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Structural and kinetic studies of XPF/ERCC1-DNA complex for drug discovery

Final Report Summary - XPF-ERCC1 (Structural and kinetic studies of XPF/ERCC1-DNA complex for drug discovery)

DNA is the molecule that encodes hereditary information required for survival and development of most organisms. Given the potentially devastating effects of DNA damage, cells have evolved an intricate series of mechanisms that maintain DNA integrity. One of the most versatile DNA repair mechanisms is nucleotide excision repair. An indispensable component of the NER machinery is the XPF/ERCC1 heterodimer that incises the damaged strand 5′ to an adduct. The XPF protein possesses the nuclease activity, while the ERCC1 is thought to be responsible for DNA binding. While the efficiency of DNA repair is essential for maintaining genomic stability, it presents a problem for cancer treatment. Chemotherapy has been the mainstay of cancer therapy for decades and includes drugs that induce covalent crosslinks between DNA bases and promote cell death. It has been suggested that reducing XPF/ERCC1 activity could sensitize cancer cells to chemotherapeutic drugs improving the efficiency of the therapy. Despite the pharmacological potential of targeting XPF/ERCC1, very little progress has been achieved so far in identifying inhibitors suitable for anti-cancer combination therapies. To develop such an inhibitor, we need to understand how XPF/ERCC1 recognizes DNA targets and the mechanism of enzymatic cleavage.
To achieve this we have proposed to study the recognition of the relevant DNA substrates by XPF/ERCC1 protein using the combination of structural and kinetic methods. NMR methodology was used to probe protein-DNA interactions at the atomic level and obtain a structural model of XPF/ERCC1-DNA complex. Kinetic measurements were planned to assess binding/dissociation rates, DNA determinants of binding specificity, and evaluate structure-based mutants in DNA recognition and catalysis. The combined information was to be used in development of XPF/ERCC1 inhibitors targeting protein-DNA interactions by in silico screening.
Using the literature data on the XPF/ERCC1 heterodimer we have developed what we call a minimal XPF/ERCC1 heterodimer that consists of domains that are responsible for dimerization, DNA binding, and catalysis. Over the course of the project we have optimized the constructs that we used for expression of the minimal heterodimer by removing the flexible parts that gave high-intensity peaks in the NMR spectrum and may have interfered with crystallization of the protein. Finally, we have optimized the expression of the developed constructs both in rich media for crystallography and in labelled minimal media for NMR studies.
To obtain the pure protein samples required for crystallographic and enzymatic studies we have developed a multi-step purification protocol. The purified protein was extensively tested in enzymatic assay and was shown to exhibit only the specific enzymatic activity even when incubated with DNA substrates overnight. The purified protein did not show any extra bands when analyzed by denaturing gel electrophoresis with silver staining indicating purity suitable for enzymatic and crystallographic studies.
The purified protein was analyzed using both enzymatic and binding assays. The obtained results recapitulate the findings reported on the full-length protein and shows that the minimal heterodimer cleaves all DNA structures containing ds/ss junctions at exactly the same position in the duplex portion.
An extensive buffer conditions screening was done for the optimized minimal XPF/ERCC1 protein to increase its solubility and thus suitability for NMR and crystallographic studies where the high concentrations of a target protein are required. In this screen, a number of chemical compounds were identified that increased the maximum possible concentration of the protein heterodimer, decreased its tendency to aggregate and did not show a significant impact on DNA binding and enzymatic activities.
The initial inhibitors obtained from collaborators were tested and found to be insufficiently soluble to permit NMR studies. Thus, in collaboration with Dr. Lumir Krejci we performed a new high-throughput screen for inhibitors of nuclease activity under protein-friendly conditions. So far, we have identified a number of promising leads that are being investigated.
As it is time and resource consuming to perform a validation of small molecule inhibitors using NMR we have employed a MicroScale Thermophoresis method to measure the affinity of the identified inhibitors that are available commercially to the target protein to select ones that are suitable for NMR analysis. The method required the fluorescent labelling of the target protein, the initial labelling of the protein using cysteine residues resulted in a protein that was not able to bind a DNA substrate with expected affinity. Thus, we labelled a protein using amine residues which showed an affinity to DNA substrate similar to the unlabeled protein. Unfortunately, none of the available inhibitors showed any reasonable affinity to the target protein under NMR-friendly conditions which makes NMR analysis all but impossible.
Using the Kinetic Capillary Electrophoresis methodology we were able to measure the stability of the protein-DNA complexes by measuring the dissociation kinetics and compare it with affinity data obtained using fluoresce-anisotropy method. We have discovered that while the affinities of different substrates are similar the complex stability is not. This is important in understanding of how XPF/ERCC1 protein recognizes the DNA and determining what is the correlation between DNA binding and enzymatic activities of the protein. In collaboration with Dr. Karel Říhas group we have used our affinity data for XPF/ERCC1 protein to validate a new method to validate a new method for affinity measurements. The paper was submitted to Scientific Reports, generated favorable reviews and is currently at revision stage where the minor points raised by reviewers are being addressed.
Using the optimized expression constructs we were able to obtain NMR finger-print spectrum which was still very crowded to permit NMR-based analysis of the protein structure. To overcome this obstacle we have tried to crystallize the protein using commercially available screens. Unfortunately, so far we did not have any luck in crystallizing the protein alone as well as in the presence of its binding partners (DNA substrate, short peptide).
While the project did not achieve its ultimate goal of solving the structure of XPF/ERCC1-DNA complex, it increased our general knowledge of the protein characteristics: factors that increase the solubility of the protein and prevent aggregation, affinities to different DNA substrates and stabilities of the relevant protein-DNA complexes. The obtained data would be invaluable for current collaborators and other scientists that work with this protein.