XLF and APLF bind Ku80 at two remote sites to ensure DNA repair by non-homologous end joining

International audienceThe Ku70-Ku80 (Ku) heterodimer binds rapidly and tightly to the ends of DNA double-strand breaks and recruits factors of the non-homologous end-joining (NHEJ) repair pathway through molecular interactions that remain unclear. We have determined crystal structures of the Ku-binding motifs (KBM) of the NHEJ proteins APLF (A-KBM) and XLF (X-KBM) bound to a Ku-DNA complex. The two KBM motifs bind remote sites of the Ku80 alpha/beta domain. The X-KBM occupies an internal pocket formed by an unprecedented large outward rotation of the Ku80 alpha/beta domain. We observe independent recruitment of the APLF-interacting protein XRCC4 and of XLF to laser-irradiated sites via binding of A- and X-KBMs, respectively, to Ku80. Finally, we show that mutation of the X-KBM and A-KBM binding sites in Ku80 compromises both the efficiency and accuracy of end joining and cellular radiosensitivity. A- and X-KBMs may represent two initial anchor points to build the intricate interaction network required for NHEJ

Protein-DNA interaction study : Ku70/Ku80-DNA interactions in the NHEJ pathway and characterization of inhibitors of a target protein-DNA interaction in a drug search context

Pour qu’un organisme soit viable, il est essentiel que la molécule d’ADN soit dupliquée et transmise aux générations filles dans son intégralité avec le moins d’altérations possibles. Des cassures double-brin (CDB) peuvent apparaitre suite à des stress endogènes ou exogènes et constituent les dommages de l’ADN les plus toxiques. La radiothérapie a pour but générer de manière localisée des CDBs dans les cellules tumorales et de déclencher leur apoptose. Un enjeu majeur actuel est de comprendre le mécanisme de résistance de certaines tumeurs à ces CDBs et de caractériser de nouveaux inhibiteurs du NHEJ pour potentialiser l’effet de la radiothérapie. La voie NHEJ débute par la reconnaissance des deux extrémités de l’ADN d’une CDB par une protéine hétérodimérique Ku70/80 (Ku). Celle-ci sert de plateforme de recrutement aux autres facteurs de la voie NHEJ impliqués dans la maturation (processing) et la ligation des extrémités de l’ADN. Le premier volet de cette thèse a porté sur la caractérisation de sites d’interactions protéine-protéine de Ku et sur la caractérisation de nouvelles molécules comme médicaments potentiels dirigés contre Ku. Les sites d’interactions de deux partenaires majeurs de Ku, XLF et APLF, ont été caractérisés par calorimétrie en utilisant des peptides contenant le motif d’interaction de ces protéines appelés KBM (pour Ku-Binding Motifs) ou les protéines elles-mêmes. La structure cristallographique d’un complexe Ku-ADN avec une petite molécule, l’IP6, a été résolue. L’IP6 présente une forte affinité pour Ku avec un KD de 50 nM. Nous avons montré que cette molécule stimule la fixation de Ku sur l’ADN par trois méthodes biophysiques (calorimétrie, switchSENSE et sm-TIRF). Enfin, un criblage haut débit d’une chimiothèque de 987 molécules fragments de Sanofi a été réalisé in vitro par DSF et validé par RMN sur la protéine Ku. Ce crible a permis d’identifier 15 fragments susceptibles d’interagir avec Ku. Ces travaux ouvrent la voie pour la conception rationnelle d’inhibiteurs dérivés des peptides pXLF, pAPLF, de la molécules IP6 ou des fragments identifiés par le crible en greffant sur ces plateformes de nouvelles fonctions chimiques pour améliorer encore l’affinité et bloquer la voie NHEJ. Le second volet de cette thèse a porté sur des interactions de type protéine-ADN étudiées par Sanofi dans un projet de découverte de nouvelles drogues en oncologie et l’analyse de la valeur ajoutée d’une nouvelle approche le switchSENSE implémentée au CEA de Saclay. Nous avons analysé 4 constructions d’une protéine interagissant avec deux ADN et l’effet de 15 molécules inhibitrices identifiées en amont dans le service « Integrated Drug Discovery » de Sanofi. Les expériences de switchSENSE ont permis de caractériser les KD des 4 constructions pour l’ADN et de mesurer des constantes d’inhibition pour les inhibiteurs. Ces mesures sont en bon accord avec les mesures effectuées à Sanofi par SPR, MST et RMN et permettent de positionner le switchSENSE comme une approche prometteuse dans la validation de nouveaux inhibiteurs en particulier pour les interactions protéine-ADN.The maintenance of DNA integrity is central in all organisms to ensure successful life and reproduction.The DNA should be duplicated and transmitted to next generation cells in its entirety with a minimalamount of alteration. Double-strand breaks (DSBs) are the most toxic DNA damages and can originatefrom endogenous or exogenous stresses . In antic-cancer treatments, radiotherapy aims at generatingDSBs locally on tumor cells and thus induce their apoptosis. Current major challenge nowadays is tounderstand the mechanism of resistance of some tumors to these treatments and to characterize newinhibitors of NHEJ to potentiate the effect of radiotherapy. The NHEJ pathway begins with therecognition of CDBs extremities by a Ku70/80 (Ku) heterodimeric protein. Ku serves as a recruitmentplatform for the other factors of the NHEJ pathway involved in the processing and ligation of the DNAends. The first part of this thesis focuses on the characterization of Ku-protein interaction sites and onthe characterization of new molecules as potential drugs against Ku. The interaction sites of two majorKu partners, XLF and APLF, were characterized by calorimetry using peptides containing theinteraction motif of these proteins, called KBM (for Ku-Binding Motifs) or the proteins themselves. Thecrystallographic structure of a Ku-DNA complex with a small molecule, IP6, has been determined. TheIP6 has a strong affinity for Ku with a KD of 50 nM. We showed that this molecule stimulates Ku bindingon DNA by three biophysical methods (calorimetry, switchSENSE and sm-TIRF). Finally, a highthroughput screening of library of Sanofi fragments of 987 molecules was performed in vitro by DSFand validated by NMR on the Ku protein and identified 15 fragments that could interact with Ku. Thiswork paves the way for the rational design of inhibitors derived from the pXLF peptides, pAPLF, IP6molecules or fragments by grafting on these platforms new chemical functions to further improve theaffinity and inhibit the NHEJ pathway. The second part of this thesis focused on protein-DNAinteractions studied by Sanofi in a discovery drugs project in oncology and the analysis of the addedvalue of a new approach switchSENSE implemented at CEA Saclay. We analyzed 4 constructs of aprotein interacting with two DNAs and the effect of 15 inhibitory molecules identified upstream inSanofi's « Integrated Drug Discovery » service. The switchSENSE experiments made it possible tocharacterize the KD of the 4 constructs for DNA and to measure inhibition constants for the inhibitors.These measurements are in good agreement with the measurements made at Sanofi by SPR, MST andNMR. This study positions the switchSENSE as a promising approach in the validation of newinhibitors, in particular for protein-DNA interactions

Etude d'interactions protéine-ADN : Interactions Ku70/Ku80-ADN au sein de la voie NHEJ et caractérisation d'inhibiteurs d'une interaction protéine-ADN cible dans un contexte de recherche de médicaments

The maintenance of DNA integrity is central in all organisms to ensure successful life and reproduction.The DNA should be duplicated and transmitted to next generation cells in its entirety with a minimalamount of alteration. Double-strand breaks (DSBs) are the most toxic DNA damages and can originatefrom endogenous or exogenous stresses . In antic-cancer treatments, radiotherapy aims at generatingDSBs locally on tumor cells and thus induce their apoptosis. Current major challenge nowadays is tounderstand the mechanism of resistance of some tumors to these treatments and to characterize newinhibitors of NHEJ to potentiate the effect of radiotherapy. The NHEJ pathway begins with therecognition of CDBs extremities by a Ku70/80 (Ku) heterodimeric protein. Ku serves as a recruitmentplatform for the other factors of the NHEJ pathway involved in the processing and ligation of the DNAends. The first part of this thesis focuses on the characterization of Ku-protein interaction sites and onthe characterization of new molecules as potential drugs against Ku. The interaction sites of two majorKu partners, XLF and APLF, were characterized by calorimetry using peptides containing theinteraction motif of these proteins, called KBM (for Ku-Binding Motifs) or the proteins themselves. Thecrystallographic structure of a Ku-DNA complex with a small molecule, IP6, has been determined. TheIP6 has a strong affinity for Ku with a KD of 50 nM. We showed that this molecule stimulates Ku bindingon DNA by three biophysical methods (calorimetry, switchSENSE and sm-TIRF). Finally, a highthroughput screening of library of Sanofi fragments of 987 molecules was performed in vitro by DSFand validated by NMR on the Ku protein and identified 15 fragments that could interact with Ku. Thiswork paves the way for the rational design of inhibitors derived from the pXLF peptides, pAPLF, IP6molecules or fragments by grafting on these platforms new chemical functions to further improve theaffinity and inhibit the NHEJ pathway. The second part of this thesis focused on protein-DNAinteractions studied by Sanofi in a discovery drugs project in oncology and the analysis of the addedvalue of a new approach switchSENSE implemented at CEA Saclay. We analyzed 4 constructs of aprotein interacting with two DNAs and the effect of 15 inhibitory molecules identified upstream inSanofi's « Integrated Drug Discovery » service. The switchSENSE experiments made it possible tocharacterize the KD of the 4 constructs for DNA and to measure inhibition constants for the inhibitors.These measurements are in good agreement with the measurements made at Sanofi by SPR, MST andNMR. This study positions the switchSENSE as a promising approach in the validation of newinhibitors, in particular for protein-DNA interactions.Pour qu’un organisme soit viable, il est essentiel que la molécule d’ADN soit dupliquée et transmise aux générations filles dans son intégralité avec le moins d’altérations possibles. Des cassures double-brin (CDB) peuvent apparaitre suite à des stress endogènes ou exogènes et constituent les dommages de l’ADN les plus toxiques. La radiothérapie a pour but générer de manière localisée des CDBs dans les cellules tumorales et de déclencher leur apoptose. Un enjeu majeur actuel est de comprendre le mécanisme de résistance de certaines tumeurs à ces CDBs et de caractériser de nouveaux inhibiteurs du NHEJ pour potentialiser l’effet de la radiothérapie. La voie NHEJ débute par la reconnaissance des deux extrémités de l’ADN d’une CDB par une protéine hétérodimérique Ku70/80 (Ku). Celle-ci sert de plateforme de recrutement aux autres facteurs de la voie NHEJ impliqués dans la maturation (processing) et la ligation des extrémités de l’ADN. Le premier volet de cette thèse a porté sur la caractérisation de sites d’interactions protéine-protéine de Ku et sur la caractérisation de nouvelles molécules comme médicaments potentiels dirigés contre Ku. Les sites d’interactions de deux partenaires majeurs de Ku, XLF et APLF, ont été caractérisés par calorimétrie en utilisant des peptides contenant le motif d’interaction de ces protéines appelés KBM (pour Ku-Binding Motifs) ou les protéines elles-mêmes. La structure cristallographique d’un complexe Ku-ADN avec une petite molécule, l’IP6, a été résolue. L’IP6 présente une forte affinité pour Ku avec un KD de 50 nM. Nous avons montré que cette molécule stimule la fixation de Ku sur l’ADN par trois méthodes biophysiques (calorimétrie, switchSENSE et sm-TIRF). Enfin, un criblage haut débit d’une chimiothèque de 987 molécules fragments de Sanofi a été réalisé in vitro par DSF et validé par RMN sur la protéine Ku. Ce crible a permis d’identifier 15 fragments susceptibles d’interagir avec Ku. Ces travaux ouvrent la voie pour la conception rationnelle d’inhibiteurs dérivés des peptides pXLF, pAPLF, de la molécules IP6 ou des fragments identifiés par le crible en greffant sur ces plateformes de nouvelles fonctions chimiques pour améliorer encore l’affinité et bloquer la voie NHEJ. Le second volet de cette thèse a porté sur des interactions de type protéine-ADN étudiées par Sanofi dans un projet de découverte de nouvelles drogues en oncologie et l’analyse de la valeur ajoutée d’une nouvelle approche le switchSENSE implémentée au CEA de Saclay. Nous avons analysé 4 constructions d’une protéine interagissant avec deux ADN et l’effet de 15 molécules inhibitrices identifiées en amont dans le service « Integrated Drug Discovery » de Sanofi. Les expériences de switchSENSE ont permis de caractériser les KD des 4 constructions pour l’ADN et de mesurer des constantes d’inhibition pour les inhibiteurs. Ces mesures sont en bon accord avec les mesures effectuées à Sanofi par SPR, MST et RMN et permettent de positionner le switchSENSE comme une approche prometteuse dans la validation de nouveaux inhibiteurs en particulier pour les interactions protéine-ADN

Measurements of Protein-DNA Complexes Interactions by Isothermal Titration Calorimetry (ITC) and Microscale Thermophoresis (MST)

Interactions between protein complexes and DNA are central regulators of the cell life. They control the activation and inactivation of a large set of nuclear processes including transcription, replication, recombination, repair, and chromosome structures. In the literature, protein-DNA interactions are characterized by highly complementary approaches including large-scale studies and analyses in cells. Biophysical approaches with purified materials help to evaluate if these interactions are direct or not. They provide quantitative information on the strength and specificity of the interactions between proteins or protein complexes and their DNA substrates. Isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) are widely used and are complementary methods to characterize nucleo-protein complexes and quantitatively measure protein-DNA interactions. We present here protocols to analyze the interactions between a DNA repair complex, Ku70-Ku80 (Ku) (154 kDa), and DNA substrates. ITC is a label-free method performed with both partners in solution. It serves to determine the dissociation constant (Kd), the enthalpy (ΔH), and the stoichiometry N of an interaction. MST is used to measure the Kd between the protein or the DNA labeled with a fluorescent probe. We report the data obtained on Ku-DNA interactions with ITC and MST and discuss advantages and drawbacks of both the methods

Clickable Bambusurils to Access Multivalent Architectures

Publisher: American Chemical SocietyInternational audiencePropargylated bambus[4,6]urils were prepared by an efficient one-step condensation of dipropargylglycoluril with formaldehyde under microwave irradiation. Their functionalization by click chemistry (CuAAC) afforded new multivalent architectures decorated with 8 or 12 ligands. Grafting of glycosides provided water-soluble glycobambus[4,6]uril platforms with glucosyl12BU[6] showing good affinity toward iodide anion in aqueous medium

Structural Insights into the Intrinsically Disordered GPCR C:Terminal Region, Major Actor in Arrestin:GPCR Interaction

International audienceArrestin-dependent pathways are a central component of G protein-coupled receptor (GPCRs) signaling. However, the molecular processes regulating arrestin binding are to be further illuminated, in particular with regard to the structural impact of GPCR C-terminal disordered regions. Here, we used an integrated biophysical strategy to describe the basal conformations of the C-terminal domains of three class A GPCRs, the vasopressin V2 receptor (V2R), the growth hormone secretagogue or ghrelin receptor type 1a (GHSR) and the β2-adernergic receptor (β2AR). By doing so, we revealed the presence of transient secondary structures in these regions that are potentially involved in the interaction with arrestin. These secondary structure elements differ from those described in the literature in interaction with arrestin. This suggests a mechanism where the secondary structure conformational preferences in the C-terminal regions of GPCRs could be a central feature for optimizing arrestins recognitio

Structural and functional basis of inositol hexaphosphate stimulation of NHEJ through stabilization of Ku-XLF interaction

International audienceThe classical Non-Homologous End Joining (c-NHEJ) pathway is the predominant process in mammals for repairing endogenous, accidental or programmed DNA Double-Strand Breaks. c-NHEJ is regulated by several accessory factors, post-translational modifications, endogenous chemical agents and metabolites. The metabolite inositol-hexaphosphate (IP6) stimulates c-NHEJ by interacting with the Ku70–Ku80 heterodimer (Ku). We report cryo-EM structures of apo- and DNA-bound Ku in complex with IP6, at 3.5 Å and 2.74 Å resolutions respectively, and an X-ray crystallography structure of a Ku in complex with DNA and IP6 at 3.7 Å. The Ku-IP6 interaction is mediated predominantly via salt bridges at the interface of the Ku70 and Ku80 subunits. This interaction is distant from the DNA, DNA-PKcs, APLF and PAXX binding sites and in close proximity to XLF binding site. Biophysical experiments show that IP6 binding increases the thermal stability of Ku by 2°C in a DNA-dependent manner, stabilizes Ku on DNA and enhances XLF affinity for Ku. In cells, selected mutagenesis of the IP6 binding pocket reduces both Ku accrual at damaged sites and XLF enrolment in the NHEJ complex, which translate into a lower end-joining efficiency. Thus, this study defines the molecular bases of the IP6 metabolite stimulatory effect on the c-NHEJ repair activity

Structural and functional basis of inositol hexaphosphate stimulation of NHEJ through stabilization of Ku-XLF interaction

Acknowledgements: JBC thank the I2BC platforms for Interactions of Macromolecules (PIM) and for Crystallization supported by French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INBS-0005. We thank the HTX platform at EMBL, Grenoble. We thank Magali Aumont-Nicaise and Adrian Velazquez-Campoy for their help with ITC data. AG was supported by a CIFRE (ANRT) fellowship. JBC was supported by ANR-20-CE11-0026, ANR-17-CE12-0020 and ANR-21-CE12-0019-01. We also thank the staff from PROXIMA-1, PROXIMA-2a and SWING beamlines for their help in data collection and synchrotron SOLEIL (Saint-Aubin, France) for provision of synchrotron radiation facilities. SJ, MJM, and ER thank Mauro Modesti for providing SNAP tag Ku protein. PC, SB and PF were supported by the Ligue Contre le Cancer as Equipe Labellisée 2018 and ANR-20-CE11-0026. PC is a scientist from INSERM. We acknowledge the TRI imaging facility, member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04). We would like to thank the Imaging Core Facility TRI-IPBS, in particular Antonio Peixoto and Eve Pitot for technical assistance. A.C., A.K. and T.L.B. thank the Wellcome Trust for support of this research through the award of an Investigator Award (200814/Z/16/Z; 2016–2022).Funder: Ligue Contre le Cancer; DOI: https://doi.org/10.13039/501100004099The classical Non-Homologous End Joining (c-NHEJ) pathway is the predominant process in mammals for repairing endogenous, accidental or programmed DNA Double-Strand Breaks. c-NHEJ is regulated by several accessory factors, post-translational modifications, endogenous chemical agents and metabolites. The metabolite inositol-hexaphosphate (IP6) stimulates c-NHEJ by interacting with the Ku70–Ku80 heterodimer (Ku). We report cryo-EM structures of apo- and DNA-bound Ku in complex with IP6, at 3.5 Å and 2.74 Å resolutions respectively, and an X-ray crystallography structure of a Ku in complex with DNA and IP6 at 3.7 Å. The Ku-IP6 interaction is mediated predominantly via salt bridges at the interface of the Ku70 and Ku80 subunits. This interaction is distant from the DNA, DNA-PKcs, APLF and PAXX binding sites and in close proximity to XLF binding site. Biophysical experiments show that IP6 binding increases the thermal stability of Ku by 2°C in a DNA-dependent manner, stabilizes Ku on DNA and enhances XLF affinity for Ku. In cells, selected mutagenesis of the IP6 binding pocket reduces both Ku accrual at damaged sites and XLF enrolment in the NHEJ complex, which translate into a lower end-joining efficiency. Thus, this study defines the molecular bases of the IP6 metabolite stimulatory effect on the c-NHEJ repair activity

Structural and functional basis of inositol hexaphosphate stimulation of NHEJ through stabilization of Ku-XLF interaction.

Acknowledgements: JBC thank the I2BC platforms for Interactions of Macromolecules (PIM) and for Crystallization supported by French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INBS-0005. We thank the HTX platform at EMBL, Grenoble. We thank Magali Aumont-Nicaise and Adrian Velazquez-Campoy for their help with ITC data. AG was supported by a CIFRE (ANRT) fellowship. JBC was supported by ANR-20-CE11-0026, ANR-17-CE12-0020 and ANR-21-CE12-0019-01. We also thank the staff from PROXIMA-1, PROXIMA-2a and SWING beamlines for their help in data collection and synchrotron SOLEIL (Saint-Aubin, France) for provision of synchrotron radiation facilities. SJ, MJM, and ER thank Mauro Modesti for providing SNAP tag Ku protein. PC, SB and PF were supported by the Ligue Contre le Cancer as Equipe Labellisée 2018 and ANR-20-CE11-0026. PC is a scientist from INSERM. We acknowledge the TRI imaging facility, member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04). We would like to thank the Imaging Core Facility TRI-IPBS, in particular Antonio Peixoto and Eve Pitot for technical assistance. A.C., A.K. and T.L.B. thank the Wellcome Trust for support of this research through the award of an Investigator Award (200814/Z/16/Z; 2016–2022).Funder: Ligue Contre le Cancer; DOI: https://doi.org/10.13039/501100004099The classical Non-Homologous End Joining (c-NHEJ) pathway is the predominant process in mammals for repairing endogenous, accidental or programmed DNA Double-Strand Breaks. c-NHEJ is regulated by several accessory factors, post-translational modifications, endogenous chemical agents and metabolites. The metabolite inositol-hexaphosphate (IP6) stimulates c-NHEJ by interacting with the Ku70-Ku80 heterodimer (Ku). We report cryo-EM structures of apo- and DNA-bound Ku in complex with IP6, at 3.5 Å and 2.74 Å resolutions respectively, and an X-ray crystallography structure of a Ku in complex with DNA and IP6 at 3.7 Å. The Ku-IP6 interaction is mediated predominantly via salt bridges at the interface of the Ku70 and Ku80 subunits. This interaction is distant from the DNA, DNA-PKcs, APLF and PAXX binding sites and in close proximity to XLF binding site. Biophysical experiments show that IP6 binding increases the thermal stability of Ku by 2°C in a DNA-dependent manner, stabilizes Ku on DNA and enhances XLF affinity for Ku. In cells, selected mutagenesis of the IP6 binding pocket reduces both Ku accrual at damaged sites and XLF enrolment in the NHEJ complex, which translate into a lower end-joining efficiency. Thus, this study defines the molecular bases of the IP6 metabolite stimulatory effect on the c-NHEJ repair activity

Structural and functional basis of inositol hexaphosphate stimulation of NHEJ through stabilization of Ku-XLF interaction

Acknowledgements: JBC thank the I2BC platforms for Interactions of Macromolecules (PIM) and for Crystallization supported by French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INBS-0005. We thank the HTX platform at EMBL, Grenoble. We thank Magali Aumont-Nicaise and Adrian Velazquez-Campoy for their help with ITC data. AG was supported by a CIFRE (ANRT) fellowship. JBC was supported by ANR-20-CE11-0026, ANR-17-CE12-0020 and ANR-21-CE12-0019-01. We also thank the staff from PROXIMA-1, PROXIMA-2a and SWING beamlines for their help in data collection and synchrotron SOLEIL (Saint-Aubin, France) for provision of synchrotron radiation facilities. SJ, MJM, and ER thank Mauro Modesti for providing SNAP tag Ku protein. PC, SB and PF were supported by the Ligue Contre le Cancer as Equipe Labellisée 2018 and ANR-20-CE11-0026. PC is a scientist from INSERM. We acknowledge the TRI imaging facility, member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04). We would like to thank the Imaging Core Facility TRI-IPBS, in particular Antonio Peixoto and Eve Pitot for technical assistance. A.C., A.K. and T.L.B. thank the Wellcome Trust for support of this research through the award of an Investigator Award (200814/Z/16/Z; 2016–2022).Funder: Ligue Contre le Cancer; DOI: https://doi.org/10.13039/501100004099The classical Non-Homologous End Joining (c-NHEJ) pathway is the predominant process in mammals for repairing endogenous, accidental or programmed DNA Double-Strand Breaks. c-NHEJ is regulated by several accessory factors, post-translational modifications, endogenous chemical agents and metabolites. The metabolite inositol-hexaphosphate (IP6) stimulates c-NHEJ by interacting with the Ku70–Ku80 heterodimer (Ku). We report cryo-EM structures of apo- and DNA-bound Ku in complex with IP6, at 3.5 Å and 2.74 Å resolutions respectively, and an X-ray crystallography structure of a Ku in complex with DNA and IP6 at 3.7 Å. The Ku-IP6 interaction is mediated predominantly via salt bridges at the interface of the Ku70 and Ku80 subunits. This interaction is distant from the DNA, DNA-PKcs, APLF and PAXX binding sites and in close proximity to XLF binding site. Biophysical experiments show that IP6 binding increases the thermal stability of Ku by 2°C in a DNA-dependent manner, stabilizes Ku on DNA and enhances XLF affinity for Ku. In cells, selected mutagenesis of the IP6 binding pocket reduces both Ku accrual at damaged sites and XLF enrolment in the NHEJ complex, which translate into a lower end-joining efficiency. Thus, this study defines the molecular bases of the IP6 metabolite stimulatory effect on the c-NHEJ repair activity