Search

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]

Host mitochondrial association (HMA) is a well-known phenomenon during Toxoplasma gondii infection of the host cell. The T. gondii locus mitochondrial association factor 1 (MAF1) is required for HMA and MAF1 encodes distinct paralogs of secreted dense granule effector proteins, some of which mediate the HMA phenotype (MAF1b paralogs drive HMA; MAF1a paralogs do not). To identify host proteins required for MAF1b-mediated HMA, we performed unbiased, label-free quantitative proteomics on host cells infected with type II parasites expressing MAF1b, MAF1a, and an HMA-incompetent MAF1b mutant. Across these samples, we identified ~1,360 MAF1-interacting proteins, but only 13 that were significantly and uniquely enriched in MAF1b pull-downs. The gene products include multiple mitochondria-associated proteins, including those that traffic to the mitochondrial outer membrane. Based on follow-up endoribonuclease-prepared short interfering RNA (esiRNA) experiments targeting these candidate MAF1btargeted host factors, we determined that the mitochondrial receptor protein TOM70 and mitochondria-specific chaperone HSPA9 were essential mediators of HMA. Additionally, the enrichment of TOM70 at the parasitophorous vacuole membrane interface suggests parasite-driven sequestration of TOM70 by the parasite. These results show that the interface between the T. gondii vacuole and the host mitochondria is characterized by interactions between a single parasite effector and multiple target host proteins, some of which are critical for the HMA phenotype itself. The elucidation of the functional members of this complex will permit us to explain the link between HMA and changes in the biology of the host cell. [ABSTRACT FROM AUTHOR]

The lipid kinase PI4KB, which generates phosphatidylinositol 4‐phosphate (PI4P), is a key enzyme in regulating membrane transport and is also hijacked by multiple picornaviruses to mediate viral replication. PI4KB can interact with multiple protein binding partners, which are differentially manipulated by picornaviruses to facilitate replication. The protein c10orf76 is a PI4KB‐associated protein that increases PI4P levels at the Golgi and is essential for the viral replication of specific enteroviruses. We used hydrogen–deuterium exchange mass spectrometry to characterize the c10orf76‐PI4KB complex and reveal that binding is mediated by the kinase linker of PI4KB, with formation of the heterodimeric complex modulated by PKA‐dependent phosphorylation. Complex‐disrupting mutations demonstrate that PI4KB is required for membrane recruitment of c10orf76 to the Golgi, and that an intact c10orf76‐PI4KB complex is required for the replication of c10orf76‐dependent enteroviruses. Intriguingly, c10orf76 also contributed to proper Arf1 activation at the Golgi, providing a putative mechanism for the c10orf76‐dependent increase in PI4P levels at the Golgi. Synopsis: Enteroviruses hijack host PI4KB and GBF1 to build replication organelles. This study reveals that the direct interaction between c10orf76 and PI4KB is required for c10orf76 recruitment to the Golgi and the replication of c10orf76‐dependent enteroviruses. c10orf76 is localized to the Golgi through a direct interaction with the kinase linker of PI4KB.c10orf76 regulates GBF1 and active Arf1 dynamics that control Golgi PI4P levels.Enteroviruses that rely on c10orf76 for replication depend on formation of the c10orf76‐PI4KB complex. [ABSTRACT FROM AUTHOR]

Parasites of the phylum Apicomplexa are responsible for significant morbidity and mortality on a global scale. Central to the virulence of these pathogens are the phylum-specific, unconventional class XIV myosins that power the essential processes of parasite motility and host cell invasion. Notably, class XIV myosins differ from human myosins in key functional regions, yet they are capable of fast movement along actin filaments with kinetics rivaling previously studied myosins. Toward establishing a detailed molecular mechanism of class XIV motility, we determined the 2.6-Å resolution crystal structure of the Toxoplasma gondii MyoA (TgMyoA) motor domain. Structural analysis reveals intriguing strategies for force transduction and chemomechanical coupling that rely on a divergent SH1/SH2 region, the class-defining "HYAG"-site polymorphism, and the actin-binding surface. In vitro motility assays and hydrogen-deuterium exchange coupled with MS further reveal the mechanistic underpinnings of phosphorylation-dependent modulation of TgMyoA motility whereby localized regions of increased stability and order correlate with enhanced motility. Analysis of solvent-accessible pockets reveals striking differences between apicomplexan class XIV and human myosins. Extending these analyses to high-confidence homology models of Plasmodium and Cryptosporidium MyoA motor domains supports the intriguing potential of designing class-specific, yet broadly active, apicomplexan myosin inhibitors. The successful expression of the functional TgMyoA complex combined with our crystal structure of the motor domain provides a strong foundation in support of detailed structure-function studies and enables the development of small-molecule inhibitors targeting these devastating global pathogens. [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]

Plasmodium falciparum, the causative agent of malaria, employs a diverse array of surface displayed proteins to promote dissemination and establish infection in the human host. Of these, Pf3D7_0606800 is highly immunogenic and has been designated a potential top 10 candidate for inclusion in a multicomponent malarial vaccine. The role of Pf3D7_0606800 in parasite biology, however, is unknown and its characterization has been complicated by a lack of sequence identity with proteins of known structure or function. Towards elucidating Pf3D7_0606800 function, we determined its structure to a resolution of 2.35 Å using selenium single wavelength anomalous dispersion. A bi-lobed architecture displays the core structural hallmarks of Venus Flytrap (VFT) proteins prompting us to re-annotate Pf3D7_0606800 as PfVFT1. Structural analysis further revealed an extended inter-lobe groove that, when interrogated by molecular docking, appears well suited to bind peptide-based ligands. Collectively, our structural characterization of the highly antigenic P. falciparum surface protein PfVFT1 provides intriguing functional insight and establishes a structural template that could prove valuable for malaria vaccine engineering studies. [ABSTRACT FROM AUTHOR]