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From Proteopedia
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<StructureSection load='' size='350' side='right' caption='Human thioredoxin (PDB entry [[1ert]])' scene='43/430885/Cv/2'> | <StructureSection load='' size='350' side='right' caption='Human thioredoxin (PDB entry [[1ert]])' scene='43/430885/Cv/2'> | ||
| - | [[Thioredoxin]] (Trx) is an enzyme which facilitates, in its reduced form, the reduction of proteins by cysteine thiol-disulfide exchange<ref>PMID: 17531267</ref>. They contain a CXXC motif.<br /> | ||
| - | * '''Trx-1''' is a mammalian cellular protein.<br /> | ||
| - | * '''Trx-2''' is mitochondria-specific.<br /> | ||
| - | * '''Trx C,M,X,Y''' are found in prokaryotes.<br /> | ||
| - | * '''Trx F,H,O''' are found in eukaryotes.<br /> | ||
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== Structural highlights == | == Structural highlights == | ||
Oxidized PtGPX5 was crystallized as a dimer in the asymmetric unit, with a resolution of 2.45Å.<ref name="Koh"/> The oxidized conformation of PtGPX5 has less defined regions than the reduced form, especially between residue 77-84, where there was barely any electron density in one of the chains.<ref name="Koh"/> As with any protein model, there is some level of interpretation. These researchers took the electron density and sequence of the flexible loop (residues 73-100) from the chain they could see and applied it to the loop containing residues 77-84 which they couldn’t see, keeping allowable Ramachandran conformations in mind.<ref name="Koh"/> This inconveniently contains Cys92, a residue involved in catalysis/disulfide bond formation.<ref name="Koh"/> They also predicted the presence of five calcium ions in the oxidized form, since it was crystallized with calcium chloride.<ref name="Koh"/> <br /> | Oxidized PtGPX5 was crystallized as a dimer in the asymmetric unit, with a resolution of 2.45Å.<ref name="Koh"/> The oxidized conformation of PtGPX5 has less defined regions than the reduced form, especially between residue 77-84, where there was barely any electron density in one of the chains.<ref name="Koh"/> As with any protein model, there is some level of interpretation. These researchers took the electron density and sequence of the flexible loop (residues 73-100) from the chain they could see and applied it to the loop containing residues 77-84 which they couldn’t see, keeping allowable Ramachandran conformations in mind.<ref name="Koh"/> This inconveniently contains Cys92, a residue involved in catalysis/disulfide bond formation.<ref name="Koh"/> They also predicted the presence of five calcium ions in the oxidized form, since it was crystallized with calcium chloride.<ref name="Koh"/> <br /> | ||
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The <scene name='43/430885/Cv/4'>active site motif Cys-Gly-Pro-Cys</scene> is involved in the reduction of disulfide bonds in proteins<ref>PMID:8805557</ref> | The <scene name='43/430885/Cv/4'>active site motif Cys-Gly-Pro-Cys</scene> is involved in the reduction of disulfide bonds in proteins<ref>PMID:8805557</ref> | ||
| - | == Crystallization Methods == | ||
[[Thioredoxin 3D structures]] | [[Thioredoxin 3D structures]] | ||
</StructureSection> | </StructureSection> | ||
| - | + | == Crystallization Methods == | |
PtGPX5 was crystallized using selenomethionine methods to help with phasing issues.<ref name="Koh"/> Crystals of oxidized PtGPX5 were grown over two days in 0.1M Tris-HCl (pH 8.5), 25% (w/v) PEG 4000, and 0.2M calcium chloride, whereas crystals of the reduced form took a week to grow in 0.1M HEPES (pH 7.5), 0.05M cadmium sulphate hydrate, and 1.0M sodium acetate.<ref name="Koh"/> Oxidized PtGPX5 was trigonal and designated form II, meaning that it is part of the P3121 space group and has cell dimensions of a=71.6Å and c=48.1Å.<ref name="Koh"/> There are two subunits per asymmetric unit.<ref name="Koh"/> | PtGPX5 was crystallized using selenomethionine methods to help with phasing issues.<ref name="Koh"/> Crystals of oxidized PtGPX5 were grown over two days in 0.1M Tris-HCl (pH 8.5), 25% (w/v) PEG 4000, and 0.2M calcium chloride, whereas crystals of the reduced form took a week to grow in 0.1M HEPES (pH 7.5), 0.05M cadmium sulphate hydrate, and 1.0M sodium acetate.<ref name="Koh"/> Oxidized PtGPX5 was trigonal and designated form II, meaning that it is part of the P3121 space group and has cell dimensions of a=71.6Å and c=48.1Å.<ref name="Koh"/> There are two subunits per asymmetric unit.<ref name="Koh"/> | ||
Revision as of 20:23, 3 May 2022
Contents |
Introduction
Glutathione peroxidases are a family of enzymes that have antioxidant properties.[1] It fights against oxidative stress by removing reactive oxygen species (ROS) from the cell.[1] One example of this is how glutathione peroxidase (GPx) can convert hydrogen peroxide to water. To do this, GPx utilizes glutathione, glutathione reductase, and cofactors FAD and NADPH. Interestingly, GPx in plants typically rely on thioredoxin instead of glutathione as its electron donor to reduce ROS.[1] Being able to utilize both thioredoxin and glutathione as substrates is not very common, generally speaking, but it has been seen before in both thioredoxin-dependent and glutathione-dependent antioxidant systems (for example, thioredoxin reductase from Karenia brevis).[1],2 Additionally, plants often have cysteine in their active site instead of selenocysteine found in most other GPx homologues.[1] Having cysteine in the active site typically reduces the catalytic efficiency of the enzyme in comparison with its selenocysteine-containing counterparts. Even though plant GPxs are slower, they can reduce wider variety of ROS, as they have lower substrate specificity.[1]
This structure, 2P5R, is the oxidized form of glutathione peroxidase 5 from Populus trichocarpa x Populus deltoides (PtGPX5), from a paper entitled “Crystal Structures of a Poplar Thioredoxin Peroxidase that Exhibits the Structure of Glutathione Peroxidases: Insights into Redox-driven Conformational Changes”.[1] At the time of this publication, there were only six crystal structures of GPxs, all of which were mammalian.[1] This paper was ground-breaking, as it provided the first GPx structures not from mammals. Black cottonwood poplar was chosen as the model organism because at the time, its full genome had recently been released and it had six GPX genes.[1] PtGPX5 got classified as a GPx-5, the category of GPxs which are not selenoproteins.[1]
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Crystallization Methods
PtGPX5 was crystallized using selenomethionine methods to help with phasing issues.[1] Crystals of oxidized PtGPX5 were grown over two days in 0.1M Tris-HCl (pH 8.5), 25% (w/v) PEG 4000, and 0.2M calcium chloride, whereas crystals of the reduced form took a week to grow in 0.1M HEPES (pH 7.5), 0.05M cadmium sulphate hydrate, and 1.0M sodium acetate.[1] Oxidized PtGPX5 was trigonal and designated form II, meaning that it is part of the P3121 space group and has cell dimensions of a=71.6Å and c=48.1Å.[1] There are two subunits per asymmetric unit.[1]
Summary
Glutathione peroxidases are part of the antioxidant network that minimizes the buildup of ROS in a cell. PtGPX5 was the seventh GPx to be crystallized and to have the structure determined.[1] Although GPxs don’t typically use thioredoxin as a substrate, this one does.[1] It is structurally similar to animal GPxs and has a thioredoxin fold. Its dimerization interface remains conserved regardless of oxidation state and of crystallization methods.[1] It was easier to crystallize PtGPX5 in the oxidized state compared to the reduced state.[1] The greatest conformational changes occur when it is switching redox states, as the α2-helix completely unfolds.[1] It becomes a flexible loop when Cys44 gets oxidized and Cy92 subsequently forms a disulfide with it.[1]
References
- ↑ 1.00 1.01 1.02 1.03 1.04 1.05 1.06 1.07 1.08 1.09 1.10 1.11 1.12 1.13 1.14 1.15 1.16 1.17 1.18 1.19 1.20 1.21 1.22 1.23 1.24 1.25 1.26 1.27 1.28 1.29 1.30 1.31 1.32 1.33 1.34 1.35 1.36 1.37 1.38 1.39 1.40 1.41 1.42 1.43 1.44 1.45 1.46 1.47 Koh CS, Didierjean C, Navrot N, Panjikar S, Mulliert G, Rouhier N, Jacquot JP, Aubry A, Shawkataly O, Corbier C. Crystal structures of a poplar thioredoxin peroxidase that exhibits the structure of glutathione peroxidases: insights into redox-driven conformational changes. J Mol Biol. 2007 Jul 13;370(3):512-29. Epub 2007 Apr 19. PMID:17531267 doi:http://dx.doi.org/10.1016/j.jmb.2007.04.031
- ↑ Weichsel A, Gasdaska JR, Powis G, Montfort WR. Crystal structures of reduced, oxidized, and mutated human thioredoxins: evidence for a regulatory homodimer. Structure. 1996 Jun 15;4(6):735-51. PMID:8805557
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