ATP driven structural changes of the bacterial Mre11:Rad50 catalytic head complex

DNA double-strand breaks (DSBs) threaten genome stability in all kingdoms of life and are linked to cancerogenic chromosome aberrations in humans. The Mre11:Rad50 (MR) complex is an evolutionarily conserved complex of two Rad50 ATPases and a dimer of the Mre11 nuclease that senses and processes DSBs and tethers DNA for repair. ATP binding and hydrolysis by Rad50 is functionally coupled to DNA-binding and tethering, but also regulates Mre11's nuclease in processing DNA ends. To understand how ATP controls the interaction between Mre11 and Rad50, we determined the crystal structure of Thermotoga maritima (Tm) MR trapped in an ATP/ADP state. ATP binding to Rad50 induces a large structural change from an open form with accessible Mre11 nuclease sites into a closed form. Remarkably, the NBD dimer binds in the Mre11 DNA-binding cleft blocking Mre11's dsDNA-binding sites. An accompanying large swivel of the Rad50 coiled coil domains appears to prepare the coiled coils for DNA tethering. DNA-binding studies show that within the complex, Rad50 likely forms a dsDNA-binding site in response to ATP, while the Mre11 nuclease module retains a ssDNA-binding site. Our results suggest a possible mechanism for ATP-dependent DNA tethering and DSB processing by MR

Structural Biochemistry of ATP-Driven Dimerization and DNA-Stimulated Activation of SMC ATPases

AbstractStructural maintenance of chromosome (SMC) proteins play a central role in higher-order chromosome structure in all kingdoms of life [1–5]. SMC proteins consist of a long coiled-coil domain that joins an ATP binding cassette (ABC) ATPase domain on one side and a dimerization domain on the other side [6]. SMC proteins require ATP binding or hydrolysis to promote cohesion and condensation, which is suggested to proceed via formation of SMC rings or assemblies [7–11]. To learn more about the role of ATP in the architecture of SMC proteins, we report crystal structures of nucleotide-free and ATP bound P. furiosus SMC ATPase domains. ATP dimerizes two SMC ATPase domains by binding to opposing Walker A and signature motifs, indicating that ATP binding can directly assemble SMC proteins. DNA stimulates ATP hydrolysis in the engaged SMC ABC domains, suggesting that ATP hydrolysis can be allosterically regulated. Structural and mutagenesis data identify an SMC protein conserved-arginine finger that is required for DNA stimulation of the ATPase activity and directly connects a putative DNA interaction site to ATP. Our results suggest that stimulation of the SMC ATPase activity may be a specific feature to regulate the ATP-driven assembly and disassembly of SMC proteins

Structure of Mre11–Nbs1 complex yields insights into ataxia-telangiectasia–like disease mutations and DNA damage signaling

The Mre11–Rad50–Nbs1 (MRN) complex tethers, processes and signals DNA double strand breaks, promoting genomic stability. To understand the functional architecture of MRN, we determined the crystal structures of the Schizosaccharomyces pombe Mre11 dimeric catalytic domain alone and in complex with a fragment of Nbs1. Two Nbs1 subunits stretch around the outside of Mre11’s nuclease domains, with one subunit additionally bridging and locking the Mre11 dimer via a highly conserved asymmetrical binding motif. Our results reveal that Mre11 forms a flexible dimer and suggest that Nbs1 is not only a checkpoint adaptor, but also functionally impacts on Mre11-Rad50. Clinical mutations in Mre11 are located along the Nbs1 interaction sites and weaken the Mre11–Nbs1 interaction. However, they differentially affect DNA repair and telomere maintenance in Saccharomyces cerevisiae, potentially providing insight into their different human disease pathologies

The Mre11:Rad50 Structure Shows an ATP-Dependent Molecular Clamp in DNA Double-Strand Break Repair

SummaryThe MR (Mre11 nuclease and Rad50 ABC ATPase) complex is an evolutionarily conserved sensor for DNA double-strand breaks, highly genotoxic lesions linked to cancer development. MR can recognize and process DNA ends even if they are blocked and misfolded. To reveal its mechanism, we determined the crystal structure of the catalytic head of Thermotoga maritima MR and analyzed ATP-dependent conformational changes. MR adopts an open form with a central Mre11 nuclease dimer and two peripheral Rad50 molecules, a form suited for sensing obstructed breaks. The Mre11 C-terminal helix-loop-helix domain binds Rad50 and attaches flexibly to the nuclease domain, enabling large conformational changes. ATP binding to the two Rad50 subunits induces a rotation of the Mre11 helix-loop-helix and Rad50 coiled-coil domains, creating a clamp conformation with increased DNA-binding activity. The results suggest that MR is an ATP-controlled transient molecular clamp at DNA double-strand breaks.PaperFlic

Targeted CD47 checkpoint blockade using a mesothelin-directed antibody construct for enhanced solid tumor-specific immunotherapy

Abstract The immune checkpoint CD47 is highly upregulated in several cancers as an innate immune escape mechanism. CD47 delivers a “don’t eat me” signal to its co-receptor signal regulatory protein α (SIRPα), thereby inhibiting phagocytosis. Blocking the CD47–SIRPα axis is a promising immunotherapeutic strategy against cancer. However, early trial data has demonstrated on-target off-leukemia toxicity. In addition, the ubiquitous expression pattern of CD47 might contribute to an antigen sink. In this study, we combined low-affinity CD47 checkpoint blockade and specific tumor targeting in a multivalent and multifunctional antibody construct to prevent CD47-related toxicities. First, we established a local inhibitory checkpoint monoclonal antibody (LicMAb) by fusing two N-terminal extracellular domains of SIRPα to a full-length anti-human mesothelin (MSLN)-IgG1 antibody, a well-described tumor-associated antigen in epithelial ovarian cancer (EOC) and pancreatic ductal adenocarcinoma (PDAC). Next, we evaluated the SIRPα-αMSLN LicMAb for mediating a tumor-restricted immune response as observed by antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Our data validates CD47 and MSLN as highly upregulated targets expressed on various solid cancer entities, particularly EOC. We show tumor-specific binding and CD47 blocking by the SIRPα-αMSLN LicMAb even in the presence of healthy CD47-expressing cells. Furthermore, the LicMAb induces NK-cell-mediated cytotoxicity and improves phagocytosis of EOC and PDAC tumor cells. Moreover, cell death in EOC-derived organoids was specifically LicMAb-driven. Hence, the SIRPα-αMSLN LicMAb combines a tumor-restricted blockade of the CD47–SIRPα axis with a specific antitumor response while preventing on-target off-tumor toxicities. Our data supports the multifunctional SIRPα-αMSLN LicMAb as a promising approach to treating solid tumors. Graphical abstract The local inhibitory checkpoint monoclonal antibody (LicMAb) binds mesothelin (MSLN) with high affinity and simultaneously blocks CD47 on MSLN-expressing tumor cells to inhibit the “don’t eat me” signal. CD47 is blocked by the fused extracellular SIRPα domain that intrinsically has a low affinity. Furthermore, the SIRPα-αMSLN LicMAb is based on a human IgG1 backbone to provide an Fc receptor (FcR)-activating stimulus to enable direct NK-cell-mediated killing by granzyme B (GrzB) and perforin secretion, and an additional pro-phagocytic signal to phagocytic cells, such as macrophages (MØ). This leads to tumor-restricted antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) of cancer cells. This scheme was created with BioRender (BioRender.com/g77u465)

CD19-specific triplebody SPM-1 engages NK and γδ T cells for rapid and efficient lysis of malignant B-lymphoid cells

Triplebodies are antibody-derived recombinant proteins carrying 3 antigen-binding domains in a single polypeptide chain. Triplebody SPM-1 was designed for lysis of CD19-bearing malignant B-lymphoid cells through the engagement of CD16-expressing cytolytic effectors, including NK and γδ T cells.SPM-1 is an optimized version of triplebody ds(19-16-19) and includes humanization, disulfide stabilization and the removal of potentially immunogenic sequences. A three-step chromatographic procedure yielded 1.7 - 5.5 mg of purified, monomeric protein per liter of culture medium. In cytolysis assays with NK cell effectors, SPM-1 mediated potent lysis of cancer-derived B cell lines and primary cells from patients with various B-lymphoid malignancies, which surpassed the ADCC activity of the therapeutic antibody Rituximab. EC50-values ranged from 3 to 86 pM. Finally, in an impedance-based assay, SPM-1 mediated a particularly rapid lysis of CD19-bearing target cells by engaging and activating both primary and expanded human γδ T cells from healthy donors as effectors.These data establish SPM-1 as a useful tool for a kinetic analysis of the cytolytic reactions mediated by γδ T and NK cells and as an agent deserving further development towards clinical use for the treatment of B-lymphoid malignancies