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

Ángela Rodríguez-Cárdenas,Adriana L Rojas,Ramón Hurtado-Guerrero,Adrián Velázquez-Campoy,María Conde-Giménez,Javier Sancho,Bostjan Kobe

doi:10.1371/journal.pone.0161020

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.

Highlights

Streptococcus pneumoniae is a gram-positive bacterium that causes pneumonia, meningitis, otitis media, acute sinusitis, septicemia, and conjunctivitis, among several other diseases [1,2,3,4]
In this work we provide a detailed biophysical characterization of SpFld and report the x-ray structures of the apo and holo forms, the conformational stability of the protein and the thermodynamic characterization of the cofactor binding equilibrium
The UV-visible spectrum of apo SpFld shows a single peak with maximal absorbance at 277 nm (Fig 1a) and a completely flat spectrum beyond 350 nm, indicative of both a full removal of the cofactor and a lack of protein aggregation phenomena

Summary

Introduction

Streptococcus pneumoniae is a gram-positive bacterium that causes pneumonia, meningitis, otitis media, acute sinusitis, septicemia, and conjunctivitis, among several other diseases [1,2,3,4]. In this work we provide a detailed biophysical characterization of SpFld and report the x-ray structures of the apo and holo forms, the conformational stability of the protein and the thermodynamic characterization of the cofactor binding equilibrium. The fluorescence emission spectra of holo SpFld (Fig 1c) displays characteristic peaks of tyrosine residues and of the FMN cofactor.

Results

Conclusion