Sensor Domain of the Mycobacterium tuberculosis Receptor Ser/Thr Protein Kinase, PknD, forms a Highly Symmetric β Propeller

https://doi.org/10.1016/j.jmb.2004.03.063Get rights and content

Abstract

Diverse pathogenic bacteria produce transmembrane receptor Ser/Thr protein kinases (STPKs), but little is known about the signals mediated by these “eukaryotic-like” proteins. To explore the basis for signaling in the bacterial STPK receptor family, we determined the structure of the sensor domain of Mycobacterium tuberculosis PknD. In two crystal forms, the PknD sensor domain forms a rigid, six-bladed β-propeller with a flexible tether to the transmembrane domain. The PknD sensor domain is the most symmetric β-propeller structure described. All residues that vary most among the blade subdomains cluster in the large “cup” motif, analogous to the ligand-binding surface in many β-propeller proteins. These results suggest that PknD binds a multivalent ligand that signals by changing the quaternary structure of the intracellular kinase domain.

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

References (65)

  • T.A. Springer

    An extracellular β-propeller module predicted in lipoprotein and scavenger receptors, tyrosine kinases, epidermal growth factor precursor, and extracellular matrix components

    J. Mol. Biol.

    (1998)
  • L. Holm et al.

    Protein structure comparison by alignment of distance matrices

    J. Mol. Biol.

    (1993)
  • E.I. Scharff et al.

    Crystal structure of diisopropylfluorophosphatase from Loligo vulgaris

    Structure

    (2001)
  • A. Gaskell et al.

    The three domains of a bacterial sialidase: a β-propeller, an immunoglobulin module and a galactose-binding jelly-roll

    Structure

    (1995)
  • P.A. Norton et al.

    sevenless: Seven found?

    Cell

    (1990)
  • S.J. Crennell

    The structures of Salmonella typhimurium LT2 neuraminidase and its complexes with three inhibitors at high resolution

    J. Mol. Biol.

    (1996)
  • M.C. Lawrence

    Structure of the haemagglutinin-neuraminidase from human parainfluenza virus type III

    J. Mol. Biol.

    (2004)
  • G.D. Van Duyne et al.

    Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin

    J. Mol. Biol.

    (1993)
  • F. Jeanmougin et al.

    Multiple sequence alignment with Clustal X

    Trends Biochem. Sci.

    (1998)
  • V. Fulop et al.

    The anatomy of a bifunctional enzyme: structural basis for reduction of oxygen to water and synthesis of nitric oxide by cytochrome cd1

    Cell

    (1995)
  • D.G. Russell

    Mycobacterium tuberculosis: here today, and here tomorrow

    Nature Rev. Mol. Cell. Biol.

    (2001)
  • S.T. Cole

    Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence

    Nature

    (1998)
  • Y. Av-Gay et al.

    Expression and characterization of the Mycobacterium tuberculosis serine/threonine protein kinase PknB

    Infect. Immun.

    (1999)
  • R. Chaba et al.

    Evidence that a eukaryotic-type serine/threonine protein kinase from Mycobacterium tuberculosis regulates morphological changes associated with cell division

    Eur. J. Biochem.

    (2002)
  • A. Koul

    Serine/threonine protein kinases PknF and PknG of Mycobacterium tuberculosis: characterization and localization

    Microbiology

    (2001)
  • P. Peirs et al.

    A serine/threonine protein kinase from Mycobacterium tuberculosis

    Eur. J. Biochem.

    (1997)
  • E. Madec et al.

    Characterization of a membrane-linked Ser/Thr protein kinase in Bacillus subtilis, implicated in developmental processes

    Mol. Microbiol.

    (2002)
  • B. Boitel

    PknB kinase activity is regulated by phosphorylation in two Thr residues and dephosphorylation by PstP, the cognate phospho-Ser/Thr phosphatase, in Mycobacterium tuberculosis

    Mol. Microbiol.

    (2003)
  • T.A. Young et al.

    Structure of Mycobacterium tuberculosis PknB supports a universal activation mechanism for Ser/Thr protein kinases

    Nature Struct. Biol.

    (2003)
  • L.A. Kelley et al.

    Enhanced genome annotation using structural profiles in the program 3D-PSSM

    J. Mol. Biol.

    (2000)
  • D.P. Sun et al.

    Use of site-directed mutagenesis to obtain isomorphous heavy-atom derivatives for protein crystallography: cysteine-containing mutants of phage T4 lysozyme

    Protein Eng.

    (1987)
  • J.N. Varghese et al.

    Structure of the influenza virus glycoprotein antigen neuraminidase at 2.9 Å resolution

    Nature

    (1983)
  • Cited by (61)

    • Resurrecting the Dead (Molecules)

      2017, Computational and Structural Biotechnology Journal
      Citation Excerpt :

      An intriguing example of a hybrid approach comes from the work of Voet et al. [98], summarised in Fig. 2. They took a domain of protein kinase from Mycobacterium tuberculosis (NHL repeat structure, PDB entry 1RWL [99]) which forms a β-propeller domain made from six highly similar but not identical “blades”. The number of blades in a β-propeller protein varies depending on the protein but all blades are similar, consisting of a short sequence forming a β-sheet [100,101] with 1RWL consisting of six such blades.

    • Structural and genetic analyses of the mycobacterium tuberculosis protein kinase B sensor domain identify a potential ligand-binding site

      2016, Journal of Biological Chemistry
      Citation Excerpt :

      Residue Val-360 likely defines the beginning of the folded ECD, which is thus likely to be loosely tethered to the membrane by a six-residue peptide with amino acid sequence GGITRD. Similar arrangement of a well folded ECD tethered by a flexible linker were found during our prior investigations of PknD and PknH extracellular domains (18, 19), suggesting that ligand binding controls localization and oligomerization of the kinase domains in the membrane rather than directly signaling via conformational changes induced across the cytoplasmic membrane. The PknB sensor domain comprises four PASTA repeats, the last of which, PASTA4, contains a hydrophobic groove with conserved partially exposed aromatic residues, a signature of a ligand-binding site.

    View all citing articles on Scopus

    Present address: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143-2240, USA.

    View full text