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The structure of the antigen‐binding fragment (Fab) of mouse monoclonal antibody 7H2.2 in complex with a 15‐residue fragment from the metalloproteinase sperm acrosomal SLLP1 binding protein (SAS1B), which is a molecular and cellular candidate for both cancer therapy and female contraception, has been determined at 2.75 Å resolution by single‐crystal X‐ray diffraction. Although the crystallization conditions contained the final 148 C‐terminal residues of SAS1B, the Fab was observed to crystallize in complex with a 15‐residue fragment corresponding to one of only two elements of secondary structure that are predicted to be ordered within the C‐terminal region of SAS1B. The antigen forms an amphipathic α‐helix that binds the 7H2.2 combining site via hydrophilic residues in an epitope that spans the length of the antigen α‐helix, with only two CH–π interactions observed along the edge of the interface between the antibody and antigen. Interestingly, the paratope contains two residues mutated away from the germline (YL32F and YH58R), as well as a ProH96‐ThrH97‐AspH98‐AspH99 insertion within heavy chain CDR3. The intact 7H2.2 antibody exhibits high affinity for the SAS1B antigen, with 1:1 binding and nanomolar affinity for both the SAS1B C‐terminal construct used for crystallization (3.38 ± 0.59 nM) and a 15‐amino‐acid synthetic peptide construct corresponding to the helical antigen observed within the crystal structure (1.60 ± 0.31 nM). The SAS1B–antibody structure provides the first structural insight into any portion of the subdomain architecture of the C‐terminal region of the novel cancer‐oocyte tumor surface neoantigen SAS1B and provides a basis for the targeted use of SAS1B. [ABSTRACT FROM AUTHOR]

Summary: The Toxoplasma gondii locus mitochondrial association factor 1 (MAF1) encodes multiple paralogs, some of which mediate host mitochondrial association (HMA). Previous work showed that HMA was a trait that arose in T. gondii through neofunctionalization of an ancestral MAF1 ortholog. Structural analysis of HMA‐competent and incompetent MAF1 paralogs (MAF1b and MAF1a, respectively) revealed that both paralogs harbor an ADP ribose binding macro‐domain, with comparatively low (micromolar) affinity for ADP ribose. Replacing the 16 C‐terminal residues of MAF1b with those of MAF1a abrogated HMA, and we also show that only three residues in the C‐terminal helix are required for MAF1‐mediated HMA. Importantly these same three residues are also required for the in vivo growth advantage conferred by MAF1b, providing a definitive link between in vivo proliferation and manipulation of host mitochondria. Co‐immunoprecipitation assays reveal that the ability to interact with the mitochondrial MICOS complex is shared by HMA‐competent and incompetent MAF1 paralogs and mutants. The weak ADPr coordination and ability to interact with the MICOS complex shared between divergent paralogs may represent modular ancestral functions for this tandemly expanded and diversified T. gondii locus. [ABSTRACT FROM AUTHOR]