Oxidation Alters the Architecture of the Phenylalanyl-tRNA Synthetase Editing Domain to Confer Hyperaccuracy

High fidelity during protein synthesis is accomplished by aminoacyl-tRNA synthetases (aaRSs). These enzymes ligate an amino acid to a cognate tRNA and have proofreading and editing capabilities that ensure high fidelity. Phenylalanyl-tRNA synthetase (PheRS) preferentially ligates a phenylalanine to a tRNAPhe over the chemically similar tyrosine, which differs from phenylalanine by a single hydroxyl group. In bacteria that undergo exposure to oxidative stress such as Salmonella enterica serovar Typhimurium, tyrosine isomer levels increase due to phenylalanine oxidation. Several residues are oxidized in PheRS and contribute to hyperactive editing, including against mischarged Tyr-tRNAPhe, despite these oxidized residues not being directly implicated in PheRS activity. Here, we solve a 3.6 Å cryo-electron microscopy structure of oxidized S. Typhimurium PheRS. We find that oxidation results in widespread structural rearrangements in the β-subunit editing domain and enlargement of its editing domain. Oxidization also enlarges the phenylalanyl-adenylate binding pocket but to a lesser extent. Together, these changes likely explain why oxidation leads to hyperaccurate editing and decreased misincorporation of tyrosine. Taken together, these results help increase our understanding of the survival of S. Typhimurium during human infection

Structural analysis of cross α-helical nanotubes provides insight into the designability of filamentous peptide nanomaterials

The exquisite structure-function correlations observed in filamentous protein assemblies provide a paradigm for the design of synthetic peptide-based nanomaterials. However, the plasticity of quaternary structure in sequence-space and the lability of helical symmetry present significant challenges to the de novo design and structural analysis of such filaments. Here, we describe a rational approach to design self-assembling peptide nanotubes based on controlling lateral interactions between protofilaments having an unusual cross-α supramolecular architecture. Near-atomic resolution cryo-EM structural analysis of seven designed nanotubes provides insight into the designability of interfaces within these synthetic peptide assemblies and identifies a non-native structural interaction based on a pair of arginine residues. This arginine clasp motif can robustly mediate cohesive interactions between protofilaments within the cross-α nanotubes. The structure of the resultant assemblies can be controlled through the sequence and length of the peptide subunits, which generates synthetic peptide filaments of similar dimensions to flagella and pili

Structural and functional analysis of Cysteine loop receptors, Chorismatases and a C-type like Lectin protein

The work presented here was focused on expression, purification, functional characterization and X-ray structure determination of proteins from three different protein families - Cysteine Loopreceptors, Chorismatases and C-type like lectin proteins.Cysteine loop receptors are ligand gated ion channels and are found in the invertebrate and the mammalian nervous system such as nicotinic acetylcholine receptors (nAChRs) and γ-aminobutyric acid receptors (GABAA). As a part of doctoral thesis, a new member of Cysteine Loopreceptors was discovered from a thermophilic worm Alvinella pompejana and was named Alv-a1-pHCl. The Alv-a1-pHCl receptor was functionally characterized in Xenopus oocytes. Further, Alv-a1-pHCl protein was expressed in Sf9 cells, purified and assayed for thermostability andcrystallization attempts were made.An alpha-bungarotoxin affinity based purification method was developed and used for purification of nAChR from Torpedo californica. The stability of alpha-bungarotoxin-nAChR complex was determined in different detergents and crystallization attempts were made. Furthermore attempts were made to develop an expression system for the alpha 7 nAChR from Rattus norvegicus in Drosophila eyes, which was of no avail. The GABAA β3 receptor, an inhibitory neurotransmitter receptor from Rattus norvegicus was expressed in Sf9 cells, purified and assayed for stability in different detergents and crystallization was tried in different detergents in presence of ligands and lipids.Chorismatases are enzymes involved in chorismate metabolism. As a recently discovered family ofenzymes, no structural and functional details were available at the outset of this doctoral work.Two different homologues, FkbO and Hyg5 were expressed in E.coli, purified and crystallized at a resolution of 1 Å and 1.9 Å in a complex with the competitive inhibitor3-(2-carboxyethyl)benzoate and a functional mechanism was proposed.Proteins belonging to the C-type lectin family are involved in sugar binding and cell-cellinteractions. Proteins with a C-type lectin domain in sea urchins such as SM50 have shown to be important in biomineralization. As no structural details were available of such proteins from sea urchins, the CTL domain of SM50 protein from sea urchin Strongylocentrotus purpuratus was purified and crystallized. The crystals diffracted to a resolution of 2.8 Å.The work presented in the thesis is part of following publications.1. Puneet Juneja, Reinhold Horlacher, Daniel Bertrand, Ryoko Krause, Fabrice Marger,Wolfram WelteJ Biol Chem. 2014 May 23;289(21):15130-15140. Epub 2014 Apr 9.An internally modulated, thermostable, pH sensitive Cys-loop receptor from the hydrothermal vent worm Alvinella pompejana.2. Juneja et al, Manuscript in preparation.Stability of Alpha-Bungarotoxin affinity purified Torpedo nicotinic acetylcholine receptor in lipid based detergents.3. Puneet Juneja, Florian Hubrich, Kay Diederichs, Wolfram Welte, Jennifer N. AndexerJ Mol Biol. 2014 Jan 9;426(1):105-15. doi: 10.1016/j.jmb.2013.09.006Mechanistic implications for the chorismatase FkbO based on the crystal structure.4. Juneja et al, Manuscript in preparation.Crystal structure and mechanism of Hyg5 type chorismatase.5. Puneet Juneja, Ashit Rao, Helmut Cölfen, Kay Diederichs, Wolfram WelteActa Crystallogr F Struct Biol Commun. 2014 Feb 1;70 (Pt 2):260-2.Crystallization and preliminary X-ray analysis of the C-type lectin domain of the spicule Matrixprotein SM50 from Strongylocentrotus purpuratus

Crystallization and preliminary X-ray analysis of the C-type lectin domain of the spicule matrix protein SM50 from Strongylocentrotus purpuratus

Sea urchin spicules have a calcitic mesocrystalline architecture that is closely associated with a matrix of proteins and amorphous minerals. The mechanism underlying spicule formation involves complex processes encompassing spatio-temporally regulated organic–inorganic interactions. C-type lectin domains are present in several spicule matrix proteins in Strongylocentrotus purpuratus, implying their role in spiculogenesis. In this study, the C-type lectin domain of SM50 was overexpressed, purified and crystallized using a vapour-diffusion method. The crystal diffracted to a resolution of 2.85 Å and belonged to space group P212121, with unit-cell parameters a = 100.6, b = 115.4, c = 130.6 Å, α = β = γ = 90°. Assuming 50% solvent content, six chains are expected to be present in the asymmetric unit

Mechanistic Implications for the Chorismatase FkbO Based on the Crystal Structure

Chorismate-converting enzymes are involved in many biosynthetic pathways leading to natural products and can often be used as tools for the synthesis of chemical building blocks. Chorismatases such as FkbO from Streptomyces species catalyse the hydrolysis of chorismate yielding (dihydro)benzoic acid derivatives. In contrast to many other chorismate-converting enzymes, the structure and catalytic mechanism of a chorismatase had not been previously elucidated. Here we present the crystal structure of the chorismatase FkbO in complex with a competitive inhibitor at 1.08 Å resolution. FkbO is a monomer in solution and exhibits pseudo-3-fold symmetry; the structure of the individual domains indicates a possible connection to the trimeric RidA/YjgF family and related enzymes. The co-crystallised inhibitor led to the identification of FkbO's active site in the cleft between the central and the C-terminal domains. A mechanism for FkbO is proposed based on both interactions between the inhibitor and the surrounding amino acids and an FkbO structure with chorismate modelled in the active site. We suggest that the methylene group of the chorismate enol ether takes up a proton from an active-site glutamic acid residue, thereby initiating chorismate hydrolysis. A similar chemistry has been described for isochorismatases, albeit implemented in an entirely different protein scaffold. This reaction model is supported by kinetic data from active-site variants of FkbO derived by site-directed mutagenesis

Chorismatase Mechanisms Reveal Fundamentally Different Types of Reaction in a Single Conserved Protein Fold

Chorismatases are a class of chorismate-converting enzymes involved in the biosynthetic pathways of different natural products, many of them with interesting pharmaceutical characteristics. So far, three subfamilies of chorismatases are described that convert chorismate into different (dihydro-)benzoate derivatives (CH-FkbO, CH-Hyg5, and CH-XanB2). Until now, the detailed enzyme mechanism and the molecular basis for the different reaction products were unknown. Here we show that the CH-FkbO and CH-Hyg5 subfamilies share the same protein fold, but employ fundamentally different reaction mechanisms. While the FkbO reaction is a typical hydrolysis, the Hyg5 reaction proceeds intramolecularly, most likely via an arene oxide intermediate. Two nonconserved active site residues were identified that are responsible for the different reaction mechanisms in CH-FkbO and CH-Hyg5. Further, we propose an additional amino acid residue to be responsible for the discrimination of the CH-XanB2 subfamily, which catalyzes the formation of two different hydroxybenzoate regioisomers, likely in a single active site. A multiple sequence alignment shows that these three crucial amino acid positions are located in conserved motifs and can therefore be used to assign unknown chorismatases to the corresponding subfamily

Covalent inhibition of hAChE by organophosphates causes homodimer dissociation through long-range allosteric effects.

Acetylcholinesterase (EC 3.1.1.7), a key acetylcholine-hydrolyzing enzyme in cholinergic neurotransmission, is present in a variety of states in situ, including monomers, C-terminally disulfide-linked homodimers, homotetramers, and up to three tetramers covalently attached to structural subunits. Could oligomerization that ensures high local concentrations of catalytic sites necessary for efficient neurotransmission be affected by environmental factors? Using small-angle X-ray scattering (SAXS) and cryo-EM, we demonstrate that homodimerization of recombinant monomeric human acetylcholinesterase (hAChE) in solution occurs through a C-terminal four-helix bundle at micromolar concentrations. We show that diethylphosphorylation of the active serine in the catalytic gorge or isopropylmethylphosphonylation by the RP enantiomer of sarin promotes a 10-fold increase in homodimer dissociation. We also demonstrate the dissociation of organophosphate (OP)-conjugated dimers is reversed by structurally diverse oximes 2PAM, HI6, or RS194B, as demonstrated by SAXS of diethylphosphoryl-hAChE. However, binding of oximes to the native ligand-free hAChE, binding of high-affinity reversible ligands, or formation of an SP-sarin-hAChE conjugate had no effect on homodimerization. Dissociation monitored by time-resolved SAXS occurs in milliseconds, consistent with rates of hAChE covalent inhibition. OP-induced dissociation was not observed in the SAXS profiles of the double-mutant Y337A/F338A, where the active center gorge volume is larger than in wildtype hAChE. These observations suggest a key role of the tightly packed acyl pocket in allosterically triggered OP-induced dimer dissociation, with the potential for local reduction of acetylcholine-hydrolytic power in situ. Computational models predict allosteric correlated motions extending from the acyl pocket toward the four-helix bundle dimerization interface 25 Å away

Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer.

Neurofibromin is a tumor suppressor encoded by the NF1 gene, which is mutated in Rasopathy disease neurofibromatosis type I. Defects in NF1 lead to aberrant signaling through the RAS-mitogen-activated protein kinase pathway due to disruption of the neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of most of the neurofibromin protein; to date, biochemical and structural data exist only for its GAP domain and a region containing a Sec-PH motif. To better understand the role of this large protein, here we carried out a series of biochemical and biophysical experiments, including size-exclusion chromatography-multiangle light scattering (SEC-MALS), small-angle X-ray and neutron scattering, and analytical ultracentrifugation, indicating that full-length neurofibromin forms a high-affinity dimer. We observed that neurofibromin dimerization also occurs in human cells and likely has biological and clinical implications. Analysis of purified full-length and truncated neurofibromin variants by negative-stain EM revealed the overall architecture of the dimer and predicted the potential interactions that contribute to the dimer interface. We could reconstitute structures resembling high-affinity full-length dimers by mixing N- and C-terminal protein domains in vitro The reconstituted neurofibromin was capable of GTPase activation in vitro, and co-expression of the two domains in human cells effectively recapitulated the activity of full-length neurofibromin. Taken together, these results suggest how neurofibromin dimers might form and be stabilized within the cell