Crystal Structure of PhnF, a GntR-Family Transcriptional Regulator of Phosphate Transport in Mycobacterium smegmatis

Abstract

dBacterial uptake of phosphate is usually accomplished via high-affinity transporters that are commonly regulated by two-component systems, which are activated when the concentration of phosphate is low. Mycobacterium smegmatis possesses two such transporters, the widely distributed PstSCAB system and PhnDCE, a transporter that in other bacteria mediates the uptake of alternative phosphorus sources. We previously reported that the transcriptional regulator PhnF controls the production of the Phn system, acting as a repressor under high-phosphate conditions. Here we show that the phnDCE genes are common among environmental mycobacteria, where they are often associated with phnF-like genes. In contrast, pathogenic mycobacteria were not found to encode Phn-like systems but instead were found to possess multiple copies of the pst genes. A detailed biochemical analysis of PhnF binding to its identified binding sites in the phnD-phnF intergenic region of M. smegmatis has allowed us to propose a quantitative model for repressor binding, which shows that a PhnF dimer binds independently to each site. We present the crystal structure of M. smegmatis PhnF at 1.8-Å resolution, showing a homodimer with a helix-turn-helix N-terminal domain and a C-terminal domain with a UbiC transcription regulator-associated fold. The C-terminal domain crystallized with a bound sulfate ion instead of the so far unidentified physiological ligand, allowing the identification of residues involved in effector binding. Comparison of the positioning of the DNA binding domains in PhnF with that in homologous proteins suggests that its DNA binding activity is regulated via a conformational change in the linker region, triggering a movement of the N-terminal domains.

Allosteric control of transcription in GntR family of transcription regulators: A structural overview

“…The members of this subfamily have been implicated in processes like antibiotic production and as sensors, of the nutritional status in the environment. The crystal structure of one of the member of this family, PhnF from Mycobacterium smegmatis as well as of PhnF‐CTD has been determined upto 1.8 Å and 1.7 Å resolution respectively. PhnF regulates the bio‐degradation of phosphonates, (Pn)‐compounds having CP bonds and is predicted to interact with alkylphosphonates.…”

Crystal Structures of the Global Regulator DasR from Streptomyces coelicolor: Implications for the Allosteric Regulation of GntR/HutC Repressors

“…Although numerous crystal structures of individual EBDs of GntR/HutC family members are available from the protein data bank, only a few of these describe full-length repressors comprising both the DNA- and effector-binding domain [ 17 ]. Besides full-length PhnF from Mycobacterium smegmatis [ 18 ] and NagR (previously known as YvoA) from Bacillus subtilis [ 11 , 16 ], full-length crystal structures are available for YydK from B . subtilis (PDB-ID 3BWG; UniProtKB Q45591) and for an unnamed regulator from Streptomyces avermitilis (PDB-ID 3EET; UniProtKB Q82IF8) [ 17 , 19 ].…”

“…Only in the case of the GntR/HutC family members PhnF and NagR it was so far possible to relate observations from multiple crystal structures to different functional states [ 11 , 16 , 18 ]. The crystal structure of full-length NagR from B .…”

“…subtilis with sulphate molecules bound in the effector-binding site was determined first followed by sulphate-bound PhnF from M . smegmatis and by crystal structures of NagR bound to an idealised operator DNA sequence as well as of NagR in complex with the putative effector molecules glucosamine-6-phosphate (GlcN-6-P) and N -acetylglucosamine-6-phosphate (GlcNAc-6-P) [ 11 , 16 , 18 ]. The structures are consistent with a model wherein ligand binding to PhnF and NagR repositions two particular α-helices in the EBDs and at the same time stabilises segments that appear to display high flexibility in the absence of ligands [ 11 , 15 , 18 ].…”

“…smegmatis and by crystal structures of NagR bound to an idealised operator DNA sequence as well as of NagR in complex with the putative effector molecules glucosamine-6-phosphate (GlcN-6-P) and N -acetylglucosamine-6-phosphate (GlcNAc-6-P) [ 11 , 16 , 18 ]. The structures are consistent with a model wherein ligand binding to PhnF and NagR repositions two particular α-helices in the EBDs and at the same time stabilises segments that appear to display high flexibility in the absence of ligands [ 11 , 15 , 18 ]. A comparison between the DNA-bound and the sugar-bound structures of NagR suggested that a so-called ‘jumping jack’-like movement of the DBDs characterises the allosteric mechanism that modulates the DNA-binding affinity in NagR [ 11 , 16 ].…”