Research Article: Structure of a quinolone-stabilized cleavage complex of topoisomerase IV from Klebsiella pneumoniae and comparison with a related Streptococcus pneumoniae complex

Date Published: April 01, 2016

Publisher: International Union of Crystallography

Author(s): Dennis A. Veselkov, Ivan Laponogov, Xiao-Su Pan, Jogitha Selvarajah, Galyna B. Skamrova, Arthur Branstrom, Jana Narasimhan, Josyula V. N. Vara Prasad, L. Mark Fisher, Mark R. Sanderson.


Crystal structures of the cleavage complexes of topoisomerase IV from Gram-negative (K. pneumoniae) and Gram-positive (S. pneumoniae) bacterial pathogens stabilized by the clinically important antibacterial drug levofloxacin are presented, analysed and compared. For K. pneumoniae, this is the first high-resolution cleavage complex structure to be reported.

Partial Text

Klebsiella is a genus belonging to the Enterobacteriaceae family of Gram-negative bacilli, which is divided into seven species with demonstrated similarities in DNA homology: K. pneumoniae, K. ozaenae, K. rhinoscleromatis, K. oxytoca, K. planticola, K. terrigena and K. ornithinolytica. K. pneumoniae is the most medically important species of the genus owing to its high resistance to antibiotics. Significant morbidity and mortality has been associated with an emerging, highly drug-resistant strain of K. pneumoniae characterized as producing the carbapenemase enzyme (KPC-producing bacteria; Nordmann et al., 2009 ▸). The best therapeutic approach to KPC-producing organisms has yet to be defined. However, common treatments (based on in vitro susceptibility testing) are the polymyxins, tigecycline and, less frequently, aminoglycoside antibiotics (Arnold et al., 2011 ▸). Another effective strategy involves the limited use of certain antimicrobials, specifically fluoroquinolones and cephalo­sporins (Gasink et al., 2009 ▸). Several new antibiotics are under development for KPC producers. These include combinations of existing β-lactam antibiotics with new β-lactamase inhibitors able to circumvent KPC resistance. Neoglycosides are novel aminoglycosides that have activity against KPC-producing bacteria that are also being developed (Chen et al., 2012 ▸).

We have co-crystallized the K. pneumoniae topoisomerase IV ParC/ParE breakage-reunion domain (ParC55; residues 1–490) and ParE TOPRIM domain (ParE30; residues 390–631) with a precut 34 bp DNA duplex (the E-site), stabilized by levofloxacin. The X-ray crystal structure of the complex was determined to 3.35 Å resolution, revealing a closed ParC55 dimer flanked by two ParE30 monomers (Figs. 1 ▸, 2 ▸ and 3 ▸). The overall architecture of this complex is similar to that found for S. pneumoniae topoisomerase–DNA–drug complexes (Laponogov et al., 2009 ▸, 2010 ▸). Residues 6–30 of the N-terminal α-helix α1 of the ParC subunit again embrace the ParE subunit, ‘hugging’ the ParE subunits close to either side of the ParC dimer (Laponogov et al., 2010 ▸). Deletion of this ‘arm’ α1 results in loss of DNA-cleavage activity (Laponogov et al., 2007 ▸) and is clearly very important in complex stability (Fig. 3 ▸). This structural feature was absent in our original ParC55 structure (Laponogov et al., 2007 ▸; Sohi et al., 2008 ▸). The upper region of the topoisomerase complex consists of the E-subunit TOPRIM metal-binding domain formed of four parallel β-sheets and the surrounding α-helices. The C-subunit provides the WHD (winged-helix domain; a CAP-like structure; McKay & Steitz, 1981 ▸) and the ‘tower’ which form the U groove-shaped protein region into which the G-gate DNA binds with an induced U-shaped bend. The lower C-gate region (Fig. 3 ▸) consists of the same disposition of pairs of two long α-helices terminated by a spanning short α-helix forming a 30 Å wide DNA-accommodating cavity through which the T-gate DNA passes as found in the S. pneumoniae complex. Owing to the structural similarity, it appears that the topo­isomerases IV from K. pneumoniae and S. pneumoniae are likely to follow a similar overall topoisomerase catalytic cycle as shown in Fig. 4 ▸; we have confirmation of one intermediate from our recent structure of the full complex (the holoenzyme less the CTD β-pinwheel domain) with the ATPase domain in the open conformation (Laponogov et al., 2013 ▸).




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