Abstract
Transfer of the tumor-inducing plasmid in Agrobacterium tumefaciens is controlled by a quorum-sensing system whose main components are the transcriptional regulator TraR and its autoinducer. This system allows bacteria to synchronize infection of the host plant when a "quorum" of cells has been reached. TraM is an A. tumefaciens protein involved in the regulation of this system because it binds to TraR and prevents it from binding DNA. As a first step to understanding the molecular basis for the regulation of TraR by TraM, we have determined the crystal structure of TraM at 1.65 A resolution. This protein is packed as a dimer, with each monomer consisting mainly of two antiparallel alpha helices. Monomers are tightly associated, with a large hydrophobic area buried upon dimerization. Secondly, we characterized the TraR-TraM complex in vitro. TraM (11.4 kDa, monomer molecular mass) binds tightly TraR (27 kDa, monomer molecular mass) forming a stable oligomeric complex that likely accounts for two TraR and two TraM dimers.
Highlights
Transfer of the tumor-inducing plasmid in Agrobacterium tumefaciens is controlled by a quorum-sensing system whose main components are the transcriptional regulator TraR and its autoinducer
TraM is an A. tumefaciens protein involved in the regulation of this system because it binds to TraR and prevents it from binding DNA
Quorum-sensing is a term that reflects the ability of bacteria to control the expression of specific operons in a cell densitydependent manner and is based on the production, release, and “sensing” of small signal molecules called autoinducers that accumulate in the environment as a function of cell density
Summary
Protein Production, Crystallization, and Data Collection—TraR and TraM were purified from Escherichia coli as described previously [15, 16]. TraM crystallization has been described elsewhere [16]. Data from TraM crystals were collected at 100 K using synchrotron radiation at the beamline ID29, European Synchrotron Radiation Facility (ESRF), Grenoble. Heavy atom refinement and density modification were carried out at 2.0 Å resolution, using multiwavelength anomalous diffraction data from the peak and the remote (as the reference wavelength), in CNX (Accelrys; Pharmacopeia Inc.). The model was fed into Arp/Warp (modality WarpNTrace) [20] to combine model building with iterative. This paper is available on line at http://www.jbc.org
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