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.
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.
β2 integrins are well-known leukocyte adhesion molecules consisting of 4 members: CD11a-d. Their known biological functions range widely from leukocyte recruitment, phagocytosis, to immunological synapse formation, but the studies have been primarily focused on CD11a and CD11b. CD11c is 1 of the 4 members and is extremely homologous to CD11b. It has been well known as a dendritic cell marker, but the characterization of its function has been limited. We found that CD11c was expressed on the short-term hematopoietic stem cells and multipotent progenitor cells. The lack of CD11c did not affect the number of hematopoietic stem and progenitor cells (HSPCs) in healthy CD11c knockout mice. Different from other β2 integrin members, however, CD11c deficiency was associated with increased apoptosis and significant loss of HSPCs in sepsis and bone marrow transplantation. Although integrins are generally known for their overlapping and redundant roles, we showed that CD11c had a distinct role of regulating the expansion of HSPCs under stress. This study shows that CD11c, a well-known dendritic cell marker, is expressed on HSPCs and serves as their functional regulator. CD11c deficiency leads to the loss of HSPCs via apoptosis in sepsis and bone marrow transplantation.
Inserted (I) domains function as ligand-binding domains in adhesins that support cell adhesion and migration in many eukaryotic phyla. These adhesins include integrin αβ heterodimers in metazoans and single subunit transmembrane proteins in apicomplexans such as TRAP in and MIC2 in . Here we show that the I domain of TRAP is essential for sporozoite gliding motility, mosquito salivary gland invasion and mouse infection. Its replacement with the I domain from Toxoplasma MIC2 fully restores tissue invasion and parasite transmission, while replacement with the aX I domain from human integrins still partially restores liver infection. Mutations around the ligand binding site allowed salivary gland invasion but led to inefficient transmission to the rodent host. These results suggest that apicomplexan parasites appropriated polyspecific I domains in part for their ability to engage with multiple ligands and to provide traction for emigration into diverse organs in distant phyla.
The circumsporozoite protein (CSP) and thrombospondin-related adhesion protein (TRAP) are major targets for pre-erythrocytic malaria vaccine development. However, the CSP-based vaccine RTS,S provides only marginal protection, highlighting the need for innovative vaccine design and development. Here we design and characterize expression and folding of P. berghei (Pb) and P. falciparum (Pf) TRAP-CSP fusion proteins, and evaluate immunogenicity and sterilizing immunity in mice. TRAP N-terminal domains were fused to the CSP C-terminal αTSR domain with or without the CSP repeat region, expressed in mammalian cells, and evaluated with or without N-glycan shaving. Pb and Pf fusions were each expressed substantially better than the TRAP or CSP components alone; furthermore, the fusions but not the CSP component could be purified to homogeneity and were well folded and monomeric. As yields of TRAP and CSP fragments were insufficient, we immunized BALB/c mice with Pb TRAP-CSP fusions in AddaVax adjuvant and tested the effects of absence or presence of the CSP repeats and absence or presence of high mannose N-glycans on total antibody titer and protection from infection by mosquito bite both 2.5 months and 6 months after the last immunization. Fusions containing the repeats were completely protective against challenge and re-challenge, while those lacking repeats were significantly less effective. These results correlated with higher total antibody titers when repeats were present. Our results show that TRAP-CSP fusions increase protein antigen production, have the potential to yield effective vaccines, and also guide design of effective proteins that can be encoded by nucleic acid-based and virally vectored vaccines.
Integrin αVβ8, which like αVβ6 functions to activate TGF-βs, is atypical. Its β8 subunit binds to a distinctive cytoskeleton adaptor and does not exhibit large changes in conformation upon binding to ligand. Here, crystal structures, hydrogen-deuterium exchange dynamics, and affinity measurements on mutants are used to compare αVβ8 and αVβ6. Lack of a binding site for one of three βI domain divalent cations and a unique β6-α7 loop conformation in β8 facilitate movements of the α1 and α1' helices at the ligand binding pocket toward the high affinity state, without coupling to β6-α7 loop reshaping and α7-helix pistoning that drive large changes in βI domain-hybrid domain orientation seen in other integrins. Reciprocal swaps between β6 and β8 βI domains increase affinity of αVβ6 and decrease affinity of αVβ8 and define features that regulate affinity of the βI domain and its coupling to the hybrid domain.
CagL is an essential pilus surface component of the virulence-associated type IV secretion system (T4SS) employed by Helicobacter pylori to translocate the oncogenic effector protein CagA into human gastric epithelial cells. CagL contains an RGD motif and integrin α β is widely accepted as its host cell receptor. Here, we show that CagL binds integrin α β with substantially higher affinity and that this interaction is functionally important. Cell surface expression of α β on various cell lines correlated perfectly with cell adhesion to immobilized CagL and with binding of soluble CagL to cells. We found no such correlation for α β . The purified α β ectodomain bound CagL with high affinity. This interaction was highly specific, as the affinity of CagL for other RGD-binding integrins was two to three orders of magnitude weaker. Mutation of either conserved leucine in the CagL RGDLXXL motif, a motif that generally confers specificity for integrin α β and α β , lowered the affinity of CagL for α β . Stable expression of α β in α β -negative but α β -expressing human cells promoted two hallmarks of the functional H. pylori T4SS, namely translocation of CagA into host cells and induction of interleukin-8 secretion by host cells. These findings suggest that integrin α β , although not essential for T4SS function, represents an important host cell receptor for CagL.
Von Willebrand factor (VWF), a large multimeric blood protein, senses changes in shear stress during bleeding and responds by binding platelets to plug ruptures in the vessel wall. Molecular mechanisms underlying this dynamic process are difficult to uncover using standard approaches due to the challenge of applying mechanical forces while monitoring structure and activity. By combining single-molecule fluorescence imaging with high-pressure, rapidly switching microfluidics, we reveal the key role of electrostatic steering in accelerating the binding between flow-activated VWF and GPIbα, and in rapidly immobilizing platelets under flow. We measure the elongation and tension-dependent activation of individual VWF multimers under a range of ionic strengths and pH levels, and find that the association rate is enhanced by 4 orders of magnitude by electrostatic steering. Under supraphysiologic salt concentrations, strong electrostatic screening dramatically decreases platelet binding to VWF in flow, revealing the critical role of electrostatic attraction in VWF-platelet binding during bleeding.
D assemblies comprise half of von Willebrand factor yet are of unknown structure. D1 and D2 in the prodomain and D'D3 in mature VWF at Golgi pH form helical VWF tubules in Weibel Palade bodies and template dimerization of D3 through disulfides to form ultralong VWF concatemers. D'D3 forms the binding site for factor VIII. The crystal structure of monomeric D'D3 with cysteine residues required for dimerization mutated to alanine was determined at endoplasmic reticulum (ER)-like pH. The smaller C8-3, TIL3 and E3 modules pack through specific interfaces as they wind around the larger, N-terminal, Ca-binding VWD3 module to form a wedge shape. D' with its TIL' and E' modules projects away from D3. The two mutated cysteines implicated in D3 dimerization are buried, providing a mechanism for protecting them against premature disulfide linkage in the ER where intrachain disulfide linkages are formed. D3 dimerization requires co-association with D1 and D2, Ca, and Golgi-like acidic pH. Associated structural rearrangements in the C8-3 and TIL3 modules are required to expose cysteine residues for disulfide linkage. Our structure provides insight into many von Willebrand disease mutations including those that diminish FVIII binding, which suggest that factor VIII binds not only to the N-terminal TIL' domain of D' distal from D3 but also extends across one side of D3. The organizing principle for the D3 assembly has implications for other D assemblies and the construction of higher order, disulfide-linked assemblies in the Golgi in both VWF and mucins.
Thrombospondin type 1 repeats (TSRs), small adhesive protein domains with a wide range of functions, are usually modified with O-linked fucose, which may be extended to O-fucose-β1,3-glucose. Collision-induced dissociation (CID) spectra of O-fucosylated peptides cannot be sequenced by standard tandem mass spectrometry (MS/MS) sequence database search engines because O-linked glycans are highly labile in the gas phase and are effectively absent from the CID peptide fragment spectra, resulting in a large mass error. Electron transfer dissociation (ETD) preserves O-linked glycans on peptide fragments, but only a subset of tryptic peptides with low m/ z can be reliably sequenced from ETD spectra compared to CID. Accordingly, studies to date that have used MS to identify O-fucosylated TSRs have required manual interpretation of CID mass spectra even when ETD was also employed. In order to facilitate high-throughput, automatic identification of O-fucosylated peptides from CID spectra, we re-engineered the MS/MS sequence database search engine Comet and the MS data analysis suite Trans-Proteomic Pipeline to enable automated sequencing of peptides exhibiting the neutral losses characteristic of labile O-linked glycans. We used our approach to reanalyze published proteomics data from Plasmodium parasites and identified multiple glycoforms of TSR-containing proteins.
Mutations of the common cytokine receptor gamma chain (γc) cause Severe Combined Immunodeficiency characterized by absent T and NK cell development. Although stem cell therapy restores these lineages, residual immune defects are observed that may result from selective persistence of γc-deficiency in myeloid lineages. However, little is known about the contribution of myeloid-expressed γc to protective immune responses. Here we examine the importance of γc for myeloid dendritic cell (DC) function. We utilize a combination of DC/T-cell co-culture assays and a novel lipid bilayer system mimicking the T cell surface to delineate the role of DC-expressed γc during DC/T-cell interaction. We observed that γc in DC was recruited to the contact interface following MHCII ligation, and promoted IL-15Rα colocalization with engaged MHCII. Unexpectedly, trans-presentation of IL-15 was required for optimal CD4+T cell activation by DC and depended on DC γc expression. Neither recruitment of IL-15Rα nor IL-15 trans-signaling at the DC immune synapse (IS), required γc signaling in DC, suggesting that γc facilitates IL-15 transpresentation through induced intermolecular associations or cytoskeletal reorganization following MHCII ligation. These findings show that DC-expressed γc is required for effective antigen-induced CD4+ T cell activation. We reveal a novel mechanism for recruitment of DC IL-15/IL-15Rα complexes to the IS, leading to CD4+ T cell costimulation through localized IL-15 transpresentation that is coordinated with antigen-recognition.
HAP2 is a class II gamete fusogen in many eukaryotic kingdoms. A crystal structure of HAP2 shows a trimeric fusion state. Domains D1, D2.1 and D2.2 line the 3-fold axis; D3 and a stem pack against the outer surface. Surprisingly, hydrogen-deuterium exchange shows that surfaces of D1, D2.2 and D3 closest to the 3-fold axis are more dynamic than exposed surfaces. Three fusion helices in the fusion loops of each monomer expose hydrophobic residues at the trimer apex that are splayed from the 3-fold axis, leaving a solvent-filled cavity between the fusion loops in each monomer. At the base of the two fusion loops, Arg185 docks in a carbonyl cage. Comparisons to other structures, dynamics, and the greater effect on gamete fusion of mutation of axis-proximal than axis-distal fusion helices suggest that the apical portion of each monomer could tilt toward the 3-fold axis with merger of the fusion helices into a common fusion surface.
Extracellular proTGF-β is covalently linked to "milieu" molecules in the matrix or on cell surfaces and is latent until TGF-β is released by integrins. Here, we show that LRRC33 on the surface of microglia functions as a milieu molecule and enables highly localized, integrin-αVβ8-dependent TGF-β activation. Lrrc33 mice lack CNS vascular abnormalities associated with deficiency in TGF-β-activating integrins but have microglia with a reactive phenotype and after 2 months develop ascending paraparesis with loss of myelinated axons and death by 5 months. Whole bone marrow transplantation results in selective repopulation of Lrrc33 brains with WT microglia and halts disease progression. The phenotypes of WT and Lrrc33 microglia in the same brain suggest that there is little spreading of TGF-β activated from one microglial cell to neighboring microglia. Our results suggest that interactions between integrin-bearing cells and cells bearing milieu molecule-associated TGF-β provide localized and selective activation of TGF-β.
In aI integrins including leukocyte function-associated antigen-1 (LFA-1), ligand-binding function is delegated to the aI domain, requiring extra steps in the relay of signals that activate ligand binding and coordinate it with cytoplasmic signals. Crystal structures reveal great variation in orientation between the aI domain and the remainder of the integrin head. Here, we investigated the mechanisms involved in signal relay to the aI domain, including whether binding of the ligand intercellular adhesion molecule-1 (ICAM-1) to the aI domain is linked to headpiece opening and engenders a preferred aI domain orientation. Using small-angle Xray scattering (SAXS) and negative-stain EM we define structures of ICAM-1, LFA-1, and their complex, and the effect of activation by Mn2+. Headpiece opening was substantially stabilized by substitution of Mg2+ with Mn2+ and became complete upon ICAM-1 addition. These agents stabilized aI-headpiece orientation, resulting in a well-defined orientation of ICAM-1 such that its tandem Iglike domains pointed in the opposite direction from the β-subunit leg of LFA-1. Mutations in the integrin βI domain α1/α1` helix stabilizing either the open or the closed βI-domain conformation indicated that α1/α1` helix movements are linked to ICAM-1 binding by the aI domain and to the extended-open conformation of the ectodomain. The LFA-1--ICAM-1 orientation described here with ICAM-1 pointing anti-parallel to the LFA-1 β-subunit leg is the same orientation that would be stabilized by tensile force transmitted between the ligand and the actin cytoskeleton, and is consistent with the cytoskeletal force model of integrin activation.
We use super-resolution interferometric photoactivation and localization microscopy (iPALM) and a constrained photoactivatable fluorescent protein integrin fusion to measure the displacement of the head of integrin lymphocyte function-associated 1 (LFA-1) resulting from integrin conformational change on the cell surface. We demonstrate that the distance of the LFA-1 head increases substantially between basal and ligand-engaged conformations, which can only be explained at the molecular level by integrin extension. We further demonstrate that one class of integrin antagonist maintains the bent conformation, while another antagonist class induces extension. Our molecular scale measurements on cell-surface LFA-1 are in excellent agreement with distances derived from crystallographic and electron microscopy structures of bent and extended integrins. Our distance measurements are also in excellent agreement with a previous model of LFA-1 bound to ICAM-1 derived from the orientation of LFA-1 on the cell surface measured using fluorescence polarization microscopy.
The role of the hybrid domain in integrin affinity regulation is unknown, as is whether the kinetics of ligand binding is modulated by integrin affinity state. Here, we compare cell surface and soluble integrin αVβ6 truncation mutants for ligand-binding affinity, kinetics, and thermodynamics. Removal of the integrin transmembrane/cytoplasmic domains or lower legs has little effect on αVβ6 affinity, in contrast to β1 integrins. In integrin opening, rearrangement at the interface between the βI and hybrid domains is linked to remodeling at the ligand-binding site at the opposite end of the βI domain, which greatly increases in affinity in the open conformation. The larger size of the βI-hybrid interface in the closed state suggests that the hybrid domain stabilizes closing. In agreement, deletion of the hybrid domain raised affinity by 50-fold. Surface plasmon resonance and isothermal titration calorimetry gave similar results and the latter revealed tradeoffs between enthalpy and entropy not apparent from affinity. At extremely high affinity reached in Mn2+ with hybrid domain truncation, αVβ6 on-rate for both pro-TGF-β1 and fibronectin declined. The results suggest that the open conformation of αVβ6 has lower on-rate than the closed conformation, correlate with constriction of the ligand-binding pocket in open αVβ6 structures, and suggest that the extended-closed conformation is kinetically selected for ligand binding. Subsequent transition to the extended-open conformation is stabilized by its much higher affinity for ligand and would also be stabilized by force exerted across ligand-bound integrins by the actin cytoskeleton.
Growth differentiation factor 8 (GDF8)/myostatin is a latent TGF-β family member that potently inhibits skeletal muscle growth. Here, we compared the conformation and dynamics of precursor, latent, and Tolloid-cleaved GDF8 pro-complexes to understand structural mechanisms underlying latency and activation of GDF8. Negative stain electron microscopy (EM) of precursor and latent pro-complexes reveals a V-shaped conformation that is unaltered by furin cleavage and sharply contrasts with the ring-like, cross-armed conformation of latent TGF-β1. Surprisingly, Tolloid-cleaved GDF8 does not immediately dissociate, but in EM exhibits structural heterogeneity consistent with partial dissociation. Hydrogen-deuterium exchange was not affected by furin cleavage. In contrast, Tolloid cleavage, in the absence of prodomain-growth factor dissociation, increased exchange in regions that correspond in pro-TGF-β1 to the α1-helix, latency lasso, and β1-strand in the prodomain and to the β6'- and β7'-strands in the growth factor. Thus, these regions are important in maintaining GDF8 latency. Our results show that Tolloid cleavage activates latent GDF8 by destabilizing specific prodomain-growth factor interfaces and primes the growth factor for release from the prodomain.
Integrin αβ heterodimer cell surface receptors mediate adhesive interactions that provide traction for cell migration. Here, we test whether the integrin, when engaged to an extracellular ligand and the cytoskeleton, adopts a specific orientation dictated by the direction of actin flow on the surface of migrating cells. We insert GFP into the rigid, ligand-binding head of the integrin, model with Rosetta the orientation of GFP and its transition dipole relative to the integrin head, and measure orientation with fluorescence polarization microscopy. Cytoskeleton and ligand-bound integrins orient in the same direction as retrograde actin flow with their cytoskeleton-binding β-subunits tilted by applied force. The measurements demonstrate that intracellular forces can orient cell surface integrins and support a molecular model of integrin activation by cytoskeletal force. Our results place atomic, Å-scale structures of cell surface receptors in the context of functional and cellular, μm-scale measurements.
Why do integrins differ in basal activity, and how does affinity for soluble ligand correlate with cellular adhesiveness? We show that basal conformational equilibrium set points for integrin α4β1 are cell type specific and differ from integrin α5β1 when the two integrins are coexpressed on the same cell. Although α4β1 is easier to activate, its high-affinity state binds vascular cell adhesion molecule and fibronectin 100- to 1,000-fold more weakly than α5β1 binds fibronectin. Furthermore, the difference in affinity between the high- and low-affinity states is more compressed in α4β1 (600- to 800-fold) than in α5β1 (4,000- to 6,000-fold). α4β1 basal conformational equilibria differ among three cell types, define affinity for soluble ligand and readiness for priming, and may reflect differences in interactions with intracellular adaptors but do not predict cellular adhesiveness for immobilized ligand. The measurements here provide a necessary framework for understanding integrin activation in intact cells, including activation of integrin adhesiveness by application of tensile force by the cytoskeleton, across ligand-integrin-adaptor complexes.