Streptococcus pneumoniae TIGR4 Flavodoxin: Structural and Biophysical Characterization of a Novel Drug Target

PLoS One. 2016 Sep 20;11(9):e0161020. doi: 10.1371/journal.pone.0161020. eCollection 2016.

Abstract

Streptococcus pneumoniae (Sp) strain TIGR4 is a virulent, encapsulated serotype that causes bacteremia, otitis media, meningitis and pneumonia. Increased bacterial resistance and limited efficacy of the available vaccine to some serotypes complicate the treatment of diseases associated to this microorganism. Flavodoxins are bacterial proteins involved in several important metabolic pathways. The Sp flavodoxin (Spfld) gene was recently reported to be essential for the establishment of meningitis in a rat model, which makes SpFld a potential drug target. To facilitate future pharmacological studies, we have cloned and expressed SpFld in E. coli and we have performed an extensive structural and biochemical characterization of both the apo form and its active complex with the FMN cofactor. SpFld is a short-chain flavodoxin containing 146 residues. Unlike the well-characterized long-chain apoflavodoxins, the Sp apoprotein displays a simple two-state thermal unfolding equilibrium and binds FMN with moderate affinity. The X-ray structures of the apo and holo forms of SpFld differ at the FMN binding site, where substantial rearrangement of residues at the 91-100 loop occurs to permit cofactor binding. This work will set up the basis for future studies aiming at discovering new potential drugs to treat S. pneumoniae diseases through the inhibition of SpFld.

MeSH terms

  • Apoproteins / chemistry*
  • Apoproteins / genetics
  • Apoproteins / metabolism*
  • Cloning, Molecular
  • Crystallography, X-Ray
  • Flavin Mononucleotide / metabolism*
  • Flavodoxin / chemistry*
  • Flavodoxin / genetics
  • Flavodoxin / metabolism*
  • Humans
  • Models, Molecular
  • Pneumococcal Infections / microbiology
  • Protein Binding
  • Protein Conformation
  • Protein Stability
  • Protein Unfolding
  • Streptococcus pneumoniae / chemistry
  • Streptococcus pneumoniae / genetics
  • Streptococcus pneumoniae / metabolism*
  • Temperature

Substances

  • Apoproteins
  • Flavodoxin
  • apoflavodoxin
  • Flavin Mononucleotide

Grants and funding

We acknowledge financial support from BFU2010-16297 and BFU2010-19504 [Ministerio de Ciencia e Innovación Spain], BFU2013-47064-P, BIO2014-57314-REDT and CTQ2013-44367-C2-2-P [Ministerio de Economía y Competitividad, Spain], and DGA (Protein Targets B89). We also thank synchrotron radiation sources DLS (Oxford), and in particular beamline I04-1 (experiment number MX8035-3 and MX8035-11). The research leading to these results has also received funding from the FP7 (2007-2013) under BIOSTRUCTX-7687. A.R.C. was funded by a Banco Santander Central Hispano/Universidad de Zaragoza predoctoral fellowship. M. C-G was recipient of a predoctoral fellowship from the Government of Aragón. We thank Dr. Ana Sánchez for help with the dithionite titration.