Friend or Foe: Protein Inhibitors of DNA Gyrase

Abstract

DNA gyrase is essential for the successful replication of circular chromosomes, such as those found in most bacterial species, by relieving topological stressors associated with unwinding the double-stranded genetic material. This critical central role makes gyrase a valued target for antibacterial approaches, as exemplified by the highly successful fluoroquinolone class of antibiotics. It is reasonable that the activity of gyrase could be intrinsically regulated within cells, thereby helping to coordinate DNA replication with doubling times. Numerous proteins have been identified to exert inhibitory effects on DNA gyrase, although at lower doses, it can appear readily reversible and therefore may have regulatory value. Some of these, such as the small protein toxins found in plasmid-borne addiction modules, can promote cell death by inducing damage to DNA, resulting in an analogous outcome as quinolone antibiotics. Others, however, appear to transiently impact gyrase in a readily reversible and non-damaging mechanism, such as the plasmid-derived Qnr family of DNA-mimetic proteins. The current review examines the origins and known activities of protein inhibitors of gyrase and highlights opportunities to further exert control over bacterial growth by targeting this validated antibacterial target with novel molecular mechanisms. Furthermore, we are gaining new insights into fundamental regulatory strategies of gyrase that may prove important for understanding diverse growth strategies among different bacteria.

Keywords: DNA gyrase; GyrA; GyrB; antibiotics; gyrase inhibitors; gyrase regulation; mobile genetic elements; quinolones; target protection.

Figure 2

ParE toxins have canonical structural features, but the molecular mechanism of gyrase inhibition remains unclear. ParE toxins can be distinguished from other super-family members by the extra turns in the helix–turn–helix motif (dashed lines, left panel). The toxicity of ParE proteins appears dependent on the C-terminus. C-terminal amino acids too disordered to place in the crystal structures are indicated with “Δ”. Note the ParE shown in blue has a shorter C-terminus and has been published as having attenuated toxicity. Superposed structures: PDB 3G5O, M. tuberculosis RelE2 [66], purple; PDB 3KXE, Caulobacter crescentus ParE1 [67], green; PDB 6XRW, P. aeruginosa ParE1 [65], blue; PDB 7R5A, V. cholerae ParE2 (Garcia-Rodriguez et al., unpublished), dark cyan; M. tuberculosis ParE2 Alpha Fold model (AF-P9WHG5-F1_v4), dark grey.

Figure 3

CcdB toxins are dimeric and bind to the bottom “gate” in GyrA and block the release of either DNA segment (only the cleaved segment is shown, “G” in Figure 1), resulting in an accumulation of dsDNA breaks. Binding can only occur when gyrase is midway through the catalytic cycle, as GyrA dimers open at the site of dsDNA cleavage to allow transit of the top “T” segment of DNA (not shown here, see Figure 1). A peptide mimic, corresponding to the yellow helix (insert) of CcdB, has shown antibacterial efficacy in a mouse infection model [77]. Mutations at gyrase Arg462 (shown as sticks in the inset) impart resistance to CcdB toxicity. In available holo-gyrase structures, all GyrA are in the “closed” conformation correlating to the conformational state before strand passage. When the full gyrase structure is superposed with the CcdB structures (right, GyrA blue surface, GyrB green surface), a steric clash is evident (marked by a yellow asterisk). Superposed structures: PDB 1X75, E. coli GyrA and F plasmid CcdB [82], PDB 4ELY, Shigella flexneri GyrA and V. fischeri CcdB, and PDB 4ELZ, V. fischeri GyrA and CcdB [78]; these are superposed onto PDB 6RKW, E. coli GyrA and GyrB bound to a 130 bp DNA [87].

Figure 1

Schematic of the catalytic cycle of DNA gyrase. Step 1: Two segments of double-stranded DNA are captured on, for example, an underwound circular DNA molecule, and these are referred to as the “T”, or transfer segment, and the “G”, or gate segment. Note how the G segment is wrapped around the C-terminal domains (CTD) of GyrA. This unique arrangement is specific to gyrase enzymes. Step 2: a. The binding of two molecules of ATP locks the GyrB domains around the T segment. b. A double-stranded break is made in the G segment. This is mediated by the GyrA active site using a tyrosine amino acid, resulting in a covalent DNA-protein intermediate. Step 3: The hydrolysis of one ATP is coupled with large conformational changes, resulting in passing the T segment through the DNA break and into the central part of GyrA. Step 4: a. The DNA break is ligated, reforming the intact DNA molecule. b. Concomitant with ligation, hydrolysis of the second ATP molecule triggers complete DNA release, and Step 5: resets the gyrase enzyme for another catalytic cycle by triggering the re-opening of the GyrB domains.

Figure 4

TsbT toxins are predicted to form a helical structure (orange) containing two residues (E27, D37, red boxes) critical for toxicity. This helix superposes relatively well with the second helix of ParE toxins (blue, amino acids denoted along the bottom in blue font), which has a relatively higher sequence conservation in the ParE protein family.

Figure 5

The pentapeptide repeat protein MfpA protein binds to GyrB and mimics DNA. The conserved dimeric solenoid structure and charge distribution (surface of MfpA, right) makes similar interactions as double-stranded DNA. The length of MfrP extends down GyrB (green, one GyrB hidden to allow viewing of MfpA) into the active site of GyrA (blue, gray), suggesting a mechanism for relieving fluoroquinolone poisoning of gyrase. Note that the “closed” form of GyrB in the complete gyrase structure has steric clashes with MfpA; for this reason, one GyrB chain has been removed from the depicted view. Superposed structures: PDB 6ZT5 MfpA with M. smegmatis GyrB N-terminal domain (only MfpA is shown here) [133]; PDB 6RKW, E. coli GyrA and GyrB bound to a 130 bp DNA [87].

Figure 6

YacG protein binds to GyrB with the C-terminal region interacting with GyrB and GyrA regions. The small YacG protein inserts into openings among GyrB domains and extends into the central region adjacent to the GyrA active site. The tail of YacG can be visualized as likely clashing with DNA binding. Superposed structures: PDB 4TMA, YacG bound to a truncated E. coli GyrA GyrB [137], PDB 6RKW, E. coli GyrA and GyrB bound to a 130 bp DNA [87].

Figure 7

Schematic of the proposed regulatory link mediated by MurI to maintain a balance between gyrase activity and cell replication. The continued production of peptidoglycan precursors may be temporally regulated to occur when DNA replication is not active, such that the activity of gyrase is no longer needed.

Figure 8

Structure of GyrI (PDB 1JYH, [157]) with corresponding gyrase-inhibiting peptides indicated. Peptides 89–96 (yellow) binds to gyrase approx. 10-fold more tightly than a peptide corresponding to 33–48 (orange). The yellow peptide is inhibitory when included in in vitro gyrase activity assays.