity not by modifying its sugar-binding site, but instead by evolving substrate-gating dynamics.

Catalytic Conversion of Lipophilic Substrates by Phase constrained Enzymes in the Aqueous or in the Membrane Phase.

Both soluble and membrane-bound enzymes can catalyze the conversion of lipophilic substrates. The precise substrate access path, with regard to phase, has however, until now relied on conjecture from enzyme structural data only (certainly giving credible and valuable hypotheses). Alternative methods have been missing. To obtain the first experimental evidence directly determining the access paths (of lipophilic substrates) to phase constrained enzymes we here describe the application of a BODIPY-derived substrate (PS1). Using this tool, which is not accessible to cytosolic enzymes in the presence of detergent and, by contrast, not accessible to membrane embedded enzymes in the absence of detergent, we demonstrate that cytosolic and microsomal glutathione transferases (GSTs), both catalyzing the activation of PS1, do so only within their respective phases. This approach can serve as a guideline to experimentally validate substrate access paths, a fundamental property of phase restricted enzymes. Examples of other enzyme classes with members in both phases are xenobiotic-metabolizing sulphotransferases/UDP-glucuronosyl transferases or epoxide hydrolases. Since specific GSTs have been suggested to contribute to tumor drug resistance, PS1 can also be utilized as a tool to discriminate between phase constrained members of these enzymes by analyzing samples in the absence and presence of Triton X-100.

A dynamic Asp-Arg interaction is essential for catalysis in microsomal prostaglandin E2 synthase.

Microsomal prostaglandin E2 synthase type 1 (mPGES-1) is responsible for the formation of the potent lipid mediator prostaglandin E2 under proinflammatory conditions, and this enzyme has received considerable attention as a drug target. Recently, a high-resolution crystal structure of human mPGES-1 was presented, with Ser-127 being proposed as the hydrogen-bond donor stabilizing thiolate anion formation within the cofactor, glutathione (GSH). We have combined site-directed mutagenesis and activity assays with a structural dynamics analysis to probe the functional roles of such putative catalytic residues. We found that Ser-127 is not required for activity, whereas an interaction between Arg-126 and Asp-49 is essential for catalysis. We postulate that both residues, in addition to a crystallographic water, serve critical roles within the enzymatic mechanism. After characterizing the size or charge conservative mutations Arg-126-Gln, Asp-49-Asn, and Arg-126-Lys, we inferred that a crystallographic water acts as a general base during GSH thiolate formation, stabilized by interaction with Arg-126, which is itself modulated by its respective interaction with Asp-49. We subsequently found hidden conformational ensembles within the crystal structure that correlate well with our biochemical data. The resulting contact signaling network connects Asp-49 to distal residues involved in GSH binding and is ligand dependent. Our work has broad implications for development of efficient mPGES-1 inhibitors, potential anti-inflammatory and anticancer agents.

Structural insights into omega-class glutathione transferases: a snapshot of enzyme reduction and identification of a non-catalytic ligandin site.

Glutathione transferases (GSTs) are dimeric enzymes containing one active-site per monomer. The omega-class GSTs (hGSTO1-1 and hGSTO2-2 in humans) are homodimeric and carry out a range of reactions including the glutathione-dependant reduction of a range of compounds and the reduction of S-(phenacyl)glutathiones to acetophenones. Both types of reaction result in the formation of a mixed-disulfide of the enzyme with glutathione through the catalytic cysteine (C32). Recycling of the enzyme utilizes a second glutathione molecule and results in oxidized glutathione (GSSG) release. The crystal structure of an active-site mutant (C32A) of the hGSTO1-1 isozyme in complex with GSSG provides a snapshot of the enzyme in the process of regeneration. GSSG occupies both the G (GSH-binding) and H (hydrophobic-binding) sites and causes re-arrangement of some H-site residues. In the same structure we demonstrate the existence of a novel "ligandin" binding site deep within in the dimer interface of this enzyme, containing S-(4-nitrophenacyl)glutathione, an isozyme-specific substrate for hGSTO1-1. The ligandin site, conserved in Omega class GSTs from a range of species, is hydrophobic in nature and may represent the binding location for tocopherol esters that are uncompetitive hGSTO1-1 inhibitors.

Structural insights into the dehydroascorbate reductase activity of human omega-class glutathione transferases.

The reduction of dehydroascorbate (DHA) to ascorbic acid (AA) is a vital cellular function. The omega-class glutathione transferases (GSTs) catalyze several reductive reactions in cellular biochemistry, including DHA reduction. In humans, two isozymes (GSTO1-1 and GSTO2-2) with significant DHA reductase (DHAR) activity are found, sharing 64% sequence identity. While the activity of GSTO2-2 is higher, it is significantly more unstable in vitro. We report the first crystal structures of human GSTO2-2, stabilized through site-directed mutagenesis and determined at 1.9 Å resolution in the presence and absence of glutathione (GSH). The structure of a human GSTO1-1 has been determined at 1.7 Å resolution in complex with the reaction product AA, which unexpectedly binds in the G-site, where the glutamyl moiety of GSH binds. The structure suggests a similar mode of ascorbate binding in GSTO2-2. This is the first time that a non-GSH-based reaction product has been observed in the G-site of any GST. AA stacks against a conserved aromatic residue, F34 (equivalent to Y34 in GSTO2-2). Mutation of Y34 to alanine in GSTO2-2 eliminates DHAR activity. From these structures and other biochemical data, we propose a mechanism of substrate binding and catalysis of DHAR activity.

Novel folding and stability defects cause a deficiency of human glutathione transferase omega 1.

The polymorphic deletion of Glu-155 from human glutathione transferase omega1 (GSTO1-1) occurs in most populations. Although the recombinant ΔGlu-155 enzyme expressed in Escherichia coli is active, the deletion causes a deficiency of the active enzyme in vivo. The crystal structure and the folding/unfolding kinetics of the ΔGlu-155 variant were determined in order to investigate the cause of the rapid loss of the enzyme in human cells. The crystal structure revealed altered packing around the Glu-155 deletion, an increase in the predicted solvent-accessible area and a corresponding reduction in the buried surface area. This increase in solvent accessibility was consistent with an elevated Stern-Volmer constant. The unfolding of both the wild type and ΔGlu-155 enzyme in urea is best described by a three-state model, and there is evidence for the more pronounced population of an intermediate state by the ΔGlu-155 enzymes. Studies using intrinsic fluorescence revealed a free energy change around 14.4 kcal/mol for the wild type compared with around 8.6 kcal/mol for the ΔGlu-155 variant, which indicates a decrease in stability associated with the Glu-155 deletion. Urea induced unfolding of the wild type GSTO1-1 was reversible through an initial fast phase followed by a second slow phase. In contrast, the ΔGlu-155 variant lacks the slow phase, indicating a refolding defect. It is possible that in some conditions in vivo, the increased solvent-accessible area and the low stability of the ΔGlu-155 variant may promote its unfolding, whereas the refolding defect limits its refolding, resulting in GSTO1-1 deficiency.