Publications by Year: 2021

Structural basis of malaria transmission blockade by a monoclonal antibody to gamete fusogen HAP2
Feng, J., et al. Structural basis of malaria transmission blockade by a monoclonal antibody to gamete fusogen HAP2. Elife 10, (2021).Abstract
HAP2 is a transmembrane gamete fusogen found in multiple eukaryotic kingdoms and is structurally homologous to viral class II fusogens. Studies in Plasmodium have suggested that HAP2 is an attractive target for vaccines that block transmission of malaria. HAP2 has three extracellular domains, arranged in the order D2, D1, and D3. Here, we report monoclonal antibodies against the D3 fragment of Plasmodium berghei HAP2 and crystal structures of D3 in complex with Fab fragments of two of these antibodies, one of which blocks fertilization of Plasmodium berghei in vitro and transmission of malaria in mosquitoes. We also show how this Fab binds the complete HAP2 ectodomain with electron microscopy. The two antibodies cross-react with HAP2 among multiple plasmodial species. Our characterization of the Plasmodium D3 structure, HAP2 ectodomain architecture, and mechanism of inhibition provide insights for the development of a vaccine to block malaria transmission.
Low affinity integrin states have faster ligand binding kinetics than the high affinity state
Li, J., Yan, J. & Springer, T. Low affinity integrin states have faster ligand binding kinetics than the high affinity state. eLife 10, e73359, (2021). Publisher's VersionAbstract
Integrin conformational ensembles contain two low-affinity states, bent-closed and extended-closed, and an active, high-affinity, extended-open state. It is widely thought that integrins must be activated before they bind ligand; however, one model holds that activation follows ligand binding. As ligand-binding kinetics are not only rate limiting for cell adhesion but also have important implications for the mechanism of activation, we measure them here for integrins α4β1 and α5β1 and show that the low-affinity states bind substantially faster than the high-affinity state. On and off-rates are similar for integrins on cell surfaces and as ectodomain fragments. Although the extended-open conformation's on-rate is ~20-fold slower, its off-rate is ~25,000-fold slower, resulting in a large affinity increase. The tighter ligand-binding pocket in the open state may slow its on-rate. Low affinity integrin states not only bind ligand more rapidly, but are also more populous on the cell surface than high affinity states. Thus, our results suggest that integrin binding to ligand may precede, rather than follow, activation by "inside-out signaling".
Congenital X-linked neutropenia with myelodysplasia and somatic tetraploidy due to a germline mutation in SEPT6
Renella, R., et al. Congenital X-linked neutropenia with myelodysplasia and somatic tetraploidy due to a germline mutation in SEPT6. Am J Hematol (2021).Abstract
Septins play key roles in mammalian cell division and cytokinesis but have not previously been implicated in a germline human disorder. A male infant with severe neutropenia and progressive dysmyelopoiesis with tetraploid myeloid precursors was identified. No known genetic etiologies for neutropenia or bone marrow failure were found. However, next-generation sequencing of germline samples from the patient revealed a novel, de novo germline stop-loss mutation in the X-linked gene SEPT6 that resulted in reduced SEPT6 staining in bone marrow granulocyte precursors and megakaryocytes. Patient skin fibroblast-derived induced pluripotent stem cells (iPSCs) produced reduced myeloid colonies, particularly of the granulocyte lineage. CRISPR/Cas9 knock-in of the patient's mutation or complete knock-out of SEPT6 was not tolerated in non-patient-derived iPSCs or human myeloid cell lines, but SEPT6 knock-out was successful in an erythroid cell line and resulting clones revealed a propensity to multinucleation. In silico analysis predicts that the mutated protein hinders the dimerization of SEPT6 coiled-coils in both parallel and antiparallel arrangements, which could in turn impair filament formation. These data demonstrate a critical role for SEPT6 in chromosomal segregation in myeloid progenitors that can account for the unusual predisposition to aneuploidy and dysmyelopoiesis.
Single-molecule imaging of von Willebrand factor reveals tension-dependent self-association
Fu, H., Jiang, Y., Wong, W. & Springer, T.A. Single-molecule imaging of von Willebrand factor reveals tension-dependent self-association. Blood 138, 23, 2425-2434 (2021). Publisher's VersionAbstract
Von Willebrand factor (VWF) is an ultra-long concatemeric protein important in hemostasis and thrombosis. VWF molecules can associate with other VWF molecules, but little is known about the mechanism. Hydrodynamic drag exerts tensile force on surface-tethered VWF that extends it and is maximal at the tether point and declines linearly to zero at the downstream, free end. Using single-molecule fluorescence microscopy, we directly visualize the kinetics of binding of free VWF in flow to surface-tethered single VWF molecules and show that self-association requires elongation of tethered VWF and that association increases with tension in tethered VWF, reaches half maximum at a characteristic tension of ~10 pN, and plateaus above ~ 25 pN. Association is reversible and hence noncovalent; a sharp decrease in shear flow results in rapid dissociation of bound VWF. Tethered, primary VWF molecules can recruit more than their own mass of secondary VWF from the flow stream. Kinetics show that instead of accelerating, the rate of accumulation decreases with time, revealing an inherently self-limiting self-association mechanism. We propose that this may be because multiple tether points between secondary and primary VWF result in lower tension on the secondary VWF, which shields more highly tensioned primary VWF from further association. GPIbα binding and VWF self-association occur in the same region of high tension in tethered VWF concatemers; however, the half-maximal tension required for activation of GPIbα is higher, suggesting differences in molecular mechanisms. These results have important implications for the mechanism of platelet plug formation in hemostasis and thrombosis.
Complement Receptor 3 Forms a Compact High-Affinity Complex with iC3b
Jensen, R.K., et al. Complement Receptor 3 Forms a Compact High-Affinity Complex with iC3b. J Immunol (2021).Abstract
Complement receptor 3 (CR3, also known as Mac-1, integrin αMβ2, or CD11b/CD18) is expressed on a subset of myeloid and certain activated lymphoid cells. CR3 is essential for the phagocytosis of complement-opsonized particles such as pathogens and apoptotic or necrotic cells opsonized with the complement fragment iC3b and, to a lesser extent, C3dg. Although the interaction between the iC3b thioester domain and the ligand binding CR3 αM I-domain is structurally and functionally well characterized, the nature of additional CR3-iC3b interactions required for phagocytosis of complement-opsonized objects remains obscure. In this study, we analyzed the interaction between iC3b and the 150-kDa headpiece fragment of the CR3 ectodomain. Surface plasmon resonance experiments demonstrated a 30 nM affinity of the CR3 headpiece for iC3b compared with 515 nM for the iC3b thioester domain, whereas experiments monitoring binding of iC3b to CR3-expressing cells suggested an affinity of 50 nM for the CR3-iC3b interaction. Small angle x-ray scattering analysis revealed that iC3b adopts an extended but preferred conformation in solution. Upon interaction with CR3, iC3b rearranges to form a compact receptor-ligand complex. Overall, the data suggest that the iC3b-CR3 interaction is of high affinity and relies on minor contacts formed between CR3 and regions outside the iC3b thioester domain. Our results rationalize the more efficient phagocytosis elicited by iC3b than by C3dg and pave the way for the development of specific therapeutics for the treatment of inflammatory and neurodegenerative diseases that do not interfere with the recognition of noncomplement CR3 ligands.