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PDBsum entry 3crt

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Transferase PDB id
3crt
Jmol
Contents
Protein chain
214 a.a.
Ligands
GSH
Waters ×158

References listed in PDB file
Key reference
Title Using affinity chromatography to engineer and characterize ph-Dependent protein switches.
Authors M.Sagermann, R.R.Chapleau, E.Delorimier, M.Lei.
Ref. Protein Sci., 2009, 18, 217-228. [DOI no: 10.1002/pro.23]
PubMed id 19177365
Abstract
Conformational changes play important roles in the regulation of many enzymatic reactions. Specific motions of side chains, secondary structures, or entire protein domains facilitate the precise control of substrate selection, binding, and catalysis. Likewise, the engineering of allostery into proteins is envisioned to enable unprecedented control of chemical reactions and molecular assembly processes. We here study the structural effects of engineered ionizable residues in the core of the glutathione-S-transferase to convert this protein into a pH-dependent allosteric protein. The underlying rational of these substitutions is that in the neutral state, an uncharged residue is compatible with the hydrophobic environment. In the charged state, however, the residue will invoke unfavorable interactions, which are likely to induce conformational changes that will affect the function of the enzyme. To test this hypothesis, we have engineered a single aspartate, cysteine, or histidine residue at a distance from the active site into the protein. All of the mutations exhibit a dramatic effect on the protein's affinity to bind glutathione. Whereas the aspartate or histidine mutations result in permanently nonbinding or binding versions of the protein, respectively, mutant GST50C exhibits distinct pH-dependent GSH-binding affinity. The crystal structures of the mutant protein GST50C under ionizing and nonionizing conditions reveal the recruitment of water molecules into the hydrophobic core to produce conformational changes that influence the protein's active site. The methodology described here to create and characterize engineered allosteric proteins through affinity chromatography may lead to a general approach to engineer effector-specific allostery into a protein structure.
Figure 1.
Ribbon representation of the overall structure of wild-type GST in the presence of glutathione (GSH, shown as stick model in purple). The smaller amino terminal domain of the protein is colored in yellow with Leu50 highlighted in red. This residue is pointing toward the hydrophobic core of this smaller domain of the protein. It is located at [similar]10 A distance from the substrate binding site. Any pH-sensitive effects of the Asp50, Cys50, or the His50 mutants, therefore, must be transmitted through conformational changes to the binding site. This figure as well as Figures 4 Figure 4-and and55 Figure 5-were prepared with the program PyMol (DeLano Scientific LLC, http://pymol.sourceforge.net).
Figure 4.
Ribbon diagram of the crystal structures of the wild-type protein and the GST50H mutant. The rainbow color-coding represents the B value distribution of the backbone atoms of the structures (blue = low B values, red = high B values). In the crystal structures, the highest conformational flexibility is observed in Helix B. The average B value for the main-chain atoms of this structural segment averages to about B = 60 A^2 (see also Fig. 7 Figure 7-). With the introduction of a histidine at position 50, the mobility of this helix is dramatically increased by almost a factor of 2 (see also Fig. 7 Figure 7-). Despite this structural flexibility, the affinity of this mutant to bind GSH has increased dramatically as well. The corresponding density of the substrate was clearly visible in the electron density maps and could be refined to full occupancy.
The above figures are reprinted from an Open Access publication published by the Protein Society: Protein Sci (2009, 18, 217-228) copyright 2009.
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