Abstract
BackgroundThe first report on the transferable, plasmid-mediated quinolone-resistance determinant qnrA1 was in 1998. Since then, qnr alleles have been discovered worldwide in clinical strains of Gram-negative bacilli. Qnr proteins confer quinolone resistance, and belong to the pentapeptide repeat protein (PRP) family. Several PRP crystal structures have been solved, but little is known about the functional significance of their structural arrangement.ResultsWe conducted random and site-directed mutagenesis on qnrA1 and on qnrC, a newly identified quinolone-resistance gene from Proteus mirabilis. Many of the Qnr mutants lost their quinolone resistance function. The highly conserved hydrophobic Leu or Phe residues at the center of the pentapeptide repeats are known as i sites, and loss-of-function mutations included replacement of the i site hydrophobic residues with charged residues, replacing the i-2 site, N-terminal to the i residues, with bulky side-chain residues, introducing Pro into the β-helix coil, deletion of the N- and C-termini, and excision of a central coil. Molecular dynamics simulations and homology modeling demonstrated that QnrC overall adopts a stable β-helix fold and shares more similarities with MfpA than with other PRP structures. Based on homology modeling and molecular dynamics simulation, the dysfunctional point mutations introduced structural deformations into the quadrilateral β-helix structure of PRPs. Of the pentapeptides of QnrC, two-thirds adopted a type II β-turn, while the rest adopted type IV turns. A gap exists between coil 2 and coil 3 in the QnrC model structure, introducing a structural flexibility that is similar to that seen in MfpA.ConclusionThe hydrophobic core and the β-helix backbone conformation are important for maintaining the quinolone resistance property of Qnr proteins. QnrC may share structural similarity with MfpA.
Highlights
Introduction of prolineAll random mutants lost activity when Pro was introduced into the pentapeptide repeat sequence in the i, i-1 or i+2 regions, as seen in mutations L38P, S116P, and S153P (Table 1).3) Cys to Ser mutationsCys residues are abundant in Qnr proteins, relative to other pentapeptide repeat protein (PRP)
Combined with computational simulations and homology modeling, we found that the hydrophobic core and the b-helix backbone conformation are important for the quinolone resistance function of QnrC and QnrA1
Ciprofloxacin minimal inhibitory concentration (MIC) values showed that increased susceptibility resulted from removal of residues 2-21, 2-10, and 11-20 at or near the N-terminus of the Qnr proteins; residues 187-218, 207218 and 216-218 at the C-terminus; residues 41-56, 4955, and 51-56 around the G56 region; or removal of residues 77-96 and 137-156, corresponding to the intact coils of the b-helix (Table 1). 5) Other mutations The D188V mutation proximal to the C-terminus of QnrA1 conferred increased susceptibility to ciprofloxacin
Summary
Cys residues are abundant in Qnr proteins, relative to other PRPs. We introduced single point mutations of. Ciprofloxacin MIC values showed that increased susceptibility resulted from removal of residues 2-21, 2-10, and 11-20 at or near the N-terminus of the Qnr proteins; residues 187-218, 207218 and 216-218 at the C-terminus; residues 41-56, 4955, and 51-56 around the G56 region; or removal of residues 77-96 and 137-156, corresponding to the intact coils of the b-helix (Table 1). Qnr proteins confer quinolone resistance, and belong to the pentapeptide repeat protein (PRP) family. The Qnr proteins belong to the pentapeptide repeat protein (PRP) family. Among the resolved structures of PRPs, MfpA [[11], 2bm4] and EfsQnr [[15], 2w7z] present a unique rodshaped dimer form, in which the two monomers associate through their C-terminal helices This dimer assembly was proposed to be a DNA mimic, and shown to be capable of binding to DNA gyrase in vitro [11]. The sequence and structural characteristic of PRPs have been determined, the relationship between their structure and function remains elusive
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