Structural principles of peptide-centric chimeric antigen receptor recognition guide therapeutic expansion.

Peptide-centric chimeric antigen receptors (PC-CARs) recognize oncoprotein epitopes displayed by cell-surface human leukocyte antigens (HLAs) and offer a promising strategy for targeted cancer therapy. We have previously developed a PC-CAR targeting a neuroblastoma-associated PHOX2B peptide, leading to robust tumor cell lysis restricted by two common HLA allotypes. Here, we determine the 2.1-angstrom crystal structure of the PC-CAR-PHOX2B-HLA-A*24:02-ß2m complex, which reveals the basis for antigen-specific recognition through interactions with CAR complementarity-determining regions (CDRs). This PC-CAR adopts a diagonal docking mode, where interactions with both conserved and polymorphic HLA framework residues permit recognition of multiple HLA allotypes from the A9 serological cross-reactive group, covering a combined global population frequency of up to 46.7%. Biochemical binding assays, molecular dynamics simulations, and structural and functional analyses demonstrate that high-affinity PC-CAR recognition of cross-reactive pHLAs necessitates the presentation of a specific peptide backbone, where subtle structural adaptations of the peptide are critical for high-affinity complex formation, and CAR T cell killing. Our results provide a molecular blueprint for engineering CARs with optimal recognition of tumor-associated antigens in the context of different HLAs, while minimizing cross-reactivity with self-epitopes.

Structural principles of peptide-centric Chimeric Antigen Receptor recognition guide therapeutic expansion.

Peptide-Centric Chimeric Antigen Receptors (PC-CARs), which recognize oncoprotein epitopes displayed by human leukocyte antigens (HLAs) on the cell surface, offer a promising strategy for targeted cancer therapy 1 . We have previously developed a PC-CAR targeting a neuroblastoma- associated PHOX2B peptide, leading to robust tumor cell lysis restricted by two common HLA allotypes 2 . Here, we determine the 2.1 Å structure of the PC-CAR:PHOX2B/HLA-A*24:02/ß2m complex, which reveals the basis for antigen-specific recognition through interactions with CAR complementarity-determining regions (CDRs). The PC-CAR adopts a diagonal docking mode, where interactions with both conserved and polymorphic HLA framework residues permit recognition of multiple HLA allotypes from the A9 serological cross-reactivity group, covering a combined American population frequency of up to 25.2%. Comprehensive characterization using biochemical binding assays, molecular dynamics simulations, and structural and functional analyses demonstrate that high-affinity PC-CAR recognition of cross-reactive pHLAs necessitates the presentation of a specific peptide backbone, where subtle structural adaptations of the peptide are critical for high-affinity complex formation and CAR-T cell killing. Our results provide a molecular blueprint for engineering CARs with optimal recognition of tumor-associated antigens in the context of different HLAs, while minimizing cross-reactivity with self-epitopes.

Decoupling peptide binding from T cell receptor recognition with engineered chimeric MHC-I molecules.

Major Histocompatibility Complex class I (MHC-I) molecules display self, viral or aberrant epitopic peptides to T cell receptors (TCRs), which employ interactions between complementarity-determining regions with both peptide and MHC-I heavy chain 'framework' residues to recognize specific Human Leucocyte Antigens (HLAs). The highly polymorphic nature of the HLA peptide-binding groove suggests a malleability of interactions within a common structural scaffold. Here, using structural data from peptide:MHC-I and pMHC:TCR structures, we first identify residues important for peptide and/or TCR binding. We then outline a fixed-backbone computational design approach for engineering synthetic molecules that combine peptide binding and TCR recognition surfaces from existing HLA allotypes. X-ray crystallography demonstrates that chimeric molecules bridging divergent HLA alleles can bind selected peptide antigens in a specified backbone conformation. Finally, in vitro tetramer staining and biophysical binding experiments using chimeric pMHC-I molecules presenting established antigens further demonstrate the requirement of TCR recognition on interactions with HLA framework residues, as opposed to interactions with peptide-centric Chimeric Antigen Receptors (CARs). Our results underscore a novel, structure-guided platform for developing synthetic HLA molecules with desired properties as screening probes for peptide-centric interactions with TCRs and other therapeutic modalities.

Differential recognition of canonical NF-κB dimers by Importin α3.

Nuclear translocation of the p50/p65 heterodimer is essential for NF-κB signaling. In unstimulated cells, p50/p65 is retained by the inhibitor IκBα in the cytoplasm that masks the p65-nuclear localization sequence (NLS). Upon activation, p50/p65 is translocated into the nucleus by the adapter importin α3 and the receptor importin ß. Here, we describe a bipartite NLS in p50/p65, analogous to nucleoplasmin NLS but exposed in trans. Importin α3 accommodates the p50- and p65-NLSs at the major and minor NLS-binding pockets, respectively. The p50-NLS is the predominant binding determinant, while the p65-NLS induces a conformational change in the Armadillo 7 of importin α3 that stabilizes a helical conformation of the p65-NLS. Neither conformational change was observed for importin α1, which makes fewer bonds with the p50/p65 NLSs, explaining the preference for α3. We propose that importin α3 discriminates between the transcriptionally active p50/p65 heterodimer and p50/p50 and p65/65 homodimers, ensuring fidelity in NF-κB signaling.

Biophysical analysis of Pseudomonas-phage PaP3 small terminase suggests a mechanism for sequence-specific DNA-binding by lateral interdigitation.

The genome packaging motor of tailed bacteriophages and herpesviruses is a powerful nanomachine built by several copies of a large (TerL) and a small (TerS) terminase subunit. The motor assembles transiently at the portal vertex of an empty precursor capsid (or procapsid) to power genome encapsidation. Terminase subunits have been studied in-depth, especially in classical bacteriophages that infect Escherichia coli or Salmonella, yet, less is known about the packaging motor of Pseudomonas-phages that have increasing biomedical relevance. Here, we investigated the small terminase subunit from three Podoviridae phages that infect Pseudomonas aeruginosa. We found TerS is polymorphic in solution but assembles into a nonamer in its high-affinity heparin-binding conformation. The atomic structure of Pseudomonas phage PaP3 TerS, the first complete structure for a TerS from a cos phage, reveals nine helix-turn-helix (HTH) motifs asymmetrically arranged around a ß-stranded channel, too narrow to accommodate DNA. PaP3 TerS binds DNA in a sequence-specific manner in vitro. X-ray scattering and molecular modeling suggest TerS adopts an open conformation in solution, characterized by dynamic HTHs that move around an oligomerization core, generating discrete binding crevices for DNA. We propose a model for sequence-specific recognition of packaging initiation sites by lateral interdigitation of DNA.

Molecular Architecture of the Inositol Phosphatase Siw14.

Siw14 is a recently discovered inositol phosphatase implicated in suppressing prion propagation in Saccharomyces cerevisiae. In this paper, we used hybrid structural methods to decipher Siw14 molecular architecture. We found the protein exists in solution as an elongated monomer that is ∼140 Šin length, containing an acidic N-terminal domain and a basic C-terminal dual-specificity phosphatase (DSP) domain, structurally similar to the glycogen phosphatase laforin. The two domains are connected by a protease susceptible linker and do not interact in vitro. The crystal structure of Siw14-DSP reveals a highly basic phosphate-binding loop and an ∼10 Šdeep substrate-binding crevice that evolved to dephosphorylate pyro-phosphate moieties. A pseudoatomic model of the full-length phosphatase generated from solution, crystallographic, biochemical, and modeling data sheds light on the interesting zwitterionic nature of Siw14, which we hypothesized may play a role in discriminating negatively charged inositol phosphates.

Synergy of two low-affinity NLSs determines the high avidity of influenza A virus nucleoprotein NP for human importin α isoforms.

The influenza A virus nucleoprotein (NP) is an essential multifunctional protein that encapsidates the viral genome and functions as an adapter between the virus and the host cell machinery. NPs from all strains of influenza A viruses contain two nuclear localization signals (NLSs): a well-studied monopartite NLS1 and a less-characterized NLS2, thought to be bipartite. Through site-directed mutagenesis and functional analysis, we found that NLS2 is also monopartite and is indispensable for viral infection. Atomic structures of importin α bound to two variants of NLS2 revealed NLS2 primarily binds the major-NLS binding site of importin α, unlike NLS1 that associates with the minor NLS-pocket. Though peptides corresponding to NLS1 and NLS2 bind weakly to importin α, the two NLSs synergize in the context of the full length NP to confer high avidity for importin α7, explaining why the virus efficiently replicates in the respiratory tract that exhibits high levels of this isoform. This study, the first to functionally characterize NLS2, demonstrates NLS2 plays an important and unexpected role in influenza A virus infection. We propose NLS1 and NLS2 form a bipartite NLS in trans, which ensures high avidity for importin α7 while preventing non-specific binding to viral RNA.