1H, 13C, 15N backbone resonance assignment for the 1–164 construct of human XRCC4

DNA double-strand breaks (DSBs) represent the most cytotoxic DNA lesions, as—if mis- or unrepaired—they can cause cell death or lead to genome instability, which in turn can cause cancer. DSBs are repaired by two major pathways termed homologous recombination and non-homologous end-joining (NHEJ). NHEJ is responsible for repairing the vast majority of DSBs arising in human cells. Defects in NHEJ factors are also associated with microcephaly, primordial dwarfism and immune deficiencies. One of the key proteins important for mediating NHEJ is XRCC4. XRCC4 is a dimer, with the dimer interface mediated by an extended coiled-coil. The N-terminal head domain forms a mixed alpha–beta globular structure. Numerous factors interact with the C-terminus of the coiled-coil domain, which is also associated with significant self-association between XRCC4 dimers. A range of construct lengths of human XRCC4 were expressed and purified, and the 1–164 variant had the best NMR properties, as judged by consistent linewidths, and chemical shift dispersion. In this work we report the 1H, 15 N and 13C backbone resonance assignments of human XRCC4 in the solution form of the 1–164 construct. Assignments were obtained by heteronuclear multidimensional NMR spectroscopy. In total, 156 of 161 assignable residues of XRCC4 were assigned to resonances in the TROSY spectrum, with an additional 11 resonances assigned to His-Tag residues. Prediction of solution secondary structure from a chemical shift analysis using the TALOS + webserver is in good agreement with the published X-ray crystal structures of this protein.

Solution structure of Gaussia Luciferase with five disulfide bonds and identification of a putative coelenterazine binding cavity by heteronuclear NMR

Gaussia luciferase (GLuc) is a small luciferase (18.2 kDa; 168 residues) and is thus attracting much attention as a reporter protein, but the lack of structural information is hampering further application. Here, we report the first solution structure of a fully active, recombinant GLuc determined by heteronuclear multidimensional NMR. We obtained a natively folded GLuc by bacterial expression and efficient refolding using a Solubility Enhancement Petide (SEP) tag. Almost perfect assignments of GLuc’s 1H, 13C and 15N backbone signals were obtained. GLuc structure was determined using CYANA, which automatically identified over 2500 NOEs of which > 570 were long-range. GLuc is an all-alpha-helix protein made of nine helices. The region spanning residues 10–18, 36–81, 96–145 and containing eight out of the nine helices was determined with a Cα-atom RMSD of 1.39 Å ± 0.39 Å. The structure of GLuc is novel and unique. Two homologous sequential repeats form two anti-parallel bundles made by 4 helices and tied together by three disulfide bonds. The N-terminal helix 1 is grabbed by these 4 helices. Further, we found a hydrophobic cavity where several residues responsible for bioluminescence were identified in previous mutational studies, and we thus hypothesize that this is a catalytic cavity, where the hydrophobic coelenterazine binds and the bioluminescence reaction takes place.

Solution structure of Gaussia Luciferase with five disulfide bonds and identification of a putative coelenterazine binding cavity by heteronuclear NMR

Gaussia luciferase (GLuc) is the smallest luciferase (18.2kDa; 168 residues) reported so far and is thus attracting much attention as a reporter protein, but the lack of structural information is hampering further application. Here, we report the first solution structure of a fully active, recombinant GLuc determined by heteronuclear multidimensional NMR. We obtained a natively folded GLuc by bacterial expression and efficient refolding using a solubility tag. Almost perfect assignments of GLuc’s 1H, 13C and 15N backbone signals were obtained. GLuc structure was determined using CYANA, which automatically identified over 2500 NOEs of which > 570 were long-range. GLuc is an all-alpha-helix protein made of nine helices. The region spanning residues 10–18, 36-81, 96-145 and containing eight out of the nine helices was determined with a Cα-atom RMSD of 1.39 ű 0.39 Å. The structure of GLuc is novel and unique. Two homologous sequential repeats form two anti-parallel bundles made by 4 helices and tied together by three disulfide bonds. The N-terminal helix 1 is grabbed by these 4 helices. Further, we found a hydrophobic cavity where several residues responsible for bioluminescence were identified in previous mutational studies, and we thus hypothesize that this is a catalytic cavity, where the hydrophobic coelenterazine binds and the bioluminescence reaction takes place.