Crystallization and preliminary crystallographic analysis of the global nitrogen regulator AmtR fromCorynebacterium glutamicum

Abstract

AmtR, a member of the TetR family of transcription regulators, is a global regulator of nitrogen control in Corynebacterium glutamicum. Unlike other TetR-family members, which are regulated by small-molecule effectors, AmtR is regulated by a protein called GlnK. It has been shown that a GlnK trimer has to become adenylylated prior to formation of a complex with AmtR. The physiological function of AmtR has been very well studied, but structural characterization of the mechanistic aspects of AmtR-regulated transcription has yet to be accomplished. AmtR has successfully been crystallized in space group P2(1)2(1)2, with six molecules in the asymmetric unit and unit-cell parameters a = 153.34, b = 163.10, c = 51.93 angstrom . Preliminary phases were obtained using Se-SAD.

Common patterns – unique features: nitrogen metabolism and regulation in Gram-positive bacteria

“…Instead, binding to promoters of nitrogen‐dependent genes is regulated by a specific protein–protein interaction: at decreasing ammonium concentrations, AmtR is released from the DNA by interaction with the P II ‐type signal transduction protein GlnK (Beckers et al ., 2005). To elucidate the interaction domains, which are still unclear, crystallization of AmtR (Hasselt et al ., 2009) and GlnK is in progress. However, it is known that as a prerequisite for this interaction, the trimeric GlnK complex has to be adenylylated at tyrosyl residue 51 located in the T‐loop of the protein.…”

Adaptation of AmtR-controlled gene expression by modulation of AmtR binding activity in Corynebacterium glutamicum

“…For corynebacteria, the TetR family protein AmtR was identified as master regulator of nitrogen regulation on the transcriptional level (Walter et al, 2007). The AmtR protein and AmtR-dependent regulation were most intensively studied in the biotechnologically important amino acid producer Corynebacterium glutamicum (Jakoby et al, 2000;Beckers et al, 2005;Buchinger et al, 2009;Hasselt et al, 2009;Muhl et al, 2009). By a combination of bioinformatic tools, transcriptome and proteome analyses as well as DNA binding studies we could show that 35 genes are directly regulated by AmtR (Beckers et al, 2005;Buchinger et al, 2009).…”

Similarities in the structure of the transcriptional repressor AmtR in two different space groups suggest a model for the interaction with GlnK

“…In a previous report from our group, the crystallization of AmtR, data collection from native and selenomethioninederivatized crystals as well as the phasing of these crystals belonging to space group P2 1 2 1 2 using the Se-SAD method was reported (Hasselt et al, 2009). Here, the structure of AmtR that was obtained after model building using the aboveobtained phases and following refinement against a newly collected native data set that extends to 2.1 Å resolution is described.…”