High Resolution X-ray Structures of Different Metal-Substituted Forms of Phosphotriesterase from Pseudomonas diminuta ,

Ligand-gated ion channel receptors mediate the response of fast neurotransmitters by opening in less than a millisecond. Here, we investigated the activation mechanism of a serotonin-gated receptor (5-HT 3A ) by systematically introducing cysteine substitutions throughout the pore-lining M1-M2 loop and M2 transmembrane domain. We hypothesized that multiple cysteines in the narrowest region of the pore, which together can form a high affinity binding site for metal cations, would reveal changes in pore structure during gating. Using cadmium (Cd 2؉ ) as a probe, two cysteine substitutions in the cytoplasmic selectivity filter, S2 C and, to a lesser extent, G-2 C, showed high affinity inhibition with Cd 2؉ Ligand-gated ion channel (LGIC) 1 receptors are responsible for rapid chemical transmission between neurons in the nervous system (1). Alterations in the response of ligand-gated receptors have profound effects on neuronal activity in the brain. For example, mutations in nicotinic acetylcholine-gated receptors (nAChRs) and glycine-gated receptors have been linked to certain forms of epilepsy and startle disease, respectively (2-4).LGIC receptors that respond to acetylcholine, ␥-aminobutyric acid, glycine, or serotonin (5-HT) possess a conserved pair of extracellular cysteines in the N-terminal domain ("Cys loop" receptors) and are composed of five subunits, each containing four putative transmembrane domains (5). Of these four domains, the second transmembrane domain (M2) lines the majority of the water-filled pore (Fig. 1A).The rapid response of LGIC receptors is achieved by the energetic coupling of agonist binding in the extracellular Nterminal domain with a "gate" located 60 Å away in the receptor pore (M2 domain) (6). Several studies with LGIC receptors suggest that the gate is situated in the middle of the M2 (6 -12) (but see Ref. 13). The primary determinants of selectivity appear to be located below the gate, near the cytoplasmic membrane surface. Mutations in the cytoplasmic end of the M2 alter ion selectivity (14 -17), and several recent studies show that by mutating sites in this region, cation-conducting LGIC can be converted to anion-conducting channels and vice versa (18 -23). Because permeability of like-charged molecules in LGIC appears to be largely dependent on size, it is believed that the narrowest region of the open LGIC pore coincides with the channel selectivity filter, which is formed by a portion of the M1-M2 loop and the cytoplasmic side of the M2 helix (16,24). Consistent with this region being narrow, site-specific mutations in the filter alter conductivity in a manner that depends upon side-chain volume (8,24). Thus, the pore of the open receptor can be envisaged as a funnel, with a wide extracellular vestibule that tapers to a constriction at the cytoplasmic side of the membrane, where ion selectivity is determined (25).What is known about the extent of movement in the cytoplasmic selectivity filter? The 9-Å three-dimensional structures of open and closed Torpedo nAChRs derived fro...

Section: Discussionmentioning

confidence: 99%

Section: High Affinity CD 2ϩmentioning

confidence: 99%

Minimal Structural Rearrangement of the Cytoplasmic Pore during Activation of the 5-HT3A Receptor

Panicker

Cruz

Arrabit

et al. 2004

“…Second, the putative active site water molecule, which is likely to be present at the product acetate ACT1 O 1 position, lies significantly closer to the zinc ion at the ␣ 1 subsite (1.95 Å) than that at the ␤ site (2.84 Å). This is one striking difference between the active site here and those in other binuclear zinc hydrolases such as the ␣␤ members, where the water/hydroxide ion is almost symmetrically located between the two metal ions (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Therefore, the inhibitory metal ion at the ␣ site may hold the active site water tightly and thus perturb the stereochemical arrangements required for enzyme catalysis as it does in bovine carboxypeptidase A (38).…”

Section: Resultsmentioning

confidence: 88%

“…Based on the binding site(s) of the catalytically essential metal ion(s), we classify these members into four types: ␣␤-binuclear (␣␤), ␣-mononuclear (␣), ␤-mononuclear (␤), and metal-independent subsets. Phosphotriesterase (homology protein), urease, dihydroorotase, renal dipeptidase, isoaspartyl didpeptidase, and dihydropyrimidinase comprise the ␣␤ subset (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Murine adenosine deaminase and Escherichia coli cytosine deaminase belong to the ␣ subset (16,17).…”

mentioning

confidence: 99%

The Functional Role of the Binuclear Metal Center in d-Aminoacylase

Lai¹,

Chou²,

Ting³

et al. 2004

Our structural comparison of the TIM barrel metal-dependent hydrolase(-like) superfamily suggests a classification of their divergent active sites into four types: ␣␤-binuclear, ␣-mononuclear, ␤-mononuclear, and metal-independent subsets. The D-aminoacylase from Alcaligenes faecalis DA1 belongs to the ␤-mononuclear subset due to the fact that the catalytically essential , revealing that elimination of the ␤ site changes the coordination geometry of the ␣ ion with an enhanced affinity. Kinetic studies of the metal ligand mutants such as C96D indicate the uniqueness of the unusual bridging cysteine and its involvement in catalysis. Therefore, the two metal-binding sites in the Daminoacylase are interactive with partially mutual exclusion, thus resulting in widely different affinities for the activation/attenuation mechanism, in which the enzyme is activated by the metal ion at the ␤ site, but inhibited by the subsequent binding of the second ion at the ␣ site.D-Aminoacylase (EC 3.5.1.81) is an attractive candidate for production of D-amino acids through its catalysis of the deacetylation of N-acetyl-D-amino acids. Since D-amino acids are intermediates in the preparation of antibiotics, pesticides, and bioactive peptides, D-aminoacylase has commercial importance for the optical resolution of N-acetyl-DL-amino acids (1-2). The crystal structure of the 483-residue D-aminoacylase from Alcaligenes faecalis DA1 revealed that the enzyme is comprised of a small ␤ barrel and a catalytic TIM barrel with a unique 63-residue insertion (3). The structural similarity and the conserved metal ligands of four histidines and one aspartate suggest that D-aminoacylase belongs to the TIM barrel metal-dependent hydrolase superfamily. This superfamily includes a variety of enzymes with highly diverse substrates and has been coined an "evolutionary treasure" (4).To date, crystal structures of 14 members in the superfamily have been determined. As more structures are solved, more divergences in the metal centers and in the ␤-strand packing of the TIM barrel are observed (Fig. 1). Based on the binding site(s) of the catalytically essential metal ion(s), we classify these members into four types: ␣␤-binuclear (␣␤), ␣-mononuclear (␣), ␤-mononuclear (␤), and metal-independent subsets. Phosphotriesterase (homology protein), urease, dihydroorotase, renal dipeptidase, isoaspartyl didpeptidase, and dihydropyrimidinase comprise the ␣␤ subset (5-15). Murine adenosine deaminase and Escherichia coli cytosine deaminase belong to the ␣ subset (16, 17). D-Aminoacylase belongs to the ␤ subset due to the essential Zn 2ϩ binding tightly at the ␤ site, even though it possesses a weak ␣ binding site (3). In contrast, the activity assay and the crystal structure of uronate isomerase show that this enzyme does not display any specific metal requirement and thus belongs to the metal-independent subset (18,19).The functional requirement for the different metal centers is still unclear. Interestingly, Bacillus subtilis N-acetylglucosamine-6-phosphate deacetylase ...

“…In bacteria these usually have either a single transmembrane domain at the C terminus (35) or an N-terminal non-cleaved twin-arginine signal anchor sequence (37). However, bioinformatic analysis supported by structural studies show that OPH has no C-terminal hydrophobic helical domain (38) and the N-terminal signal peptide of OPH is cleaved off during biosynthesis and therefore cannot serve as a signal anchor (15).…”

Section: Oph Is Tightly Bound To the B Diminuta Inner Membrane-mentioning

confidence: 99%

Organophosphate Hydrolase Is a Lipoprotein and Interacts with Pi-specific Transport System to Facilitate Growth of Brevundimonas diminuta Using OP Insecticide as Source of Phosphate

Parthasarathy

Parapatla

Nandavaram

et al. 2016

Organophosphate hydrolase (OPH), encoded by the organophosphate degradation (opd) island, hydrolyzes the triester bond found in a variety of organophosphate insecticides and nerve agents. OPH is targeted to the inner membrane of Brevundimonas diminuta in a pre-folded conformation by the twin arginine transport (Tat) pathway. The OPH signal peptide contains an invariant cysteine residue at the junction of the signal peptidase (Spase) cleavage site along with a well conserved lipobox motif. Treatment of cells producing native OPH with the signal peptidase II inhibitor globomycin resulted in accumulation of most of the pre-OPH in the cytoplasm with negligible processed OPH detected in the membrane. Substitution of the conserved lipobox cysteine to serine resulted in release of OPH into the periplasm, confirming that OPH is a lipoprotein. Analysis of purified OPH revealed that it was modified with the fatty acids palmitate and stearate. Membrane-bound OPH was shown to interact with the outer membrane efflux protein TolC and with PstS, the periplasmic component of the ABC transporter complex (PstSACB) involved in phosphate transport. Interaction of OPH with PstS appears to facilitate transport of P i generated from organophosphates due to the combined action of OPH and periplasmically located phosphatases. Consistent with this model, opd null mutants of B. diminuta failed to grow using the organophosphate insecticide methyl parathion as sole source of phosphate.