RECQL5-11-526 (construct used for APO crystals), RECQL5-11-453 (construct used for ADP crystals), RECQL5-nanobody D6 (binder), RECQL5-nanobody D10 (binder), RECQL5-nanobody D9 (inhibitor).
The first ever structures of human RECQL5 are presented as part of the TEP. The structures were determined in two distinctly different conformations in the absence and presence of ADP/Mg2+ to 3.2Å and 1.8Å respectively. Prominent differences can be seen between the two with maximal displacements of equivalent residues of up to 30Å (Fig. 1). Solution studies, comparisons with other enzymes and site directed mutagenesis have established that both conformations are relevant in solution and represent distinct states in the RECQL5 catalytic cycle. Thus molecules preventing this transition may be of therapeutic interest.
The primary assay suitable for use in high throughput screening is measurement of DNA stimulated ATPase activity using the readily commercially available ADP-Glo system (Promega). This assay features very high signal to noise (Z-factor >0.6), low compound interference and protein consumption (screening possible at just 20nM) (Fig. 2). Due to the stimulation of ATPase activity by (>100 fold for RECQL5) this assay is also sensitive to compounds interfering with DNA binding, although we also provide an orthogonal DNA binding assay in the form of fluorescence polarization assays which also features high signal to noise and are suitable for high throughput. We have also developed conditions suitable for testing higher order functions of RECQL5 such as helicase and strand annealing activity (Fig. 2). These assays are better suited as low throughput orthogonal assays for verification and characterization of potential inhibitors found in high throughput screening.
In addition to the ADP/Mg2+ bound to the ADP form crystals at 1.8Å, we have also initiated a crystallographic fragment screening campaign to identify possible additional chemical matter. Due to the low resolution of the APO form crystals it was decided to focus on the ADP from with a view to identifying possible allosteric binders. So far a single fragment hit has been obtained at 2.4Å (Fig.3) which localizes to a deep pocket formed by the cleft between the N and C-terminal helicase lobes. This pocket is extensive, with a relatively hydrophobic character (DMSO also binds to the pocket in some datasets), and importantly is the closed APO form this pocket is no longer present. Potential steric clashes with main chain and side chain residues indicate that binding to this site would prevent the transition from open to closed forms, potentially inhibiting the enzyme.
IMPORTANT: Please note that the existence of a small molecule within this TEP indicates that chemical matter can bind to the protein in a functionally relevant pocket. As such these molecules should not be used as tools for functional studies of the protein unless otherwise stated as they are not sufficiently potent or well-characterised to be used in cellular studies. The small molecule ligands are intended to be used as the basis for future chemistry optimisation to increase potency and selectivity and yield a chemical probe or lead series.
We also include three RECQL5 specific single domain camelid antibodies “nanobodies” as part of the package that we have validated as potent binders of RECQL5 catalytic domain. In addition to use as reagents in pull down experiments, one of these nanobodies has been demonstrated to be a potent inhibitor (inhibition is retained down to limit of enzyme concentration used in assay) of RECQL5 ATPase activity (Fig. 4), and thus may also be useful as a tool compound to investigate inhibition of RECQL5 and its complexes in vitro.
We intend to continue to work on RECQL5 with a view to finding more fragment hits and verify fragment binding using biophysical assays and investigate effect of compounds binding to the allosteric site on enzyme activity. We also intend to initiate enzymatic screening against helicase focussed compound libraries, with a view to developing RECQL5 specific small molecule inhibitors.