Antibody fragments are frequently used as a "crystallization chaperone" to aid structural analysis of complex macromolecules that are otherwise crystallization resistant, but conventional fragment formats have not been designed for this particular application. By fusing an anti-parallel coiled-coil structure derived from the SARAH domain of human Mst1 kinase to the variable region of an antibody, we succeeded in creating a novel chimeric antibody fragment of ∼37 kDa, termed "Fv-clasp," which exhibits excellent crystallization compatibility while maintaining the binding ability of the original IgG molecule. The "clasp" and the engineered disulfide bond at the bottom of the Fv suppressed the internal mobility of the fragment and shielded hydrophobic residues, likely contributing to the high heat stability and the crystallizability of the Fv-clasp. Finally, Fv-clasp antibodies showed superior "chaperoning" activity over conventional Fab fragments, and facilitated the structure determination of an ectodomain fragment of integrin α6β1.

Lasso-grafting (LG) technology is a method for generating de novo biologics (neobiologics) by genetically implanting macrocyclic peptide pharmacophores, which are selected in vitro against a protein of interest, into loops of arbitrary protein scaffolds. In this study, we have generated a neo-capsid that potently binds the hepatocyte growth factor receptor MET by LG of anti-MET peptide pharmacophores into a circularly permuted variant of Aquifex aeolicus lumazine synthase (AaLS), a self-assembling protein nanocapsule. By virtue of displaying multiple-pharmacophores on its surface, the neo-capsid can induce dimerization (or multimerization) of MET, resulting in phosphorylation and endosomal internalization of the MET-capsid complex. This work demonstrates the potential of the LG technology as a synthetic biology approach for generating capsid-based neobiologics capable of activating signaling receptors.

Image reconstruction by integrating exchangeable single-molecule localization (IRIS) achieves multiplexed super-resolution imaging by high-density labeling with fast exchangeable fluorescent probes. However, previous methods to develop probes for individual targets required a great amount of time and effort. Here, we introduce a method for generating recombinant IRIS probes with a new mutagenesis strategy that can be widely applied to existing antibody sequences. Several conserved tyrosine residues at the base of complementarity-determining regions were identified as candidate sites for site-directed mutagenesis. With a high probability, mutations at candidate sites accelerated the off rate of recombinant antibody-based probes without compromising specific binding. We were able to develop IRIS probes from five monoclonal antibodies and three single-domain antibodies. We demonstrate multiplexed localization of endogenous proteins in primary neurons that visualizes small synaptic connections with high binding density. It is now practically feasible to generate fast-dissociating fluorescent probes for multitarget super-resolution imaging.

Integrin α5β1 is a major fibronectin receptor critical for cell migration. Upon complex formation, fibronectin and α5β1 undergo conformational changes. While this is key for cell-tissue connections, its mechanism is unknown. Here, we report cryo-electron microscopy structures of native human α5β1 with fibronectin to 3.1-angstrom resolution, and in its resting state to 4.6-angstrom resolution. The α5β1-fibronectin complex revealed simultaneous interactions at the arginine-glycine-aspartate loop, the synergy site, and a newly identified binding site proximal to adjacent to metal ion-dependent adhesion site, inducing the translocation of helix α1 to secure integrin opening. Resting α5β1 adopts an incompletely bent conformation, challenging the model of integrin sharp bending inhibiting ligand binding. Our biochemical and structural analyses showed that affinity of α5β1 for fibronectin is increased with manganese ions (Mn2+) while adopting the half-bent conformation, indicating that ligand-binding affinity does not depend on conformation, and α5β1 opening is induced by ligand-binding.

Despite recent efforts to dissect the inter-tumor heterogeneity of pancreatic ductal adenocarcinoma (PDAC) by determining prognosis-predictive gene expression signatures for specific subtypes, their functional differences remain elusive. Here, we established a pancreatic tumor organoid library encompassing 39 patient-derived PDACs and identified 3 functional subtypes based on their stem cell niche factor dependencies on Wnt and R-spondin. A Wnt-non-producing subtype required Wnt from cancer-associated fibroblasts, whereas a Wnt-producing subtype autonomously secreted Wnt ligands and an R-spondin-independent subtype grew in the absence of Wnt and R-spondin. Transcriptome analysis of PDAC organoids revealed gene-expression signatures that associated Wnt niche subtypes with GATA6-dependent gene expression subtypes, which were functionally supported by genetic perturbation of GATA6. Furthermore, CRISPR-Cas9-based genome editing of PDAC driver genes (KRAS, CDKN2A, SMAD4, and TP53) demonstrated non-genetic acquisition of Wnt niche independence during pancreas tumorigenesis. Collectively, our results reveal functional heterogeneity of Wnt niche independency in PDAC that is non-genetically formed through tumor progression.

The SORL1 gene has recently emerged as a strong Alzheimer's Disease (AD) risk gene. Over 500 different variants have been identified in the gene and the contribution of individual variants to AD development and progression is still largely unknown. Here, we describe a family consisting of 2 parents and 5 offspring. Both parents were affected with dementia and one had confirmed AD pathology with an age of onset > 75 years. All offspring were affected with AD with ages at onset ranging from 53 years to 74 years. DNA was available from the parent with confirmed AD and 5 offspring. We identified a coding variant, p.(Arg953Cys), in SORL1 in 5 of 6 individuals affected by AD. Notably, variant carriers had severe AD pathology, and the SORL1 variant segregated with TDP-43 pathology (LATE-NC). We further characterized this variant and show that this Arginine substitution occurs at a critical position in the YWTD-domain of the SORL1 translation product, SORL1. Functional studies further show that the p.R953C variant leads to retention of the SORL1 protein in the endoplasmic reticulum which leads to decreased maturation and shedding of the receptor and prevents its normal endosomal trafficking. Together, our analysis suggests that p.R953C is a pathogenic variant of SORL1 and sheds light on mechanisms of how missense SORL1 variants may lead to AD.

Integrins are important adhesion receptors in all Metazoa that transmit conformational change bidirectionally across the membrane. Integrin alpha and beta subunits form a head and two long legs in the ectodomain and span the membrane. Here, we define with crystal structures the atomic basis for allosteric regulation of the conformation and affinity for ligand of the integrin ectodomain, and how fibrinogen-mimetic therapeutics bind to platelet integrin alpha(IIb)beta3. Allostery in the beta3 I domain alters three metal binding sites, associated loops and alpha1- and alpha7-helices. Piston-like displacement of the alpha7-helix causes a 62 degrees reorientation between the beta3 I and hybrid domains. Transmission through the rigidly connected plexin/semaphorin/integrin (PSI) domain in the upper beta3 leg causes a 70 A separation between the knees of the alpha and beta legs. Allostery in the head thus disrupts interaction between the legs in a previously described low-affinity bent integrin conformation, and leg extension positions the high-affinity head far above the cell surface.

Recent genome-wide association studies (GWAS) have revealed strong association of hypercholesterolemia and myocardial infarction with SNPs on human chromosome 1p13.3. This locus covers three genes: SORT1, CELSR2, and PSRC1. We demonstrate that sortilin, encoded by SORT1, is an intracellular sorting receptor for apolipoprotein (apo) B100. It interacts with apoB100 in the Golgi and facilitates the formation and hepatic export of apoB100-containing lipoproteins, thereby regulating plasma low-density lipoprotein (LDL) cholesterol. Absence of sortilin in gene-targeted mice reduces secretion of lipoproteins from the liver and ameliorates hypercholesterolemia and atherosclerotic lesion formation in LDL receptor-deficient animals. In contrast, sortilin overexpression stimulates hepatic release of lipoproteins and increases plasma LDL levels. Our data have uncovered a regulatory pathway in hepatic lipoprotein export and suggest a molecular explanation for the cardiovascular risk being associated with 1p13.3.

Fibronectin (FN) is secreted as a disulfide-bonded FN dimer. Each subunit contains three types of repeating modules: FN-I, FN-II, and FN-III. The interactions of alpha5beta1 or alphav integrins with the RGD motif of FN-III repeat 10 (FN-III10) are considered an essential step in the assembly of FN fibrils. To test this hypothesis in vivo, we replaced the RGD motif with the inactive RGE in mice. FN-RGE homozygous embryos die at embryonic day 10 with shortened posterior trunk, absent tail bud-derived somites, and severe vascular defects resembling the phenotype of alpha5 integrin-deficient mice. Surprisingly, the absence of a functional RGD motif in FN did not compromise assembly of an FN matrix in mutant embryos or on mutant cells. Matrix assembly assays and solid-phase binding assays reveal that alphavbeta3 integrin assembles FN-RGE by binding an isoDGR motif in FN-I5, which is generated by the nonenzymatic rearrangement of asparagines (N) into an iso-aspartate (iso-D). Our findings demonstrate that FN contains a novel motif for integrin binding and fibril formation whose activity is controlled by amino acid modification.

The large extracellular glycoprotein reelin directs neuronal migration during brain development and plays a fundamental role in layer formation. It is composed of eight tandem repeats of an approximately 380-residue unit, termed the reelin repeat, which has a central epidermal growth factor (EGF) module flanked by two homologous subrepeats with no obvious sequence similarity to proteins of known structure. The 2.05 A crystal structure of the mouse reelin repeat 3 reveals that the subrepeat assumes a beta-jelly-roll fold with unexpected structural similarity to carbohydrate-binding domains. Despite the interruption by the EGF module, the two subdomains make direct contact, resulting in a compact overall structure. Electron micrographs of a four-domain fragment encompassing repeats 3-6, which is capable of inducing Disabled-1 phosphorylation in neurons, show a rod-like shape. Furthermore, a three-dimensional molecular envelope of the fragment obtained by single-particle tomography can be fitted with four concatenated repeat 3 atomic structures, providing the first glimpse of the structural unit for this important signaling molecule.

Loss of the Sortilin-related receptor 1 (SORL1) gene seems to act as a causal event for Alzheimer's disease (AD). Recent studies have established that loss of SORL1, as well as mutations in autosomal dominant AD genes APP and PSEN1/2, pathogenically converge by swelling early endosomes, AD's cytopathological hallmark. Acting together with the retromer trafficking complex, SORL1 has been shown to regulate the recycling of the amyloid precursor protein (APP) out of the endosome, contributing to endosomal swelling and to APP misprocessing. We hypothesized that SORL1 plays a broader role in neuronal endosomal recycling and used human induced pluripotent stem cell-derived neurons (hiPSC-Ns) to test this hypothesis. We examined endosomal recycling of three transmembrane proteins linked to AD pathophysiology: APP, the BDNF receptor Tropomyosin-related kinase B (TRKB), and the glutamate receptor subunit AMPA1 (GLUA1).

The Wnt signaling pathway plays a critical role in the developmental and physiological processes of metazoans. We previously reported that the Frizzled4 (FZD4) linker domain plays an important role in Norrin binding and signaling. However, the question remains whether the FZD linker contributes to Wnt signaling in general. Here, we show that the FZD linker is involved in Wnt binding and affects downstream Wnt signaling. A FZD4 chimera, in which the linker was swapped with that of the non-canonical receptor FZD6, impairs the binding with WNT3A and suppresses the recruitment of LRP6 and Disheveled, resulting in reduced canonical signaling. A similar effect was observed for non-canonical signaling. A FZD6 chimera containing the FZD1 linker showed reduced WNT5A binding and impaired signaling in ERK, JNK, and AKT mediated pathways. Altogether, our results suggest that the FZD linker plays an important role in specific Wnt binding and intracellular Wnt signaling.

SARS-CoV-2 is a novel coronavirus responsible for the COVID-19 pandemic. Its high pathogenicity is due to SARS-CoV-2 spike protein (S protein) contacting host-cell receptors. A critical hallmark of COVID-19 is the occurrence of coagulopathies. Here, we report the direct observation of the interactions between S protein and platelets. Live imaging showed that the S protein triggers platelets to deform dynamically, in some cases, leading to their irreversible activation. Strikingly, cellular cryo-electron tomography revealed dense decorations of S protein on the platelet surface, inducing filopodia formation. Hypothesizing that S protein binds to filopodia-inducing integrin receptors, we tested the binding to RGD motif-recognizing platelet integrins and found that S protein recognizes integrin α v β 3 . Our results infer that the stochastic activation of platelets is due to weak interactions of S protein with integrin, which can attribute to the pathogenesis of COVID-19 and the occurrence of rare but severe coagulopathies.

Short half-lives in circulation and poor transport across the blood-brain barrier limit the utility of cytokines and growth factors acting as receptor agonists. Here we show that surrogate receptor agonists with longer half-lives in circulation and enhanced transport rates across the blood-brain barrier can be generated by genetically inserting macrocyclic peptide pharmacophores into the structural loops of the fragment crystallizable (Fc) region of a human immunoglobulin. We used such 'lasso-grafting' approach, which preserves the expression levels of the Fc region and its affinity for the neonatal Fc receptor, to generate Fc-based protein scaffolds with macrocyclic peptides binding to the receptor tyrosine protein kinase Met. The Met agonists dimerized Met, inducing biological responses that were similar to those induced by its natural ligand. Moreover, lasso-grafting of the Fc region of the mouse anti-transferrin-receptor antibody with Met-binding macrocyclic peptides enhanced the accumulation of the resulting Met agonists in brain parenchyma in mice. Lasso-grafting may allow for designer protein therapeutics with enhanced stability and pharmacokinetics.

Recognition of laminin by integrin receptors is central to the epithelial cell adhesion to basement membrane, but the structural background of this molecular interaction remained elusive. Here, we report the structures of the prototypic laminin receptor α6β1 integrin alone and in complex with three-chain laminin-511 fragment determined via crystallography and cryo-electron microscopy, respectively. The laminin-integrin interface is made up of several binding sites located on all five subunits, with the laminin γ1 chain C-terminal portion providing focal interaction using two carboxylate anchor points to bridge metal-ion dependent adhesion site of integrin β1 subunit and Asn189 of integrin α6 subunit. Laminin α5 chain also contributes to the affinity and specificity by making electrostatic interactions with large surface on the β-propeller domain of α6, part of which comprises an alternatively spliced X1 region. The propeller sheet corresponding to this region shows unusually high mobility, suggesting its unique role in ligand capture.

Ectodomain shedding (shedding) is a post-translational modification, which liberates the extracellular domain of membrane proteins through juxtamembrane processing executed mainly by the ADAM (a disintegrin and metalloprotease) family of metalloproteases. Because shedding alters characteristics of cells in a rapid and irreversible manner, it should be strictly regulated. However, the molecular mechanisms determining membrane protein susceptibility to shedding (shedding susceptibility) are largely unknown. Here we report that alternative splicing can give rise to both shedding-susceptible and shedding-resistant CADM1 (cell adhesion molecule 1) variant proteins. We further show that O-glycans adjacent to the shedding cleavage site interfere with CADM1 shedding, and the only 33-bp alternative exon confers shedding susceptibility to CADM1 by inserting five non-glycosylatable amino acids between interfering O-glycans and the shedding cleavage site. These results demonstrate that shedding susceptibility of membrane protein can be determined at two different levels of its biosynthesis pathway, alternative splicing and O-glycosylation.

A point mutation in isocitrate dehydrogenase 1 (IDH1) and IDH2 is directly linked to the pathogenesis of certain types of tumors. To detect this mutation, several antibodies that can distinguish between mutant and wild-type enzymes have been established. One of which, MsMab-1, has a unique multi-specific character against several types of mutated IDH1/2. This promiscuous character is in remarkable contrast to the highly specific antigen recognition typically observed with a monoclonal antibody. We solved the crystal structure of MsMab-1 Fab fragment in complex with either IDH1 or IDH2-derived peptides. Based on the structure, it became clear that the peptide-binding pocket of the antibody is highly complementary to the core determinant shared between the IDH1 and IDH2, while leaving just enough space for the side chain of the pathogenic but not the wild-type amino acids located in the mutation position. Clarification of the molecular basis for the peculiar binding characteristics of MsMab-1 in atomic detail will help facilitating its diagnostic application, and may be used to develop better diagnostic reagents through structure-guided protein engineering.

The MAP tag system comprises a 14-residue peptide derived from mouse podoplanin and its high-affinity monoclonal antibody PMab-1. We determined the crystal structure of PMab-1 complexed with the MAP tag peptide and found that the recognition required only the N-terminal 8 residues of MAP tag sequence, enabling the shortening of the tag length without losing the affinity for PMab-1. Furthermore, the structure illustrated that the MAP tag adopts a U-shaped conformation when bound by PMab-1, suggesting that loop-inserted MAP tag would assume conformation compatible with the PMab-1 binding. We inserted the 8-residue MAP tag into multiple loop regions in various proteins including fibronectin type III domain and G-protein-coupled receptors and tested if they maintain PMab-1 reactivity. Despite the conformational restraints forced by the insertion position, all MAP-inserted mutants were expressed well in mammalian cells at levels comparable to the non-tagged proteins. Furthermore, the binding by PMab-1 was fully maintained even for the mutant where MAP tag was inserted at a structurally restricted β-hairpin, indicating that the MAP tag system has unique feature that allows placement in the middle of protein domain at desired locations. Our results indicate the versatile utility of the MAP tag system in 'site-specific epitope insertion' application.

Integrin activation is associated with conformational regulation. In this study, we developed a system to evaluate conformational changes in α4β7 integrin. We first inserted the PA tag into the plexin-semaphorin-integrin (PSI) domain of β7 chain, which reacted with an anti-PA tag antibody (NZ-1) in an Mn2+-dependent manner. The small GTPase Rap1 deficiency, as well as chemokine stimulation and the introduction of the active form of Rap1, Rap1V12, enhanced the binding of NZ-1 to the PA-tagged mutant integrin, and increased the binding affinity to mucosal addressing cell adhesion molecule-1 (MAdCAM-1). Furthermore, we generated two kinds of hybridomas producing monoclonal antibodies (mAbs) that recognized Mn2+-dependent epitopes of β7. Both epitopes were exposed to bind to mAbs on the cells by the introduction of Rap1V12. Although one epitope in the PSI domain of β7 was exposed on Rap1-deficienct cells, the other epitope in the hybrid domain of β7 was not. These data indicate that the conversion of Rap1-GDP to GTP exerts two distinct effects stepwise on the conformation of α4β7. The induction of colitis by Rap1-deficient CD4+ effector/memory T cells suggests that the removal of constraining effect by Rap1-GDP on α4β7 is sufficient for homing of these pathogenic T cells into colon lamina propria (LP).

Malaria is among the most serious infectious diseases affecting humans, accounting for approximately half a million deaths each year. Plasmodium falciparum causes most life-threatening cases of malaria. Acquired immunity to malaria is inefficient, even after repeated exposure to P. falciparum, but the immune regulatory mechanisms used by P. falciparum remain largely unknown. Here we show that P. falciparum uses immune inhibitory receptors to achieve immune evasion. RIFIN proteins are products of a polymorphic multigene family comprising approximately 150-200 genes per parasite genome that are expressed on the surface of infected erythrocytes. We found that a subset of RIFINs binds to either leucocyte immunoglobulin-like receptor B1 (LILRB1) or leucocyte-associated immunoglobulin-like receptor 1 (LAIR1). LILRB1-binding RIFINs inhibit activation of LILRB1-expressing B cells and natural killer (NK) cells. Furthermore, P. falciparum-infected erythrocytes isolated from patients with severe malaria were more likely to interact with LILRB1 than erythrocytes from patients with non-severe malaria, although an extended study with larger sample sizes is required to confirm this finding. Our results suggest that P. falciparum has acquired multiple RIFINs to evade the host immune system by targeting immune inhibitory receptors.