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

Abstract

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.

Automated selection of positions determining functional specificity of proteins by comparative analysis of orthologous groups in protein families

“…It was predicted to be specificity determining in Mirny and Gelfand (2002). Although it does not contact guanine in the structure of PurR, this residue is essential for binding the corepressor (Huffman et al 2002). Notably, this position is variable in four specificity groups.…”

Structure and Function of the Arginine Repressor-Operator Complex from Bacillus subtilis

“…Analysis with SHELXC, 30 however, showed no significant anomalous signal. Despite this, the structure was solved by molecular replacement using a model based on the DNA-binding domainoperator complex of the E. coli purine repressor 31 (PDB code 1JFS), which lacks a β-wing. The BrNAhrC-DNA crystal has one full complex and one half-complex in the asymmetric unit.…”

Structural and functional analyses of the cellulase transcription regulator CelR

“…The crystal structure was determined by molecular replacement using the PhaserMR program from the CCP4 program suite . Escherichia coli purine repressor, PurR (PDB ID http://www.rcsb.org/pdb/search/structidSearch.do?structureId=1JH9) was employed as the search model for CelR . Crystallographic refinement was performed using PHENIX and model building was performed using COOT .…”