1. Introduction
Duplication of chromosomal DNA prior to cell division is a fundamental process in living cells. During initiation of DNA replication in
Escherichia coli, DnaB helicase is loaded with the assistance of the helicase loader DnaC onto double-stranded DNA and unwinds it [
1,
2]. DnaB, through direct physical interaction with DnaG primase, forms the primosome [
3], which uses its primase activity to synthesize short RNA primers essential for the function of DNA polymerase III [
4,
5].
DnaG is a DNA-dependent RNA polymerase [
6]. In bacteria it is comprised of three distinct domains: an N-terminal zinc-binding domain (ZBD) responsible for DNA template recognition [
7], a central catalytic domain (RNA polymerase domain, RPD) [
8,
9], and a C-terminal helicase-binding domain (HBD or DnaGC), which is responsible for interaction with DnaB helicase and single-stranded DNA-binding protein (SSB) [
10,
11,
12]. The crystal and solution structures of DnaGC of
E. coli were determined by X-ray crystallography as a non-physiological domain-swapped dimer [
13] and as a monomer in solution by NMR spectroscopy [
14].
SSB protects single-stranded DNA during DNA replication. It is an interaction hub known to bind to more than 14 other proteins involved in various stages of DNA replication, repair, and recombination through a highly conserved C-terminal hexapeptide motif (SSB-Ct, sequence: DDDIPF) [
15,
16]. SSB’s binding partners include DnaG [
10,
12], the Pol lll χ subunit [
12,
17,
18,
19,
20,
21,
22], the PriA replication restart helicase [
23], and exonuclease I [
24,
25].
The SSB-Ct binding site in DnaGC has been identified by NMR. The binding pocket is formed by basic residues K447, R452, and K518, as well as T450, M451, I455, and L519 [
26]. Moreover, the DnaGC point mutants K447A, T450A, R452A, and K518A dramatically attenuate SSB-Ct binding. Mutagenesis and NMR experiments, in particular
15N–
1H heteronuclear single-quantum correlation (
15N–
1H HSQC) experiments suggested that the conserved R452 residue forms a salt bridge with the carboxylic acid of the C-terminal Phe residue of the SSB-Ct, whereas the other positively charged residues around the binding pocket interact with the negatively charged residues of SSB-Ct. The SSB-Ct binding pockets in other SSB-binding proteins have characteristics in common with the binding pocket in DnaGC; e.g., those in ExoI [
24], RecO [
27], Pol lll χ [
22], and PriA [
23].
The SSB-Ct binding pockets in some or all of these proteins have been suggested to be very good targets for development of new antibacterial agents because many of the interactions are essential for bacterial survival and resistance to compounds that interfere with multiple interactions could not easily develop by target mutagenesis [
16]. This argument depends critically on a single compound mimicking the SSB-Ct peptide sufficiently well that it is able to bind tightly to three or more essential binding pockets that are lined with different residues and thus have structures that are more or less distinct. The observed gross structural similarities among pockets in SSB-Ct binding partners, including the ionic interaction with the C-terminal Phe and the basic rim that interacts with the acidic residues suggest such compounds might exist, but the only useful way to establish this for sure is to quantify the binding to multiple potential targets of compounds selected against one of them.
To begin the process of determining whether SSB-Ct binding pockets are actually suitable targets, we report the use of fragment-based screening (FBS) to find compounds binding to
E. coli DnaGC. FBS uses small (<300 Da) compounds called “fragments” as starting points for drug discovery. Several biophysical methods may be used in fragment screening [
28]. Here, we report the use of surface plasmon resonance (SPR) and NMR measurements to screen for binders that target the SSB-binding pocket in DnaGC.
3. Materials and Methods
3.1. Protein Expression and Purification
A phage λ-promoter plasmid (pZX1404) that directs overexpression of a protein comprising the central and C-terminal domains of
E. coli DnaG primase (residues 111–581, here called DnaG-RCD) was constructed by cloning a PCR fragment between the
BamHI and
EcoRI sites of vector pND706 [
40]. PCR was performed using plasmid pPL195 [
41] as template and the following primers (restriction sites in italics): dnaG_RCD_F, 5′-GCGGGATCCTAAGAAGGAGATATA
CATATG ACGCTTTATCAGTTGATG; dnaG_RCD_R, 5′-GCG
GAATTCTTACTTTTTCGCCAGCTC C. The full sequence of the gene encoding RCD was then verified by nucleotide sequence determination. Another plasmid pZX1399 encoding amino acids 115–581 of
E. coli DnaG was also constructed in a similar manner. However, the protein was expressed in insoluble form, and therefore was not used. Unlabeled DnaG-RCD and unlabeled and
15N-labeled DnaGC were expressed and purified as described previously for DnaGC [
42].
3.2. Fragment Libraries
The “first pass screen” fragment library (Zenobia Therapeutics, San Diego, CA, USA) was used for the SPR competition assay. Each fragment (50 mM in DMSO) was diluted to 1 mM final concentration. Fragment library members were tested for chip surface binding to eliminate false positives.
The Monash Institute of Pharmaceutical Science (MIPS) library comprised of around 1140 fragments purchased from Maybridge was used for STD-NMR experiments. The individual fragments were diluted in
2H
6-DMSO to give ~660 mM final stock concentrations [
30]. The fragments were mixed in cocktails of up to 6 compounds with well-resolved resonances in their 1D
1H-NMR spectra.
3.3. SPR Competition Assay
SPR measurements utilized a Biacore T200 instrument (GE Healthcare, Little Chalfont, UK) at 20 °C to measure the competition of compounds for the DnaGC/SSB-Ct peptide interaction. The buffer contained 10 mM HEPES (pH 7.4), 3 mM EDTA, 100 mM NaCl, 2% DMSO, 1 mM dithiothreitol and 0.05% (v/v) surfactant P20 (GE Healthcare). An N-terminally biotinylated SSB-Ct peptide [Biotin-(Ahx)-GSAPS-NEPPMDFDDDIPF; where Ahx is an amino-hexanoate spacer, followed by the 16 C-terminal residues of SSB highlighted in bold] was immobilized onto a streptavidin (SA) chip surface. RCD at 30 μM and fragments at 1 mM concentrations were used in all SPR experiments. Each sample was mixed for approximately 15 min prior to measurements. Mixtures were injected separately onto two flow cells, one of which served as a reference.
Prior to measurements, each individual fragment was tested for solubility and non-specific binding to an unmodified surface at 1 mM concentration to eliminate false positive responses. Compounds that bound non-specifically to the chip surface were excluded from screening. A flow rate of 5 μL/min was used during the 60 s injection and 60 s dissociation phases for all experiments.
3.4. Saturation-Transfer Difference (STD) NMR Spectroscopy
STD-NMR experiments were carried out using 5 μM unlabeled DnaG-RCD and mixtures of 6 fragments in each sample, at ~250 μM for each fragment. The sample volume was 500 μL with 98–99%
2H
2O buffer containing 50 mM phosphate (pH 7.8), 50 mM NaCl and 1 mM dithiothreitol. Spectra were recorded at 283 K using a Bruker Avance 600 MHz spectrometer (Bruker, Karlsruhe, Germany) equipped with a cryoprobe. Saturation of protein was achieved with a 4 s Gaussian pulse sequence train centered at −1 ppm. For reference spectra, a similar saturation pulse was applied 20 kHz off-resonance. A 20 ms spin-lock period was applied before acquisition to allow the residual protein signals to decay. The STD experiments were recorded over 64 scans. All NMR data were processed using TOPSPIN 3.1. Relative intensities were based on the most intense STD signal (I
max) identified across all STD spectra. A positive STD signal was categorized as “strong”, “moderate” or “weak” where the intensity was >50%, >25% or <25% of I
max, respectively [
30].
3.5. 2D 15N–1H HSQC Spectra
Protein binding by compounds identified by SPR and STD screens was confirmed by recording
15N–
1H HSQC spectra on uniformly
15N-labeled DnaGC (100 μM) in the presence of 3.3 mM compounds (from
2H
6-DMSO stocks) with HSQC buffer (50 mM MES pH 6.0, 60 mM NaCl, 1 mM dithiothreitol) containing 3%
2H
2O. The final volume of each sample was 150 μL. The recording time was 30 min for each
15N–
1H HSQC experiment. A standard pulse sequence was used for data acquisition. Spectra were recorded at 298 K with a Bruker Avance 600 MHz NMR spectrometer equipped with cryoprobe and auto-sample changer. Compounds were regarded as hits if chemical shift perturbation was observed in the
15N–
1H HSQC spectra. The spectra were processed with TOPSPIN 3.1 and analyzed using CCPN [
43]. Weighted CSP values [
44] were calculated as
Binding affinities were estimated by incremental titration of fragments into protein, with recording of a 15N–1H HSQC experiment at each concentration point. Compound solubilities were tested to determine the highest concentrations of ligands used in assays. Equilibrium dissociation constants from NMR titration data were derived using the “single site-specific binding with ligand depletion” model in GraphPad Prism v.6.0 (La Jolla, CA, USA).
3.6. 19F-NMR Spectroscopy
1D 19F-NMR spectra were recorded on a Bruker Avance III 400 MHz NMR spectrometer (Bruker, Karlsruhe, Germany) equipped with the two-channel BBO probe with z-gradient at 300 K. All 19F-NMR spectra were recorded with 256 scans for fragment and complex samples sequentially. Fragments dissolved in 2H6-DMSO were diluted in HSQC buffer to give final fragment and protein concentrations of 1 mM and 50 μM, respectively.
3.7. Molecular Docking
AutoDock Tools 1.5.6 [
45] was used to prepare protein [
46] and ligand structures for docking. The protonation state of the titratable groups in the protein were assigned at pH 7.0 using PROPKA 3.1 [
47]. Polar hydrogen atoms and atom-based Gasteiger partial charges were added. Nonpolar hydrogen atoms were merged with the parent atom. The DnaGC structure was taken from the previously solved crystal structure (PDB ID: 1T3W) [
13]. The protein was treated as a rigid body. The CSP docking calculations were performed using AutoDock Vina 1.1.2 [
45]. The calculations utilized an exhaustiveness of 1024 with grid points separated by 1.0 Å and grid size large enough to include the SSB-Ct peptide binding site (16 × 16 × 14 Å). Ligand data were obtained from the
ZINC database of commercially available compounds [
38].