Journal of Molecular Biology
Sensor Domain of the Mycobacterium tuberculosis Receptor Ser/Thr Protein Kinase, PknD, forms a Highly Symmetric β Propeller
Section snippets
Results
To characterize the sensor domain, we expressed and purified Mtb PknD residues 403–664. This sequence starts at the first charged residue following the predicted transmembrane helix. The expressed protein included a C-terminal His6 tag that was not removed. This construct crystallized in two forms (Table 1). Because no suitable heavy atom derivatives of the wild-type protein were identified, we created five mutants that introduced a single cysteine, as well as a mutant that contained two
Discussion
As predicted,15 the sensor domain of PknD forms a β propeller, a widespread motif found in numerous proteins (mostly in eukaryotes) with diverse functions.20 First reported in the structure of the influenza virus neuraminidase,21 β propellers have been described containing 4–8 cyclically arranged blades, each with four β strands. In some β-propeller structures, additional β strands occur on the outside of individual blades, or unrelated globular domains replace one or more blades.22 With the
Cloning
An expression vector for PknD residues 403–664 followed by the C-terminal His6 tag, LEHHHHHH, was created with pET24b (Novagen) using sticky-end PCR47 as described.12 Mutagenesis was carried out with the QuikChange kit (Stratagene). Constructs and mutants were confirmed by DNA sequencing.
Expression and purification
The wild-type and mutant PknD sensor domains were expressed at 25 °C in BL21(DE3) Codon Plus cells (Stratagene) as described.12 The cells were harvested by centrifugation and resuspended in 50 mM Tris–HCl (pH
Acknowledgements
We thank David S. King for protein mass spectrometry measurements, and James Holton, Emmanuel Skordalakes, and Benedicte Delagoutte for help with X-ray structure determinations. We thank Kristi Pullen, Laurie Gay, and Danielle Tomkiel for stimulating discussions. We are grateful to Tom Terwilliger and the TB Structural Genomics Consortium for support. This work was supported by a grant from the NIH. The Advanced Light Source Beamline 8.3.1 was funded by the NSF, the University of California and
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Present address: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143-2240, USA.