Structural Basis of Gate-DNA Breakage and Resealing by Type II Topoisomerases

Type II DNA topoisomerases are ubiquitous enzymes with essential functions in DNA replication, recombination and transcription. They change DNA topology by forming a transient covalent cleavage complex with a gate-DNA duplex that allows transport of a second duplex though the gate. Despite its biological importance and targeting by anticancer and antibacterial drugs, cleavage complex formation and reversal is not understood for any type II enzyme. To address the mechanism, we have used X-ray crystallography to study sequential states in the formation and reversal of a DNA cleavage complex by topoisomerase IV from Streptococcus pneumoniae, the bacterial type II enzyme involved in chromosome segregation. A high resolution structure of the complex captured by a novel antibacterial dione reveals two drug molecules intercalated at a cleaved B-form DNA gate and anchored by drug-specific protein contacts. Dione release generated drug-free cleaved and resealed DNA complexes in which the DNA gate instead adopts an unusual A/B-form helical conformation with a Mg2+ ion repositioned to coordinate each scissile phosphodiester group and promote reversible cleavage by active-site tyrosines. These structures, the first for putative reaction intermediates of a type II topoisomerase, suggest how a type II enzyme reseals DNA during its normal reaction cycle and illuminate aspects of drug arrest important for the development of new topoisomerase-targeting therapeutics

The SOCS-Box of HIV-1 Vif Interacts with ElonginBC by Induced-Folding to Recruit Its Cul5-Containing Ubiquitin Ligase Complex

The HIV-1 viral infectivity factor (Vif) protein recruits an E3 ubiquitin ligase complex, comprising the cellular proteins elongin B and C (EloBC), cullin 5 (Cul5) and RING-box 2 (Rbx2), to the anti-viral proteins APOBEC3G (A3G) and APOBEC3F (A3F) and induces their polyubiquitination and proteasomal degradation. In this study, we used purified proteins and direct in vitro binding assays, isothermal titration calorimetry and NMR spectroscopy to describe the molecular mechanism for assembly of the Vif-EloBC ternary complex. We demonstrate that Vif binds to EloBC in two locations, and that both interactions induce structural changes in the SOCS box of Vif as well as EloBC. In particular, in addition to the previously established binding of Vif's BC box to EloC, we report a novel interaction between the conserved Pro-Pro-Leu-Pro motif of Vif and the C-terminal domain of EloB. Using cell-based assays, we further show that this interaction is necessary for the formation of a functional ligase complex, thus establishing a role of this motif. We conclude that HIV-1 Vif engages EloBC via an induced-folding mechanism that does not require additional co-factors, and speculate that these features distinguish Vif from other EloBC specificity factors such as cellular SOCS proteins, and may enhance the prospects of obtaining therapeutic inhibitors of Vif function

Crystals tested for diffraction in-house.

<p>Crystals tested for diffraction in-house.</p

Orthogonal views of the ParC55 biological dimer from <i>Streptococcus pneumoniae</i>.

<p>(A) Cartoon representation. The ‘towers’ and the CAP-like domains are shown in ice blue; the ‘tails’ along with adjacent helices α14, α18 and α19 are in ochre; the helix α4 in red; the helix α3 in cyan; the 100–122 loop in yellow. The active-site tyrosines are shown in green. Residues responsible for drug-resistance upon mutation are in purple. (B) Electrostatic surface representation. The negatively charged regions are in red and positively charged regions are in blue. Panels were rendered using VMD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003201#pone.0003201-Humphrey1" target="_blank">[27]</a>, Pov-Ray and PyMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003201#pone.0003201-DeLano1" target="_blank">[28]</a>.</p

The crystal forms.

<p>(A) Orthorhombic crystal of Cys426 grown from 8% PEG 20,000, 200 mM sodium chloride, 100 mM Tris-HCl, pH 5.0, 0.1% sodium azide. (B) Hexagonal crystal of ParC55 grown from 8% PEG 20,000, 200 mM sodium chloride, 100 mM Tris-HCl, pH 7.0, 0.1% sodium azide.</p

Figure 2

<p>Structure of the ParC55 dimer and illustration of structurally related <i>E. coli</i> ParC and GyrA proteins. (A) Orthogonal views of the ParC55 biological dimer from <i>S. pneumoniae</i>. (B) Structure of the N-terminal region of <i>E. coli</i> ParC (18) (1ZVU) equivalent to ParC55 fragment. (C) Structure of the N-terminal fragment (GyrA59) of <i>E. coli</i> GyrA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000301#pone.0000301-MoraisCabral1" target="_blank">[19]</a> (1AB4). In (A), (B) and (C) the ‘towers’ and the CAP-like domains are shown in ice blue; the ‘tails’ along with adjacent helices α14, α18 and α19 are in ochre; the helix α4 in red; the helix α3 in cyan and the 100-122 loop in yellow. The active-site tyrosines are shown in green. Residues Ser 79 and Asp 83 responsible for drug-resistance upon mutation are in purple. (D) Schematic conversion of ParC55 from ‘closed’ (red) to ‘open’ (blue) conformation on the basis of the <i>E. coli</i> ParC structure. Panels were rendered using VMD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000301#pone.0000301-Humphrey1" target="_blank">[50]</a> and Pov-Ray.</p

Figure 3

<p>Structural comparison between active sites of topoisomerases from gram-positive and gram-negative bacteria. (A) Stereo view of the superposition of the active site of ParC from <i>S. pneumoniae</i> (red) and ParC from <i>E. coli</i> (violet). (B) Stereo view of the superposition of the active sites of ParC from <i>S. pneumoniae</i> (red) and GyrA from <i>E. coli</i> (blue). The active-site tyrosines and arginines are represented in CPK mode. Sites responsible for drug-resistance when mutated are represented in Licorice mode. Panels were rendered using VMD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000301#pone.0000301-Humphrey1" target="_blank">[50]</a> and Pov-Ray.</p

Figure 1

<p>Comparison of type II topoisomerases from <i>S. pneumoniae</i> and <i>E. coli</i>. Schematic domain organization of (A) <i>S. pneumoniae</i> topoisomerase IV, (B) <i>S. pneumoniae</i> gyrase (with the proteolytic fragments generated by trypsin digestion superimposed) and (C) graphical representation of the sequence similarity scores for 500-residue N-terminal fragments of ParC (P72525) and GyrA (P72524) from <i>S. pneumoniae</i> and equivalent fragments of ParC (P0AFI2) and GyrA (P0AES4) from <i>E. coli</i> calculated using ClustalW <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000301#pone.0000301-Chenna1" target="_blank">[49]</a>.</p

Figure 4

<p>Electrostatic surface potential calculation. GRASP2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000301#pone.0000301-Petrey1" target="_blank">[51]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000301#pone.0000301-Warwicker1" target="_blank">[52]</a> electrostatic surface potentials within the DNA-binding grooves calculated for equivalent N-terminal fragments of: (A) <i>S. pneumoniae</i> ParC (ParC55), (B) <i>E. coli</i> ParC, (C) <i>E. coli</i> GyrA. The <i>E. coli</i> ParC structure was manually brought into the ‘closed’ conformation, but the experimentally determined fold of the active site was preserved. Negatively charged surfaces are in red and positively charged surfaces are in blue.</p