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. 2019 Nov 1;75(Pt 11):995-1002.
doi: 10.1107/S2059798319013883. Epub 2019 Oct 31.

The Evolving Story of AtzT, a Periplasmic Binding Protein

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Free PMC article

The Evolving Story of AtzT, a Periplasmic Binding Protein

Matthew L Dennis et al. Acta Crystallogr D Struct Biol. .
Free PMC article

Abstract

Atrazine is an s-triazine-based herbicide that is used in many countries around the world in many millions of tons per year. A small number of organisms, such as Pseudomonas sp. strain ADP, have evolved to use this modified s-triazine as a food source, and the various genes required to metabolize atrazine can be found on a single plasmid. The atomic structures of seven of the eight proteins involved in the breakdown of atrazine by Pseudomonas sp. strain ADP have been determined by X-ray crystallography, but the structures of the proteins required by the cell to import atrazine for use as an energy source are still lacking. The structure of AtzT, a periplasmic binding protein that may be involved in the transport of a derivative of atrazine, 2-hydroxyatrazine, into the cell for mineralization, has now been determined. The structure was determined by SAD phasing using an ethylmercury phosphate derivative that diffracted X-rays to beyond 1.9 Å resolution. `Native' (guanine-bound) and 2-hydroxyatrazine-bound structures were also determined to high resolution (1.67 and 1.65 Å, respectively), showing that 2-hydroxyatrazine binds in a similar way to the purportedly native ligand. Structural similarities led to the belief that it may be possible to evolve AtzT from a purine-binding protein to a protein that can bind and detect atrazine in the environment.

Keywords: AtzT; SAD phasing; atrazine; periplasmic binding proteins.

Figures

Figure 1
Figure 1
The image on the left is a cartoon representation coloured by Jones’ rainbow with the N-terminus in dark blue and the C-terminus in dark red. The green mesh represents the unknown difference density found after solving the protein structure sitting between the two domains. The image on the right is a cartoon representation that is coloured by secondary structure with α-helices in cyan and β-sheets in magenta. It is orientated with an approximate 90° rotation to the figure on the left.
Figure 2
Figure 2
An enlarged image of the binding site between the two domains. The image on the left shows the difference density (mF oDF c, 3σ) before guanine was placed into the model. The relevant amino acids in the binding site are shown and the backbone is coloured by Jones’ rainbow as in Fig. 1 ▸(a). The middle and right figures are rotated plus and minus approximately 90° from the figure on the left and are coloured by atom type, with C in cyan, O in red and N in dark blue. Potential hydrogen bonds are represented as black lines; approximate distances between potential hydrogen-bond acceptors and donors are shown in Å and the binding residues are labelled. Note that the Asn218 is out of plane to the guanine, so the hydrogen on the Asn218 N atom is likely to interact with the π-cloud perpendicular to the plane of the atoms.
Figure 3
Figure 3
An enlarged image of 2-hydroxyatrazine in the binding site. The difference map (mF oDF c) is set to 3σ and the compound has good density, with the exception of the isopropyl group, which can be found in more than one orientation but has clashes with the protein (for example Asn218) in all possible orientations. The same residues that make hydrogen bonds to guanine make very similar interactions with the 2-hydroxyatrazine heteroatoms and the compound is sandwiched between Tyr45 and Trp194 in a similar fashion to guanine. C atoms are in cyan, O atoms are in red and N atoms are in dark blue.

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