HDX-MS Combining Molecular Dynamics Simulation Reveals Structural Basis of Transcriptional Coactivator PC4 Binding to a Platinum Crosslinked Double-Stranded DNA

<p>Human nuclear protein positive cofactor PC4 is a DNA-binding protein, and plays an important role in the early response to DNA damage by recognizing single-stranded oligodeoxynucleotide domain and facilitating the subsequent steps of DNA repair. Our group previously discovered that PC4 selectively binds to a double-stranded oligodeoxynucleotide (dsODN) damaged by a trans-platinum anticancer complex, trans-[PtCl2(NH3)(thiazole)] (trans-PtTz). However, the molecular basis of this unique recognition and interaction remained unclear. In this work, amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) was applied to dissect the interaction interface between PC4 and a 15-mer double-stranded oligodeoxynucleotide crosslinked by trans-PtTz (trans-PtTz-dsODN). Global deuterium uptake suggested a 1:1 binding stoichiometry of PC4 to trans-PtTz-dsODN. Local deuterium uptake revealed that the flexible N-terminal loop and the β3 – β5-sheet played key roles in the recognition and interaction between PC4 and trans-PtTz-dsODN. In order to locate the key amino acid residues, molecular dynamics simulation was employed, demonstrating that PC4 binds to the trans-PtTz-dsODN at the minor groove via strong H-bonds with the nucleobases of the complementary strand. The minor groove width was broadened to adapt for the binding of PC4. Arg86 residue in PC4 was shown to dominate the recognition and interaction, which was verified by electrophoretic mobility shift assays. This work profiles the detailed interaction mechanism of PC4 with trans-PtTz damaged dsODN, and the combination use of HDX-MS and molecular dynamics simulation provides a new paradigm for the future research of the cellular response to the platinum induced DNA damage.</p>

HDX-MS Combining Molecular Dynamics Simulation Reveals Structural Basis of Transcriptional Coactivator PC4 Binding to a Platinum Crosslinked Double-Stranded DNA

<p>Human nuclear protein positive cofactor PC4 is a DNA-binding protein, and plays an important role in the early response to DNA damage by recognizing single-stranded oligodeoxynucleotide domain and facilitating the subsequent steps of DNA repair. Our group previously discovered that PC4 selectively binds to a double-stranded oligodeoxynucleotide (dsODN) damaged by a trans-platinum anticancer complex, trans-[PtCl2(NH3)(thiazole)] (trans-PtTz). However, the molecular basis of this unique recognition and interaction remained unclear. In this work, amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) was applied to dissect the interaction interface between PC4 and a 15-mer double-stranded oligodeoxynucleotide crosslinked by trans-PtTz (trans-PtTz-dsODN). Global deuterium uptake suggested a 1:1 binding stoichiometry of PC4 to trans-PtTz-dsODN. Local deuterium uptake revealed that the flexible N-terminal loop and the β3 – β5-sheet played key roles in the recognition and interaction between PC4 and trans-PtTz-dsODN. In order to locate the key amino acid residues, molecular dynamics simulation was employed, demonstrating that PC4 binds to the trans-PtTz-dsODN at the minor groove via strong H-bonds with the nucleobases of the complementary strand. The minor groove width was broadened to adapt for the binding of PC4. Arg86 residue in PC4 was shown to dominate the recognition and interaction, which was verified by electrophoretic mobility shift assays. This work profiles the detailed interaction mechanism of PC4 with trans-PtTz damaged dsODN, and the combination use of HDX-MS and molecular dynamics simulation provides a new paradigm for the future research of the cellular response to the platinum induced DNA damage.</p>

Assessing Structure-Activity Relationships in FVIII By Integration of Structural, Biophysical, and Biochemical Data Obtained with Anti-FVIII Antibodies

Introduction Anti-factor VIII (FVIII) antibodies are valuable probes for evaluating structure-activity relationships in FVIII. In this study, we structurally and functionally characterized a panel of anti-FVIII antibodies comprising 36 mouse monoclonal antibodies and 1 camelid nanobody with specificities that span all 5 structural domains of FVIII. Integration of these data into a comprehensive functional interaction map reveals regions that are essential for the function of FVIII and provides a useful guide for researchers engaged in the engineering, quantitation, and purification of FVIII and variants thereof. Materials and Methods Monoclonal mouse IgGs were raised by conventional hybridoma methods or obtained from commercial sources (Green Mountain Antibodies, American Diagnostica). Recombinant B domain-deleted human FVIII (BDD-rFVIII), consisting of a mixture of proteolytically processed forms and dual-chain forms, was generated by transfection of HEK293 cells and purified by immuno-affinity chromatography to >95% purity. Human plasma-derived von Willebrand factor (VWF) was purchased from Haematologic Technologies. The chain and domain specificities of each antibody and competition with VWF for FVIII binding were evaluated by biolayer interferometry (BLI; ForteBio Octet). Pairwise epitope overlap analysis (binning) was performed by both BLI and multiplexed surface plasmon resonance (SPR; BioRad ProteOn). The affinity of each antibody for FVIII was determined by SPR (ProteOn, Biacore T200), and the inhibitory activity of each antibody was determined by the standard Bethesda assay using normal human plasma. The ability of antibodies to inhibit the thrombin-mediated cleavage of FVIII was determined by resolving samples from a digestion time-course on SDS-PAGE and subsequent quantitation by gel densitometry. The morphology of FVIII-Fab fragment complexes was determined by negative stain electron microscopy (EM) with images classified into 25 groups. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was used to localize antibody epitopes on FVIII by evaluating deuterium uptake in the presence and absence of selected antibodies. Differential deuterium uptake over four hours was evaluated for over 500 unique FVIII-derived peptides that resulted from cleavage with pepsin, with greater than 95% sequence coverage of BDD-FVIII. Results and Conclusions Antibodies in this panel were characterized with respect to FVIII chain and domain specificity. Fifteen antibodies were mapped to the FVIII heavy chain (3 in the A1 domain, 12 in the A2 domain), and 22 antibodies were mapped to the FVIII light chain (8 in A3/C1 domains, 14 in C2 domain). Affinities for FVIII varied widely, with KD values ranging from ~10 nM to ~10 pM. Bethesda inhibitory activities also varied widely, from undetectable levels to greater than 40,000 Bethesda units (BU) per milligram. No correlation was observed between antibody affinity and inhibitory activity, but inhibitory activity was strongly associated with distinct clusters of antibodies having overlapping epitopes. Nine antibodies competed with VWF for binding to FVIII (2 in C1 domain, 7 in C2 domain), and these antibodies were distributed among 3 distinct epitope clusters, indicating that the VWF binding interface is broadly distributed across the C1 and C2 domains. Three C2 domain-specific antibodies that compete with VWF for binding to FVIII also interfered with the liberation of the acidic a3 peptide by thrombin in the absence of VWF. Thrombin-mediated cleavage at other sites in FVIII was not affected by these antibodies, suggesting the presence of a thrombin binding site within the C2 domain that is required specifically for cleavage by thrombin at the N-terminus of the FVIII light chain. The structures of FVIII in complex with 15 individual antibody Fab fragments were determined by negative stain EM for both inhibitory and non-inhibitory antibodies, and precise epitope mapping of selected antibodies was achieved by HDX-MS. Collectively, these data were used to generate an FVIII-antibody interaction map that integrates activity data with structural data spanning the atomic level to the gross morphological level. This integrated map can serve as a resource for the evaluation of structure-activity relationships in FVIII. Disclosures Liu: Biogen Idec: Employment. Bou-Assaf:Biogen Idec: Employment. Zhang:Biogen Idec: Employment. Goodman:Biogen Idec: Employment. Walz:Biogen Idec: Honoraria, Research Funding. Peters:Biogen Idec: Employment. Kulman:Biogen Idec: Employment.