Insights into the structural variation between pentapeptide repeat proteins--crystal structure of Rfr23 from Cyanothece 51142

Cyanothece sp. PCC 51142 contains 35 pentapeptide repeat proteins (PRPs), proteins that contain a minimum of eight tandem repeated five-residues (Rfr) of the general consensus sequence A[N/D]LXX. Published crystal structures of PRPs show that the tandem pentapeptide repeats adopt a type of right-handed quadrilateral beta-helix called an Rfr-fold. To characterize how structural features of Rfr-folds might vary with different amino acid sequences, the crystal structure of Cyanothece Rfr23 (174 residues) was determined at 2.4A resolution. The structure is dominated by an Rfr-fold capped at the N-terminus with a nine-residue alpha-helix (M26(*)-E34). The Rfr-fold of Rfr23 contains four structural features previously unobserved in Rfr-folds. First, Rfr23 is composed entirely of type II beta-turns. Second, the pentapeptide repeats are not consecutive in the primary amino acid sequence. Instead, Rfr23 contains 24-residues protruding outside one corner of the first complete N-terminal coil of the Rfr-fold (L56-P79) (24-residue insertion). Third, a disulfide bond between C39 and C42 bridges the beta-turn between the first and second pentapeptide repeats in the first coil (disulfide bracket). NMR spectroscopy indicates that the reduction of the disulfide bracket with the addition of DTT destroys the entire Rfr-fold. Fourth, a single-residue perturbs the Rfr-fold slightly in the last coil between the C-terminal two pentapeptide repeats (single-residue bulge).     

Structural dynamics of pentapeptide repeat proteins

Pentapeptide repeat proteins (PRPs) represent a large superfamily with more than 38 000 sequences in nearly 3500 species, the majority belonging to cyanobacteria but represented among all branches of life. PRPs contain at least eight consecutive pentapeptide repeats with the consensus (A/C/S/V/T/L/I)(D/N/S/K/E/I/R)(L/F)(S/T/R/E/Q/K/V/D)(G/D/E/N/R/Q/K). PRPs fold into right-handed quadrilateral β helices, also known as repeat-five-residue (Rfr)-folds, with four consecutive pentapeptide repeats comprising a single coil, the ~90° change in polypeptide direction in square-shaped coils achieved by type I, II and IV β turns, and hydrogen bonds between coils establishing β ladders on each Rfr-fold face. PRPs are broadly categorized into group 1 and 2 involved in antibiotic resistance and group 3 currently having unknown functions. Motivated by their intriguing structures, we are investigating PRP biophysical characteristics, including Rfr-fold thermal stability, β turn and β ladder hydrogen bond amide exchange rates and backbone dynamics. Here, we present analysis of 20 ns molecular dynamics (MD) simulations and all atom normal mode analysis (aaNMA) calculations for four group 1 and group 2 and four group 3 PRPs whose structures have been determined by X-ray crystallography. The MD cross-correlation matrices and aaNMA indicated strong correlated motion between adjacent coils and weak coupled motion between coils separated by one or more intervening coils. Slow anticorrelated motions were detected between adjacent coils in aaNMA modes that we hypothesize are requisite to access exchange-competent states necessary to permit solvent exchange of amide hydrogens involved in β-ladder and β-turns hydrogen bonds, which can have lifetimes on the order of months.     

Structural characterization of the fusion of two pentapeptide repeat proteins, Np275 and Np276, from Nostoc punctiforme: resurrection of an ancestral protein

The Nostoc punctiforme genes Np275 and Np276 are two adjacently encoded proteins of 98 and 75 amino acids in length and exhibit sequences composed of tandem pentapeptide repeats. The structures of Np275 and a fusion of Np275 and Np276 were determined to 2.1 and 1.5 A, respectively. The two Nostoc proteins fold as highly symmetric right-handed quadrilateral beta-helices similar to the mycobacterial protein MfpA implicated in fluoroquinolone resistance and DNA gyrase inhibition. The sequence composition of the intervening coding region and the ability to express a fused protein by removing the stop codon for Np275 suggests Np275 and Np276 were recently part of a larger ancestral pentapeptide repeat protein.     

Solution structure and refolding of the Mycobacterium tuberculosis pentapeptide repeat protein MfpA

The pentapeptide repeat is a recently discovered protein fold. Mycobacterium tuberculosis MfpA is a founding member of the pentapeptide repeat protein (PRP) family that confers resistance to the antibiotic fluoroquinolone by binding to DNA gyrase and inhibiting its activity. The size, shape, and surface potential of MfpA mimics duplex DNA. As an initial step in a comprehensive biophysical analysis of the role of PRPs in the regulation of cellular topoisomerase activity and conferring antibiotic resistance, we have explored the solution structure and refolding of MfpA by fluorescence spectroscopy, CD, and analytical centrifugation. A unique CD spectrum for the pentapeptide repeat fold is described. This spectrum reveals a native structure whose beta-strands and turns within the right-handed quadrilateral beta-helix that define the PRP fold differ from canonical secondary structure types. MfpA refolded from urea or guanidium by dialysis or dilution forms stable aggregates of monomers whose secondary and tertiary structure are not native. In contrast, MfpA refolded using a novel "time-dependent renaturation" protocol yields protein with native secondary, tertiary, and quaternary structure. The generality of "time-dependent renaturation" to other proteins and denaturation methods is discussed.     

Crystal structure of Alr1298, a pentapeptide repeat protein from the cyanobacterium Nostoc sp. PCC 7120, determined at 2.1 Å resolution

Nostoc sp. PCC 7120 are filamentous cyanobacteria capable of both oxygenic photosynthesis and nitrogen fixation, with the latter taking place in specialized cells known as heterocysts that terminally differentiate from vegetative cells under conditions of nitrogen starvation. Cyanobacteria have existed on earth for more than 2 billion years and are thought to be responsible for oxygenation of the earth's atmosphere. Filamentous cyanobacteria such as Nostoc sp. PCC 7120 may also represent the oldest multicellular organisms on earth that undergo cell differentiation. Pentapeptide repeat proteins (PRPs), which occur most abundantly in cyanobacteria, adopt a right-handed quadrilateral β-helical structure, also referred to as a repeat five residue (Rfr) fold, with four-consecutive pentapeptide repeats constituting a single coil in the β-helical structure. PRPs are predicted to exist in all compartments within cyanobacteria including the thylakoid and cell-wall membranes as well as the cytoplasm and thylakoid periplasmic space. Despite their intriguing structure and importance to understanding ancient cyanobacteria, the biochemical function of PRPs in cyanobacteria remains largely unknown. Here we report the crystal structure of Alr1298, a PRP from Nostoc sp. PCC 7120 predicted to reside in the cytoplasm. The structure displays the typical right-handed quadrilateral β-helical structure and includes a four-α-helix cluster capping the N-terminus and a single α-helix capping the C-terminus. A gene cluster analysis indicated that Alr1298 may belong to an operon linked to cell proliferation and/or thylakoid biogenesis. Elevated alr1298 gene expression following nitrogen starvation indicates that Alr1298 may play a role in response to nitrogen starvation and/or heterocyst differentiation.     

Pentapeptide repeat proteins

The pentapeptide repeat protein (PRP) family has more than 500 members in the prokaryotic and eukaryotic kingdoms. These proteins are composed of, or contain domains composed of, tandemly repeated amino acid sequences with a consensus sequence of [S,T,A,V][D,N][L,F][S,T,R][G]. The biochemical function of the vast majority of PRP family members is unknown. The three-dimensional structure of the first member of the PRP family was determined for the fluoroquinolone resistance protein (MfpA) from Mycobacterium tuberculosis. The structure revealed that the pentapeptide repeats encode the folding of a novel right-handed quadrilateral beta-helix. MfpA binds to DNA gyrase and inhibits its activity. The rod-shaped, dimeric protein exhibits remarkable similarity in size, shape, and electrostatics to DNA.     

Structure and distribution of pentapeptide repeats in bacteria.

We report the discovery of a novel family of proteins, each member contains tandem pentapeptide (five residue) repeats, described by the motif A(D/N)LXX. Members of this family are both membrane bound and cytoplasmic. The function of these repeats is uncertain, but they may have a targeting or structural function rather than enzymatic activity. This family is most common in cyanobacteria, suggesting a function related to cyanobacterial-specific metabolism. Although no experimental information is available for the structure of this family, it is predicted that the tandem pentapeptide repeats will form a right-handed beta-helical structure. A structural model of the pentapeptide repeats is presented.     

Factors involved in the stability of isolated beta-sheets: Turn sequence, beta-sheet twisting, and hydrophobic surface burial

We have recently reported on the design of a 20-residue peptide able to form a significant population of a three-stranded up-and-down antiparallel beta-sheet in aqueous solution. To improve our beta-sheet model in terms of the folded population, we have modified the sequences of the two 2-residue turns by introducing the segment DPro-Gly, a sequence shown to lead to more rigid type II' beta-turns. The analysis of several NMR parameters, NOE data, as well as Deltadelta(CalphaH), DeltadeltaC(beta), and Deltadelta(Cbeta) values, demonstrates that the new peptide forms a beta-sheet structure in aqueous solution more stable than the original one, whereas the substitution of the DPro residues by LPro leads to a random coil peptide. This agrees with previous results on beta-hairpin-forming peptides showing the essential role of the turn sequence for beta-hairpin folding. The well-defined beta-sheet motif calculated for the new designed peptide (pair-wise RMSD for backbone atoms is 0.5 +/- 0.1 A) displays a high degree of twist. This twist likely contributes to stability, as a more hydrophobic surface is buried in the twisted beta-sheet than in a flatter one. The twist observed in the up-and-down antiparallel beta-sheet motifs of most proteins is less pronounced than in our designed peptide, except for the WW domains. The additional hydrophobic surface burial provided by beta-sheet twisting relative to a "flat" beta-sheet is probably more important for structure stability in peptides and small proteins like the WW domains than in larger proteins for which there exists a significant contribution to stability arising from their extensive hydrophobic cores.     

Stability, denaturation and refolding of Mycobacterium tuberculosis MfpA, a DNA mimicking protein that confers antibiotic resistance

MfpA from Mycobacterium tuberculosis is a founding member of the pentapeptide repeat class of proteins (PRP) that is believed to confer bacterial resistance to the drug fluoroquinolone by mimicking the size, shape and surface charge of duplex DNA. We show that phenylalanine side chain stacking stabilizes the N-terminus of MfpA's pentapeptide thus extending the DNA mimicry analogy. The Lumry-Eyring model was applied to multiple spectral measures of MfpA denaturation revealing that the MfpA dimer dissociates to monomers which undergo a structural transition that leads to aggregation. MfpA retains high secondary and tertiary structure content under denaturing conditions. Dimerization stabilizes MfpA's pentapeptide repeat fold. The high Arrhenius activation energy of the barrier to aggregate formation rationalizes its stability. The mechanism of MfpA denaturation and refolding is a ‘double funnel’ energy landscape where the ‘native’ and ‘aggregate’ funnels are separated by the high barrier that is not overcome during in vitro refolding.     

Structure of QnrB1, a plasmid-mediated fluoroquinolone resistance factor

QnrB1 is a plasmid-encoded pentapeptide repeat protein (PRP) that confers a moderate degree of resistance to fluoroquinolones. Its gene was cloned into an expression vector with an N-terminal polyhistidine tag, and the protein was purified by nickel affinity chromatography. The structure of QnrB1 was determined by a combination of trypsinolysis, surface mutagenesis, and single anomalous dispersion phasing. QnrB1 folds as a right-handed quadrilateral β-helix with a highly asymmetric dimeric structure typical of PRP-topoisomerase poison resistance factors. The threading of pentapeptides into the β-helical fold is interrupted by two noncanonical PRP sequences that produce outward projecting loops that interrupt the regularity of the PRP surface. Deletion of the larger upper loop eliminated the protective effect of QnrB1 on DNA gyrase toward inhibition by quinolones, whereas deletion of the smaller lower loop drastically reduced the protective effect. These loops are conserved among all plasmid-based Qnr variants (QnrA, QnrC, QnrD, and QnrS) and some chromosomally encoded Qnr varieties. A mechanism in which PRP-topoisomerase poison resistance factors bind to and disrupt the quinolone-DNA-gyrase interaction is proposed.     

Alternative type I and I' turn conformations in the beta8/beta9 beta-hairpin of human acidic fibroblast growth factor

Human acidic fibroblast growth factor (FGF-1) has a beta-trefoil structure, one of the fundamental protein superfolds. The X-ray crystal structures of wild-type and various mutant forms of FGF-1 have been solved in five different space groups: C2, C222(1), P2(1) (four molecules/asu), P2(1) (three molecules/asu), and P2(1)2(1)2(1). These structures reveal two characteristically different conformations for the beta8/beta9 beta-hairpin comprising residue positions 90-94. This region in the wild-type FGF-1 structure (P2(1), four molecules/asu), a his-tagged His93-->Gly mutant (P2(1), three molecules/asu) and a his-tagged Asn106-->Gly mutant (P2(1)2(1)2(1)) adopts a 3:5 beta-hairpin known as a type I (1-4) G1 beta-bulge (containing a type I turn). However, a his-tagged form of wild-type FGF-1 (C222(1)) and a his-tagged Leu44-->Phe mutant (C2) adopt a 3:3 beta-hairpin (containing a type I' turn) for this same region. A feature that distinguishes these two types of beta-hairpin structures is the number and location of side chain positions with eclipsed C(beta) and main-chain carbonyl oxygen groups (Psi is equivalent to +60 degrees). The effects of glycine mutations upon stability, at positions within the hairpin, have been used to identify the most likely structure in solution. Type I' turns in the structural data bank are quite rare, and a survey of these turns reveals that a large percentage exhibit crystal contacts within 3.0 A. This suggests that many of the type I' turns in X-ray structures may be adopted due to crystal packing effects.     

Sequence swapping does not result in conformation swapping for the beta4/beta5 and beta8/beta9 beta-hairpin turns in human acidic fibroblast growth factor

The beta-turn is the most common type of nonrepetitive structure in globular proteins, comprising ~25% of all residues; however, a detailed understanding of effects of specific residues upon beta-turn stability and conformation is lacking. Human acidic fibroblast growth factor (FGF-1) is a member of the beta-trefoil superfold and contains a total of five beta-hairpin structures (antiparallel beta-sheets connected by a reverse turn). beta-Turns related by the characteristic threefold structural symmetry of this superfold exhibit different primary structures, and in some cases, different secondary structures. As such, they represent a useful system with which to study the role that turn sequences play in determining structure, stability, and folding of the protein. Two turns related by the threefold structural symmetry, the beta4/beta5 and beta8/beta9 turns, were subjected to both sequence-swapping and poly-glycine substitution mutations, and the effects upon stability, folding, and structure were investigated. In the wild-type protein these turns are of identical length, but exhibit different conformations. These conformations were observed to be retained during sequence-swapping and glycine substitution mutagenesis. The results indicate that the beta-turn structure at these positions is not determined by the turn sequence. Structural analysis suggests that residues flanking the turn are a primary structural determinant of the conformation within the turn.     

Turn stability in beta-hairpin peptides: Investigation of peptides containing 3:5 type I G1 bulge turns

The turn-forming ability of a series of three-residue sequences was investigated by substituting them into a well-characterized beta-hairpin peptide. The starting scaffold, bhpW, is a disulfide-cyclized 10-residue peptide that folds into a stable beta-hairpin with two antiparallel strands connected by a two-residue reverse turn. Substitution of the central two residues with the three-residue test sequences leads to less stable hairpins, as judged by thiol-disulfide equilibrium measurements. However, analysis of NMR parameters indicated that each molecule retains a significant folded population, and that the type of turn adopted by the three-residue sequence is the same in all cases. The solution structure of a selected peptide with a PDG turn contained an antiparallel beta-hairpin with a 3:5 type I + G1 bulge turn. Analysis of the energetic contributions of individual turn residues in the series of peptides indicates that substitution effects have significant context dependence, limiting the predictive power of individual amino acid propensities for turn formation. The most stable and least stable sequences were also substituted into a more stable disulfide-cyclized scaffold and a linear beta-hairpin scaffold. The relative stabilities remained the same, suggesting that experimental measurements in the bhpW context are a useful way to evaluate turn stability for use in protein design projects. Moreover, these scaffolds are capable of displaying a diverse set of turns, which can be exploited for the mimicry of protein loops or for generating libraries of reverse turns.     

Introduction of a new scheme for classifying β-turns in protein structures

Protein β-turn classification remains an area of ongoing development in structural biology research. While the commonly used nomenclature defining type I, type II and type IV β-turns was introduced in the 1970s and 1980s, refinements of β-turn type definitions have been introduced as recently as 2019 by Dunbrack, Jr and co-workers who expanded the number of β-turn types to 18 (Shapovalov et al, PLOS Computat. Biol., 15, e1006844, 2019). Based on their analysis of 13 030 turns from 1074 ultrahigh resolution (≤1.2 Å) protein structures, they used a new clustering algorithm to expand the definitions used to classify protein β-turns and introduced a new nomenclature system. We recently encountered a specific problem when classifying β-turns in crystal structures of pentapeptide repeat proteins (PRPs) determined in our lab that are largely composed of β-turns that often lie close to, but just outside of, canonical β-turn regions. To address this problem, we devised a new scheme that merges the Klyne-Prelog stereochemistry nomenclature and definitions with the Ramachandran plot. The resulting Klyne-Prelog-modified Ramachandran plot scheme defines 1296 distinct potential β-turn classifications that cover all possible protein β-turn space with a nomenclature that indicates the stereochemistry of i + 1 and i + 2 backbone dihedral angles. The utility of the new classification scheme was illustrated by re-classification of the β-turns in all known protein structures in the PRP superfamily and further assessed using a database of 16 657 high-resolution protein structures (≤1.5 Å) from which 522 776 β-turns were identified and classified.     

Importance of secondary structural specificity determinants in protein folding: insertion of a native beta-sheet sequence into an alpha-helical coiled-coil

To examine how a short secondary structural element derived from a native protein folds when in a different protein environment, we inserted an 11-residue beta-sheet segment (cassette) from human immunoglobulin fold, Fab new, into an alpha-helical coiled-coil host protein (cassette holder). This de novo design protein model, the structural cassette mutagenesis (SCM) model, allows us to study protein folding principles involving both short- and long-range interactions that affect secondary structure stability and conformation. In this study, we address whether the insertion of this beta-sheet cassette into the alpha-helical coiled-coil protein would result in conformational change nucleated by the long-range tertiary stabilization of the coiled-coil, therefore overriding the local propensity of the cassette to form beta-sheet, observed in its native immunoglobulin fold. The results showed that not only did the nucleating helices of the coiled-coil on either end of the cassette fail to nucleate the beta-sheet cassette to fold with an alpha-helical conformation, but also the entire chimeric protein became a random coil. We identified two determinants in this cassette that prevented coiled-coil formation: (1) a tandem dipeptide NN motif at the N-terminal of the beta-sheet cassette, and (2) the hydrophilic Ser residue, which would be buried in the hydrophobic core if the coiled-coil structure were to fold. By amino acid substitution of these helix disruptive residues, that is, either the replacement of the NN motif with high helical propensity Ala residues or the substitution of Ser with Leu to enhance hydrophobicity, we were able to convert the random coil chimeric protein into a fully folded alpha-helical coiled-coil. We hypothesized that this NN motif is a "secondary structural specificity determinant" which is very selective for one type of secondary structure and may prevent neighboring residues from adopting an alternate protein fold. These sequences with secondary structural specificity determinants have very strong local propensity to fold into a specific secondary structure and may affect overall protein folding by acting as a folding initiation site.     

Analysis of the factors that stabilize a designed two-stranded antiparallel beta-sheet

Autonomously folding beta-hairpins (two-strand antiparallel beta-sheets) have become increasingly valuable tools for probing the forces that control peptide and protein conformational preferences. We examine the effects of variations in sequence and solvent on the stability of a previously designed 12-residue peptide (1). This peptide adopts a beta-hairpin conformation containing a two-residue loop (D-Pro-Gly) and a four-residue interstrand sidechain cluster that is observed in the natural protein GB1. We show that the conformational propensity of the loop segment plays an important role in beta-hairpin stability by comparing 1 with (D)P--> N mutant 2. In addition, we show that the sidechain cluster contributes both to conformational stability and to folding cooperativity by comparing 1 with mutant 3, in which two of the four cluster residues have been changed to serine. Thermodynamic analysis suggests that the high loop-forming propensity of the (D)PG segment decreases the entropic cost of beta-hairpin formation relative to the more flexible NG segment, but that the conformational rigidity of (D)PG may prevent optimal contacts between the sidechains of the GB1-derived cluster. The enthalpic favorability of folding in these designed beta-hairpins suggests that they are excellent scaffolds for studying the fundamental mechanisms by which amino acid sidechains interact with one another in folded proteins.     

Effects of turn residues in directing the formation of the beta-sheet and in the stability of the beta-sheet

The designed peptide (denoted 20-mer, sequence VFITS(D)PGKTYTEV(D)PGOKILQ) has been shown to form a three-strand antiparallel beta-sheet. It is generally believed that the (D)Pro-Gly segment has the propensity to adopt a type II' beta-turn, thereby promoting the formation of this beta-sheet. Here, we replaced (D)Pro-Gly with Asp-Gly, which should favor a type I' turn, to examine the influence of different type of turns on the stability of the beta-sheet. Contrary to our expectation, the mutant peptide, denoted P6D, forms a five-residue type I turn plus a beta-bulge between the first two strands due to a one amino-acid frameshift in the hydrogen bonding network and side-chain inversion of the first beta-strand. In contrast, the same kind of substitution at (D)Pro-14 in the double mutant, denoted P6DP14D, does not yield the same effect. These observations suggest that the SDGK sequence disfavors the type I' conformation while the VDGO sequence favors a type I' turn, and that the frameshift in the first strand provides a way for the peptide to accommodate a disfavored turn sequence by protruding a bulge in the formation of the beta-hairpin. Thus, different types of turns can affect the stability of a beta-structure.     

Mimicry by asx- and ST-turns of the four main types of beta-turn in proteins

Hydrogen-bonded beta-turns in proteins occur in four categories: type I (the most common), type II, type II', and type I'. Asx-turns resemble beta-turns, in that both have an NH. . .OC hydrogen bond forming a ring of 10 atoms. Serine and threonine side chains also commonly form hydrogen-bonded turns, here called ST-turns. Asx-turns and ST-turns can be categorized into four classes, based on side chain rotamers and the conformation of the central turn residue, which are geometrically equivalent to the four types of beta-turns. We propose asx- and ST-turns be named using the type I, II, I', and II' beta-turn nomenclature. Using this, the frequency of occurrence of both asx- and ST-turns is: type II' > type I > type II > type I', whereas for beta-turns it is type I > type II > type I' > type II'. Almost all type II asx-turns occur as a recently described three residue feature named an asx-nest.     

Structure, organization, and regulation of a hamster proline-rich protein gene. A multigene family

Genomic DNA fragments bearing proline-rich protein (PRP) genes expressed specifically in hamster parotid glands have been isolated and characterized. Complete exonic sequences as well as intronic and a considerable portion of the flanking sequences are reported for a PRP gene, H29. H29 is interrupted by three intervening sequences, with consensus splice junctions, and it likely encodes the acidic hamster PRP Hp43a. Exceedingly high homology of the 5'-untranslated region and the sequence encoding the signal peptide is observed with other PRPs of all species studied. Significant homology was also detected among the repetitive sequences of the mature acidic PRPs from human, mouse, hamster, and rat. This conservation of the internal repeats of the PRPs suggested that proline-rich protein gene evolution involved intragenic duplication of internal repeats and gene duplication and conversion. Both hamster and mouse PRP genes (H29 and mouse proline-rich protein gene, respectively) share considerable sequence similarity in the 5'-flanking regions for about 100 base pairs upstream. The remainder of the upstream sequences were heterologous except for three oligonucleotide regions with 60-70% sequence conservation. These three regions are thought to be involved in the regulation of the tissue-specific PRP gene induction.     

Pentapeptide repeat protein QnrB1 requires ATP hydrolysis to rejuvenate poisoned gyrase complexes

DNA gyrase, a type II topoisomerase found predominantly in bacteria, is the target for a variety of ‘poisons’, namely natural product toxins (e.g. albicidin, microcin B17) and clinically important synthetic molecules (e.g. fluoroquinolones). Resistance to both groups can be mediated by pentapeptide repeat proteins (PRPs). Despite long-term studies, the mechanism of action of these protective PRPs is not known. We show that a PRP, QnrB1 provides specific protection against fluoroquinolones, which strictly requires ATP hydrolysis by gyrase. QnrB1 binds to the GyrB protein and stimulates ATPase activity of the isolated N-terminal ATPase domain of GyrB (GyrB43). We probed the QnrB1 binding site using site-specific incorporation of a photoreactive amino acid and mapped the crosslinks to the GyrB43 protein. We propose a model in which QnrB1 binding allosterically promotes dissociation of the fluoroquinolone molecule from the cleavage complex.