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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]

The phylogenetically divergent spirochete bacterium Treponema pallidum subsp. pallidum is the causative agent of syphilis. Central to the capacity of T. pallidum to establish infection is the ability of the pathogen to attach to a diversity of host cells. Many pathogenic bacteria employ leucine‐rich repeat (LRR) domain‐containing proteins to mediate protein–protein interactions, including attachment to host components and establishment of infection. Intriguingly, T. pallidum expresses only one putative LRR domain‐containing protein (Tp0225) with an unknown function. In an effort to ascribe a function to Tp0225, a comprehensive phylogenetic analysis was first performed; this investigation revealed that Tp0225 clusters with the pathogenic clade of treponemes. Its crystal structure was then determined to 2.0 Å resolution using Pt SAD phasing, which revealed a noncanonical architecture containing a hexameric LRR core with a discontinuous β‐sheet bridged by solvent molecules. Furthermore, a surface‐exposed, hydrophobic pocket, which was found in Tp0225 but is largely absent in canonical LRR domains from other pathogenic bacteria, may serve to coordinate a hydrophobic ligand. Overall, this study provides the first structural characterization of the sole LRR domain‐containing protein from T. pallidum and offers insight into the unique molecular landscape of this important human pathogen. [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]

African trypanosomiasis, caused by parasites of the genus Trypanosoma, is a complex of devastating vector-borne diseases of humans and livestock in sub-Saharan Africa. Central to the pathogenesis of African trypanosomes is their transmission by the arthropod vector, Glossina spp. (tsetse fly). Intriguingly, the efficiency of parasite transmission through the vector is reduced following depletion of Trypanosoma brucei Procyclic-Specific Surface Antigen-2 ( TbPSSA-2). To investigate the underlying molecular mechanism of TbPSSA-2, we determined the crystal structures of its ectodomain and that of its homolog T. congolense Insect Stage Antigen ( TcISA) to resolutions of 1.65 Å and 2.45 Å, respectively using single wavelength anomalous dispersion. Both proteins adopt a novel bilobed architecture with the individual lobes displaying rotational flexibility around the central tether that suggest a potential mechanism for coordinating a binding partner. In support of this hypothesis, electron density consistent with a bound peptide was observed in the inter-lob cleft of a TcISA monomer. These first reported structures of insect stage transmembrane proteins expressed by African trypanosomes provide potentially valuable insight into the interface between parasite and tsetse vector. [ABSTRACT FROM AUTHOR]

S100A7 (psoriasin) is a calcium- and zinc-binding protein implicated in breast cancer. We have shown previously that S100A7 enhances survival mechanisms in breast cells through an interaction with c-jun activation domain binding protein 1 (Jab1), and an engineered S100A7 triple mutant (Asp56Gly, Leu78Met, and Gln88Lys-S100A73) ablates Jab1 binding. We extend these results to include defined breast cancer cell lines and demonstrate a disrupted S100A73/Jab1 phenotype is maintained. To establish the basis for the abrogated Jab1 binding, we have recombinantly produced S100A73, demonstrated that it retains the ability to form an exceptionally thermostable dimer, and solved the three dimensional crystal structure to 1.6 Å. Despite being positioned at the dimer interface, the Leu78Met mutation is easily accommodated and contributes to a methionine-rich pocket formed by Met12, Met15, and Met34. In addition to altering the surface charge, the Gln88Lys mutation results in a nearby rotameric shift in Tyr85, leading to a substantially reorganized surface cavity and may influence zinc binding. The final mutation of Asp56 to Gly results in the largest structural perturbation shortening helix IV by one full turn. It is noteworthy that position 56 lies in one of two divergent clusters between S100A7 and the functionally distinct yet highly homologous S100A15. The structure of S100A73 provides a unique perspective from which to characterize the S100A7-Jab1 interaction and better understand the distinct functions between S100A7, and it is closely related paralog S100A15. [ABSTRACT FROM AUTHOR]

Unlike the heme cd1-based nitrite reductase enzymes, the molecular mechanism of copper-containing nitrite reductases remains controversial. A key source of controversy is the productive binding mode of nitrite in the active site. To identify and characterize the molecular determinants associated with nitrite binding, we applied a combinatorial mutagenesis approach to generate a small library of six variants at position 257 in nitrite reductase from Alcaligenes faecalis S-6. The activities of these six variants span nearly two orders of magnitude with one variant, I257V, the only observed natural substitution for Ile257, showing greater activity than the native enzyme. High-resolution (> 1.8 Å) nitrite-soaked crystal structures of these variants display different modes of nitrite binding that correlate well with the altered activities. These studies identify for the first time that the highly conserved Ile257 in the native enzyme is a key molecular determinant in directing a catalytically competent mode of nitrite binding in the active site. The O-coordinate bidentate binding mode of nitrite observed in native and mutant forms with high activity supports a catalytic model distinct from the heme cd1 NiRs. (The atomic coordinates for I257V[NO2−], I257L[NO2−], I257A[NO2−], I257T[NO2−], I257M[NO2−] and I257G[NO2−] AfNiR have been deposited in the Protein Data Bank [PDB identification codes are listed in Table 2].) [ABSTRACT FROM AUTHOR]