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Three-dimensional structure of a truncated phosphoribosylanthranilate isomerase (residues 255-384) from Escherichia coli

The University of Sydney
Joel Mackay (Associated with, Aggregated by)
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ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Adc&rfr_id=info%3Asid%2FANDS&rft_id=info:doi10.2210/pdb2kzh/pdb&rft.title=Three-dimensional structure of a truncated phosphoribosylanthranilate isomerase (residues 255-384) from Escherichia coli&rft.identifier=https://mds.sydney.edu.au/redbox/published/detail/60f2728ab135582a7a3bac46f88f5962&rft.publisher=The University of Sydney&rft.description=Materials and Methods Expression and purification The construction of the trPRAI expression plasmid, pWP107His, was described previously.25 The plasmid was used to transform E. coli BL21 (DE3) cells, which were grown in minimal medium supplemented with 15NH4Cl and/or [13C]glucose. Expression of trPRAI was induced at 25 °C for ∼ 12 h using isopropyl-β-d-thiogalactopyranoside, and cells were lysed by sonication. The His6-tagged recombinant protein was purified from the soluble cell lysate using Ni-NTA agarose (GE Healthcare). Further purification was performed on an FPLC system (BioRad), using HiLoad™ 16/60 Superdex™ 75 (GE Healthcare). The trPRAI-containing fractions were concentrated to 1 mM in buffer containing 20 mM Hepes (pH 7.0), 100 mM NaCl and 1 mM DTT in either 5% 2D2O or 100% 2D2O as required. 4,4-Dimethyl-4-silapentane-1-sulfonic acid was added to the concentrated samples as an internal reference. Size-exclusion chromatography–multiple-angle laser light scattering SEC–MALLS studies were conducted using an AKTA-BASIC FPLC system (GE Healthcare) with in-line MALLS and in-line refractive index detectors (Wyatt Technology Corp., California). Samples of trPRAI (10 mg mL− 1 or 2 mg mL− 1) were loaded onto a Superose 12 10/300 column equilibrated in 20 mM Hepes (pH 7.0), 100 mM NaCl and 1 mM DTT. Light-scattering data were collected and analyzed using the ASTRA software package. NMR spectroscopy NMR spectra were acquired at 298 K on Bruker Avance III 600-MHz or 800-MHz spectrometers. Assignments were made using standard triple resonance techniques.52 NOE-derived distance restraints were obtained from three-dimensional 15N-separated and 13C-separated NOE spectroscopy spectra (τm = 150 ms) and from a homonuclear two-dimensional NOE spectroscopy (τm = 150 ms) spectrum. Backbone ϕ and ψ dihedral angle restraints were derived from the assigned backbone chemical shifts (HN, N, Cα, Cβ and CO) using TALOS+,30 and coupling constant restraints were obtained from an HNHA spectrum.5315N relaxation data were acquired using standard Bruker pulse programs. Processing and analysis of NMR relaxation data Longitudinal (T1) relaxation data were recorded using 15N relaxation delays of 12, 60, 150, 300, 600, 1000, 1400 and 2000 ms. Similarly, transverse (T2) relaxation measurements were performed using 15N relaxation delays of 1, 2, 4, 6, 8, 10, 14, 20, 48 and 80 ms. T1 and T2 values were derived by fitting the relaxation data to single-exponential decay functions by using the relaxation peak heights extension program provided in SPARKY †. The uncertainties in the fitted relaxation times were used as standard errors. To measure the 1H,15N hetNOE, we recorded two experiments, one with 1H saturation and one without. The error in the NOE was estimated from assuming that the uncertainty in the peak heights equals the rms noise in each of those two spectra. The 1H,15N hetNOE values were then calculated as fractional enhancements according to the equation: fI{S}=(Isat−Iref)/Iref where Isat and Iref are cross-peak intensities in the presence and in the absence of 1H saturation, respectively. Structure calculations NMR spectra were analyzed using SPARKY. Structure calculations were performed using CYANA 3.0 and CNS version 1.21,29 including a final water refinement stage according to the standard RECOORD protocol.54 The 20 lowest-energy structures out of the 100 structures calculated using CNS were selected under the following criteria: no NOE violations > 0.5 Å, no dihedral angle violations > 5° and low value for Etot. Abstract: The (βα)(8) barrel is one of the most common protein folds, and enzymes with this architecture display a remarkable range of catalytic activities. Many of these functions are associated with ancient metabolic pathways, and phylogenetic reconstructions suggest that the (βα)(8) barrel was one of the very first protein folds to emerge. Consequently, there is considerable interest in understanding the evolutionary processes that gave rise to this fold. In particular, much attention has been focused on the plausibility of (βα)(8) barrel evolution from homodimers of half barrels. However, we previously isolated a three-quarter-barrel-sized fragment of a (βα)(8) barrel, termed truncated phosphoribosylanthranilate isomerase (trPRAI), that is soluble and almost as thermostable as full-length N-(5'-phosphoribosyl)anthranilate isomerase (PRAI). Here, we report the NMR-derived structure of trPRAI. The subdomain is monomeric, is well ordered and adopts a native-like structure in solution. Side chains from strands β(1) (Glu3 and Lys5), β(2) (Tyr25) and β(6) (Lys122) of trPRAI repack to shield the hydrophobic core from the solvent. This result demonstrates that three-quarter barrels were viable intermediates in the evolution of the (βα)(8) barrel fold. We propose a unified model for (βα)(8) barrel evolution that combines our data, previously published work and plausible scenarios for the emergence of (initially error-prone) genetic systems. In this model, the earliest proto-cells contained diverse pools of part-barrel subdomains. Combinatorial assembly of these subdomains gave rise to many distinct lineages of (βα)(8) barrel proteins, that is, our model excludes the possibility that there was a single (βα)(8) barrel from which all present examples are descended. &rft.creator=Joel Mackay&rft.date=2014&rft.relation=http://dx.doi.org/10.2210/pdb2kzh/pdb&rft.relation=http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=21354426&rft.relation=http://dx.doi.org/10.1016/j.jmb.2011.02.048&rft_subject=Aldose-Ketose Isomerases&rft_subject=Chemical&rft_subject=Protein Conformation&rft_subject=Protein Folding&rft_subject=Catalytic Domain&rft_subject=Chromatography&rft_subject=Gel&rft_subject=Escherichia Coli&rft_subject=Evolution&rft_subject=Molecular&rft_subject=Magnetic Resonance Spectroscopy&rft_subject=Models&rft.type=dataset&rft.language=English Go to Data Provider

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Please forward data access requests to School of Molecular Biosciences, University of Sydney, NSW 2006, Australia. Tel.: 61-2-9351-3906; E-mail: joel.mackay@sydney.edu.au.

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The University of Sydney

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Materials and Methods
Expression and purification
The construction of the trPRAI expression plasmid, pWP107His, was described previously.25 The plasmid was used to transform E. coli BL21 (DE3) cells, which were grown in minimal medium supplemented with 15NH4Cl and/or [13C]glucose. Expression of trPRAI was induced at 25 °C for ∼ 12 h using isopropyl-β-d-thiogalactopyranoside, and cells were lysed by sonication. The His6-tagged recombinant protein was purified from the soluble cell lysate using Ni-NTA agarose (GE Healthcare). Further purification was performed on an FPLC system (BioRad), using HiLoad™ 16/60 Superdex™ 75 (GE Healthcare). The trPRAI-containing fractions were concentrated to 1 mM in buffer containing 20 mM Hepes (pH 7.0), 100 mM NaCl and 1 mM DTT in either 5% 2D2O or 100% 2D2O as required. 4,4-Dimethyl-4-silapentane-1-sulfonic acid was added to the concentrated samples as an internal reference.

Size-exclusion chromatography–multiple-angle laser light scattering
SEC–MALLS studies were conducted using an AKTA-BASIC FPLC system (GE Healthcare) with in-line MALLS and in-line refractive index detectors (Wyatt Technology Corp., California). Samples of trPRAI (10 mg mL− 1 or 2 mg mL− 1) were loaded onto a Superose 12 10/300 column equilibrated in 20 mM Hepes (pH 7.0), 100 mM NaCl and 1 mM DTT. Light-scattering data were collected and analyzed using the ASTRA software package.

NMR spectroscopy
NMR spectra were acquired at 298 K on Bruker Avance III 600-MHz or 800-MHz spectrometers. Assignments were made using standard triple resonance techniques.52 NOE-derived distance restraints were obtained from three-dimensional 15N-separated and 13C-separated NOE spectroscopy spectra (τm = 150 ms) and from a homonuclear two-dimensional NOE spectroscopy (τm = 150 ms) spectrum. Backbone ϕ and ψ dihedral angle restraints were derived from the assigned backbone chemical shifts (HN, N, Cα, Cβ and CO) using TALOS+,30 and coupling constant restraints were obtained from an HNHA spectrum.5315N relaxation data were acquired using standard Bruker pulse programs.

Processing and analysis of NMR relaxation data
Longitudinal (T1) relaxation data were recorded using 15N relaxation delays of 12, 60, 150, 300, 600, 1000, 1400 and 2000 ms. Similarly, transverse (T2) relaxation measurements were performed using 15N relaxation delays of 1, 2, 4, 6, 8, 10, 14, 20, 48 and 80 ms. T1 and T2 values were derived by fitting the relaxation data to single-exponential decay functions by using the relaxation peak heights extension program provided in SPARKY †. The uncertainties in the fitted relaxation times were used as standard errors. To measure the 1H,15N hetNOE, we recorded two experiments, one with 1H saturation and one without. The error in the NOE was estimated from assuming that the uncertainty in the peak heights equals the rms noise in each of those two spectra. The 1H,15N hetNOE values were then calculated as fractional enhancements according to the equation:

fI{S}=(Isat−Iref)/Iref

where Isat and Iref are cross-peak intensities in the presence and in the absence of 1H saturation, respectively.
Structure calculations
NMR spectra were analyzed using SPARKY. Structure calculations were performed using CYANA 3.0 and CNS version 1.21,29 including a final water refinement stage according to the standard RECOORD protocol.54 The 20 lowest-energy structures out of the 100 structures calculated using CNS were selected under the following criteria: no NOE violations > 0.5 Å, no dihedral angle violations > 5° and low value for Etot.

Abstract: The (βα)(8) barrel is one of the most common protein folds, and enzymes with this architecture display a remarkable range of catalytic activities. Many of these functions are associated with ancient metabolic pathways, and phylogenetic reconstructions suggest that the (βα)(8) barrel was one of the very first protein folds to emerge. Consequently, there is considerable interest in understanding the evolutionary processes that gave rise to this fold. In particular, much attention has been focused on the plausibility of (βα)(8) barrel evolution from homodimers of half barrels. However, we previously isolated a three-quarter-barrel-sized fragment of a (βα)(8) barrel, termed truncated phosphoribosylanthranilate isomerase (trPRAI), that is soluble and almost as thermostable as full-length N-(5'-phosphoribosyl)anthranilate isomerase (PRAI). Here, we report the NMR-derived structure of trPRAI. The subdomain is monomeric, is well ordered and adopts a native-like structure in solution. Side chains from strands β(1) (Glu3 and Lys5), β(2) (Tyr25) and β(6) (Lys122) of trPRAI repack to shield the hydrophobic core from the solvent. This result demonstrates that three-quarter barrels were viable intermediates in the evolution of the (βα)(8) barrel fold. We propose a unified model for (βα)(8) barrel evolution that combines our data, previously published work and plausible scenarios for the emergence of (initially error-prone) genetic systems. In this model, the earliest proto-cells contained diverse pools of part-barrel subdomains. Combinatorial assembly of these subdomains gave rise to many distinct lineages of (βα)(8) barrel proteins, that is, our model excludes the possibility that there was a single (βα)(8) barrel from which all present examples are descended.
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