The cryo-EM structure of the endocytic receptor DEC-205

DEC-205 (CD205), a member of the macrophage mannose receptor protein family, is the prototypic endocytic receptor of dendritic cells, whose ligands include phosphorothioated cytosine-guanosine (CpG) oligonucleotides, a motif often seen in bacterial or viral DNA. However, despite growing biological and clinical significance, little is known about the structural arrangement of this receptor or any of its family members. Here we describe the 3.2 Å cryo-EM structure of human DEC-205, thereby illuminating the structure of the mannose receptor protein family. The DEC-205 monomer forms a compact structure comprising two intercalated rings of C-type lectin-like domains, where the N-terminal cysteine-rich and fibronectin domains reside at the central intersection. We establish a pH dependant oligomerisation pathway forming tetrameric DEC-205 using solution-based techniques and ultimately solved the 4.9 Å cryo-EM structure of the DEC-205 tetramer to identify the unfurling of the second lectin ring which enables tetramer formation. Furthermore, we suggest the relevance of this oligomerisation pathway within a cellular setting, whereby CpG binding appeared to disrupt this cell-surface oligomer. Accordingly, we provide insight into the structure and oligomeric assembly of the DEC-205 receptor.

Cryo-EM analysis of human mitochondrial Hsp90 in multiple tetrameric states

Hsp90 is a ubiquitous molecular chaperone that mediates the folding and maturation of hundreds of “client” proteins. Although Hsp90s generally function as homodimers, recent discoveries suggested that the mitochondrion specific Hsp90 (TRAP1) also forms functionally relevant tetramers. The structural mechanism of tetramer formation remains elusive. Here we used a combination of solution, biochemical and cryo-electron microscopy (cryo-EM) approaches to confirm that, independent of nucleotide state, a subpopulation of TRAP1 exists as tetramers. Unexpectedly, cryo-EM reveals multiple tetramer conformations having TRAP1 dimers arranged in orthogonal, parallel, or antiparallel configurations. The cryo-EM structure of one of the orthogonal tetrameric states was determined at 3.5 Å resolution. Each of the two TRAP1 dimers is in a symmetric AMP·PNP-bound closed state with the tetramer being stabilized through three distinct dimer-dimer interaction sites. In unique ways, each of the three TRAP1 domains contributes to tetramer formation. In addition to tetramerization via direct dimer-dimer contacts, our structure suggests that additional stabilization could come from domain swapping between the dimers. These results expand our understanding of TRAP1 biology beyond the conventional view of a functional dimer and provide a platform to further explore the function and regulation of tetrameric TRAP1 in mitochondria.

CryoEM Structure of CtBP2 Confirms Tetrameric Architecture

C-terminal binding proteins 1 and 2 (CtBP1 and CtBP2) are transcriptional regulators that activate or repress many genes involved in cellular development, apoptosis and metastasis. CtBP proteins are activated under hypoxic conditions where NAD(H) levels tend to be higher. NADH-dependent activation of CtBP2 has direct implication in multiple types of cancers and poor patient prognosis. Previous studies have proposed dimeric CtBP as the relevant oligomeric state, however our studies with multi-angle light scattering have shown that the primary effect of NADH binding is to promote the assembly of two CtBP dimers into tetramers. Here, we present the cryoEM structures of two different constructs of CtBP2 corroborating that the native state of CtBP2 in the presence of NADH is indeed tetrameric. The physiological relevance of tetrameric CtBP2 was tested in HCT116; CtBP2 −/− cells transfected with tetramer destabilizing_mutants. Mutants that inhibit tetramer formation show a decrease in expression of the CtBP transcriptional target TIAM1 and exhibit a decrease in the ability to promote cell migration. Together with our cryoEM studies, these results highlight the tetramer as the functional oligomeric form of CtBP2.

Anti-parallel dimer and tetramer formation of cyclic and open structure tertiary amides, N-methyl-2-pyrrolidone and N,N-dimethylacetamide, in solution of a non-polar solvent, benzene

N-Methyl-2-pyrrolidone and N,N-dimethylacetamide form anti-parallel dimers and tetramers in a dipole configuration in the pure liquid state and solution.

Nucleic Acids Interfere with PF4 Tetramer Formation and Stabilise PF4 Monomers to Bind Non-Pathogenic HIT Antibodies: Subtypes of Non-Pathogenic Antibodies Emerge

Introduction The pathogenesis of heparin-induced thrombocytopenia (HIT) primarily involves anti-platelet factor(PF)4/heparin antibodies engaging FcγRIIa receptors on platelets to trigger a deleterious cascade of activation, thrombosis, and thrombocytopenia. Recent work with murine monoclonal antibodies has shed light on epitopic determinants that potentiate platelet activation (pathogenic KKO vs non-pathogenic RTO antibodies), but these mechanisms may not fully reflect the gamut of polyspecific antibodies seen in human clinical practice. We examined the development and characteristics of anti-PF4/heparin antibodies in patients undergoing cardiac surgery. Methods After obtaining informed consent from patients undergoing cardiac bypass surgery, we performed ELISA using: (1) commercial Diagnostica Stago Asserachrom IgG kit (Cat.Nr 00624); or (2) in-house ELISA with platelet derived native(n)PF4. ELISA performed as per manufacturer instructions and published methods. Bacterial genomic nucleic acids (NA) were extracted from overnight culture of BL21(DE3)pLysS-wtPF4 using Promega kit Wizard® SV Genomic DNA Purification System (#A2361, Promega). Cross linking assays were performed as previously described using Bis(sulfosuccinimidyl)suberate (BS3) obtained commercially (#21585, Thermo Scientific Pierce). Optical density was normalised with controls to assist interassay comparability. All samples were screened with the commercial kit day 7 post-operatively, or the nearest available serum between day 4-10. Positive samples were then examined longitudinally to chart the onset of antibody formation and their duration. Results Commercial ELISA identified 30/123 (24%) patients positive for anti-PF4/heparin antibodies following cardiac surgery, but there was no HIT observed in the complete cohort of 123 patients and therefore all 30 were false positive. Surprisingly, in-house ELISA using nPF4/heparin was negative for 22/30, but the addition of NA to PF4 rescued the OD of these to become positive again. In contrast, OD fell for all true HIT controls with the addition of NA, as they did for a subset of the false-positives 7/30 [Figure A]. Five patients had persistently raised OD despite the addition of NA, suggesting the presence of both NA/nPF4/heparin antibodies and nPF4/heparin antibodies. Overall, the incidence of false-positive anti-PF4/heparin antibodies was only 12/123 (10%) using the in-house ELISA method with nPF4. SRA was performed on all patients with nPF4/heparin antibodies, but they were all negative. Spiking nPF4 with increasing concentrations of NA caused reduction of tetramer formation and an increasing proportion of dimeric and monomeric forms seen on cross-linkage assays [Figures B and C]. Longitudinal studies demonstrated that all 13 patients positive by commercial ELISA with only low range absolute results (OD <0.4), and without increase at later timepoints, possessed only NA/nPF4/heparin antibodies. By contrast, of the 14 patients whose ELISA OD increased over time, seven had evidence of nPF4/heparin antibodies. Three patients were ELISA positive pre-operatively, and only one of these had evidence of nPF4/heparin antibodies. Conclusions Increasing amounts of NA inhibit PF4 tetramer formation, and reduce the ability for HIT antibodies to bind. Thus, NA may participate in the preferential presentation of PF4 monomers that are recognised by non-pathogenic NA/PF4 monomer/heparin antibodies, similar to RTO. There is a smaller subset of "true" non-pathogenic antibodies that bind to PF4 tetramers, and an even smaller number of patients who may possess both classes of non-pathogenic antibodies. We speculate that pre-formed antibodies to NA/PF4 monomers are responsible for the majority of false positive results from commercial ELISA kits, and these are unrelated to the pathogenic anti-PF4/heparin antibodies of HIT. Figure Caption A: Representative example of five patients with increasing ELISA OD in the presence of NA (NA1-5), two patients with OD that fell in the presence of NA (TFP1-2), and known HIT serum with reduced OD in the presence of NA. B: Reduction in PF4 tetramer formation and increasing PF4 monomers with increasing NA concentration. Lanes from left-right: control PF4 without BS3, and then increasing concentration of NA from 0, 5, 30 and 45ng/μL with BS3. C: Percentage of PF4 as tetramers or monomers with increasing concentration of NA. Disclosures No relevant conflicts of interest to declare.