Structural analysis of the purine repressor, an Escherichia coli DNA-binding protein

Purine repressor (PurR) binding to specific DNA is enhanced by complexing with purines, whereas lactose repressor (LacI) binding is diminished by interaction with inducer sugars despite 30% identity in their protein sequences and highly homologous tertiary structures. Nonetheless, in switching from low- to high-affinity DNA binding, these proteins undergo a similar structural change in which the hinge region connecting the DNA and effector binding domains folds into an alpha-helix and contacts the DNA minor groove. The differences in response to effector for these proteins should be manifest in the polyelectrolyte effect which arises from cations displaced from DNA by interaction with positively charged side chains on a protein and is quantitated by measurement of DNA binding affinity as a function of ion concentration. Consistent with structural data for these proteins, high-affinity operator DNA binding by the PurR-purine complex involved approximately 15 ion pairs, a value significantly greater than that for the corresponding state of LacI (approximately 6 ion pairs). For both proteins, however, conversion to the low-affinity state results in a decrease of approximately 2-fold in the number of cations released per dimeric DNA binding site. Heat capacity changes (DeltaC(p)) that accompany DNA binding, derived from buried apolar surface area, coupled folding, and restriction of motional freedom of polar groups in the interface, also reflect the differences between these homologous repressor proteins. DNA binding of the PurR-guanine complex is accompanied by a DeltaC(p) (-2.8 kcal mol(-1) K(-1)) more negative than that observed previously for LacI (-0.9 to -1.5 kcal mol(-1) K(-1)), suggesting that more extensive protein folding and/or enhanced structural rigidity may occur upon DNA binding for PurR compared to DNA binding for LacI. The differences between these proteins illustrate plasticity of function despite high-level sequence and structural homology and undermine efforts to predict protein behavior on the basis of such similarities.

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confidence: 74%

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confidence: 99%

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confidence: 99%

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Ion Concentration and Temperature Dependence of DNA Binding: Comparison of PurR and LacI Repressor Proteins

Moraitis

Matthews

2001

“…Crystallographic studies were undertaken to reveal the stereochemical and structural parameters that contribute to the biological and biochemical functions of each mutant. In initial studies on corepressor binding to PurR, residue W147 was hypothesized to interact with the guanine or hypoxanthine corepressor directly because its mutation to a phenylalanine resulted in weak in vivo repression and lower affinity for hypoxanthine as corepressor ( , ). Subsequently, the crystal structures of PurR in its holo and apo forms revealed that W147 is involved, but surprisingly this residue makes no direct contacts to the corepressor ( , ).…”

Section: Discussionmentioning

confidence: 99%

Role of Residue 147 in the Gene Regulatory Function of the Escherichia coli Purine Repressor

Huffman

Zalkin

et al. 2001

The crystal structures of corepressor-bound and free Escherichia coli purine repressor (PurR) have delineated the roles of several residues in corepressor binding and specificity and the intramolecular signal transduction (allosterism) of this LacI/GalR family member. From these structures, residue W147 was implicated as a key component of the allosteric response, but in many members of the LacI/GalR family, position 147 is occupied by an arginine. To understand the role of this tryptophan at position 147, three proteins, substituted by phenylalanine (W147F), alanine (W147A), or arginine (W147R), were constructed and characterized in vivo and in vitro, and their structures were determined. W147F displays a decreased affinity for corepressor and is a poor repressor in vivo. W147A and W147R, on the other hand, are super repressors and bind corepressor 13.6 and 7.9 times more tightly, respectively, than wild-type. Each mutant PurR-hypoxanthine-purF operator holo complex crystallizes isomorphously to wild-type. Whereas the apo corepressor binding domain (CBD) of W147F crystallizes under those conditions used for the wild-type protein, neither the apo CBD of W147R nor W147A crystallizes, although screened extensively for new crystal forms. Structures of the holo repressor mutants have been solved to resolutions between 2.5 and 2.9 A, and the structure of the apo CBD of W147F has been solved to 2.4 A resolution. These structures provide insight into the altered biochemical properties and physiological functions of these mutants, which appear to depend on the sometimes subtle preference for one conformation (apo vs holo) over the other.

“…On the basis of these data, we propose that Asp274 is located at the base of the inducer binding cleft in a region essential for either direct inducer binding and/or closure of the binding pocket. It is interesting to note that, in the alignments of ligand binding bacterial regulators (Weickert & Adhya, 1992;Mauzy & Hermodson, 1992;Schumacher et al, 1993), aspartate in the position corresponding to 274 in lac repressor is highly conserved. It is possible that this site is critical to structural shifts in these proteins and that any interruption of interactions in this region may affect sugar binding and/or the conformational change associated with ligand binding.…”

Section: Discussionmentioning

confidence: 99%

Identification and characterization of aspartate residues that play key roles in the allosteric regulation of a transcription factor: aspartate 274 is essential for inducer binding in lac repressor

Chang¹,

Barrera²,

Matthews³

1994

To explore the roles of three aspartate residues, Asp88, Asp130, and Asp274, found in the proposed inducer binding site of lac repressor [Sams, C. F., Vyas, N. K., Quiocho, F. A., & Matthews, K. S. (1984) Nature 310, 429-430], each site was substituted with alanine, glutamate, lysine, or asparagine by site-specific mutagenesis. The mutations at the Asp88 site resulted in a 5-13-fold decrease in inducer binding affinity, largely due to an increase in the inducer dissociation rate constants for these mutants. In addition, the mutant proteins Asp88-->Ala and Asp88-->Lys exhibited altered allosteric behavior for inducer binding. These data conflict with the original hypothesis placing Asp88 in the inducer binding site, but are in agreement with a recent model that places this amino acid close to the subunit interface involved in cooperativity associated with inducer binding [Nichols, J. C., Vyas, N. K., Quiocho, F. A., & Matthews, K. S. (1993) J. Biol. Chem. 268, 17602-17612; Chen, J., & Matthews, K. S. (1992) J. Biol. Chem. 267, 13843-13850]. Substitution at Asp130 did not alter the inducer binding affinity nor other binding activities. Thus, this amino acid is not crucial in the binding to beta-substituted monosaccharides or in protein function. In stark contrast, all mutant proteins with substitutions at the Asp274 site exhibited no detectable inducer binding. With the exception of Asp274-->Lys, the structures of these mutant proteins appear to be similar to wild-type. The data demonstrate that Asp274 plays a crucial role in inducer binding of this transcriptional regulator.