Ectodomain Conformation Research Articles

Inside-out regulation of E-cadherin conformation and adhesion

Cadherin cell-cell adhesion proteins play key roles in tissue morphogenesis and wound healing. Cadherin ectodomains bind in two conformations, X-dimers and strand-swap dimers, with different adhesive properties. However, the mechanisms by which cells regulate ectodomain conformation are unknown. Cadherin intracellular regions associate with several actin-binding proteins including vinculin, which are believed to tune cell-cell adhesion by remodeling the actin cytoskeleton. Here, we show at the single-molecule level, that vinculin association with the cadherin cytoplasmic region allosterically converts weak X-dimers into strong strand-swap dimers and that this process is mediated by myosin II-dependent changes in cytoskeletal tension. We also show that in epithelial cells, ∼70% of apical cadherins exist as strand-swap dimers while the remaining form X-dimers, providing two cadherin pools with different adhesive properties. Our results demonstrate the inside-out regulation of cadherin conformation and establish a mechanistic role for vinculin in this process.

Alternative Splicing of a Receptor Intracellular Domain Yields Different Ectodomain Conformations, Enabling Isoform-Selective Functional Ligands.

SummaryEvents at a receptor ectodomain affect the intracellular domain conformation, activating signal transduction (out-to-in conformational effects). We investigated the reverse direction (in-to-out) where the intracellular domain may impact on ectodomain conformation. The primary sequences of naturally occurring TrkC receptor isoforms (TrkC-FL and TrkC.T1) only differ at the intracellular domain. However, owing to their differential association with Protein Disulfide Isomerase the isoforms have different disulfide bonding and conformations at the ectodomain. Conformations were exploited to develop artificial ligands, mAbs, and small molecules, with isoform-specific binding and biased activation. Consistent, the physiological ligands NT-3 and PTP-sigma bind both isoforms, but NT-3 activates all signaling pathways, whereas PTP-sigma activates biased signals. Our data support an “in-to-out” model controlling receptor ectodomain conformation, a strategy that enables heterogeneity in receptors, ligands, and bioactivity. These concepts may be extended to the many wild-type or oncogenic receptors with known isoforms.

An integrin αIIbβ3 intermediate affinity state mediates biomechanical platelet aggregation.

Integrins are membrane receptors mediating cell adhesion and mechanosensing. The structure-function relationship of integrins remains incompletely understood, despite the extensive studies due to its importance to basic cell biology and translational medicine. Using fluorescence dual biomembrane force probe, microfluidics and cone-and-plate rheometry, we applied precisely-controlled mechanical stimulations to platelets and identified an intermediate state of integrin αIIbβ3, which is characterized by an ectodomain conformation, ligand affinity and bond lifetimes that are all intermediate between the well-known inactive and active states. This intermediate state is induced by ligand engagement of GPIbα via a mechano-signaling pathway and potentiates the outside-in mechano-signaling of αIIbβ3 for further transition to the active state during integrin mechanical affinity maturation. Our work reveals distinct αIIbβ3 state transitions in response to biomechanical and biochemical stimuli, and identifies a role for the αIIbβ3 intermediate state in promoting biomechanical platelet aggregation.

Biophysics of Mechanosensitive Cadherin Adhesion and its Regulation

Cadherin cell-cell adhesion proteins play critical roles in embryogenesis and in maintaining tissue integrity. Defects in cadherin adhesion occur in metastatic cancers. Cadherins mediate adhesion by binding in two conformations, X-dimers and strand-swap dimers. These binding conformations have distinctly different adhesive properties: while X-dimers form catch bonds that become longer-lived in the presence of a pulling force, strand-swap dimers form slip bonds that weaken upon pulling. It has been proposed that the cell, switches between X-dimer and strand-swap dimer conformations in order to regulate adhesion. However, little is known about the molecular mechanisms for these conformation changes and for cadherin adhesion regulation. Here, we use an integrated approach that couples live-cell, single molecule Atomic Force Microscope force measurements with precise, cell-biological manipulations of cadherin-cytoskeleton interactions, to characterize the biophysical mechanisms by which cadherins switch between alternate conformations on the cell surface and subsequently regulate adhesion. We demonstrate that cadherin interactions with the cytoskeleton tunes ectodomain conformation using an ‘inside-out’ mechanism. Our data also resolves the mechanistic details for mechanosensitive adhesion regulation.

How multi-scale structural biology elucidated context-dependent variability in ectodomain conformation along withthe ligand capture and release cycle for LDLR family members.

The low-density lipoprotein receptor (LDLR) and its homologs capture and internalize lipoproteins into the cell. Due to the fact that LDLR family members possess a modular ectodomain that undergoes dynamic conformational changes, multi-scale structural analysis has been performed so as to understand the ligand capture and release mechanism. For example, crystallographic analyses have provided models for boththe entire ectodomain and high-resolution structures of individual modules. In addition, nuclear magnetic resonance spectroscopic analyses have shown the rigidity and flexibility of inter-module linkers to restrict the mobility of ectodomain. Accumulated structural data suggest that the ectodomains of LDLR family members are flexible at the cell surface and switch between two metastable conformations, that is, the extended and contracted conformations. Recent structural analysis of ApoER2, a close homolog of LDLR, raised the possibility that the receptor binds with the ligand in the contracted conformation. After transport to an endosome by endocytosis, the receptor undergoes a conformational change to the closed conformation for completion of ligand release. In contrast, LDLR has been reported to adopt the extended conformation when it binds with a inhibitory regulator that recruits LDLR toward the degradation pathway. These findings support a mechanism of different ectodomain conformations for binding the ligand versus binding the regulatory protein. In this review, I provide an overview of studies that analyze the structural and biophysical properties of the ectodomains of LDLR family members and discuss a hypothetical model for ligand uptake and receptor recycling that integrates the known ectodomain conformational variability.

Evaluation of the contribution of the transmembrane region to the ectodomain conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein

BackgroundThe human immunodeficiency virus (HIV-1) envelope glycoprotein (Env), a Type 1 transmembrane protein, assembles into a trimeric spike complex that mediates virus entry into host cells. The high potential energy of the metastable, unliganded Env trimer is maintained by multiple non-covalent contacts among the gp120 exterior and gp41 transmembrane Env subunits. Structural studies suggest that the gp41 transmembrane region forms a left-handed coiled coil that contributes to the Env trimer interprotomer contacts. Here we evaluate the contribution of the gp41 transmembrane region to the folding and stability of Env trimers.MethodsMultiple polar/charged amino acid residues, which hypothetically disrupt the stop-transfer signal, were introduced in the proposed lipid-interactive face of the transmembrane coiled coil, allowing release of soluble cleavage-negative Envs containing the modified transmembrane region (TMmod). We also examined effects of cleavage, the cytoplasmic tail and a C-terminal fibritin trimerization (FT) motif on oligomerization, antigenicity and functionality of soluble and membrane-bound Envs.ResultsThe introduction of polar/charged amino acids into the transmembrane region resulted in the secretion of soluble Envs from the cell. However, these TMmod Envs primarily formed dimers. By contrast, control cleavage-negative sgp140 Envs lacking the transmembrane region formed soluble trimers, dimers and monomers. TMmod and sgp140 trimers were stabilized by the addition of a C-terminal FT sequence, but still exhibited carbohydrate and antigenic signatures of a flexible ectodomain structure. On the other hand, detergent-solubilized cleaved and uncleaved Envs isolated from the membranes of expressing cells exhibited "tighter” ectodomain structures, based on carbohydrate modifications. These trimers were found to be unstable in detergent solutions, but could be stabilized by the addition of a C-terminal FT moiety. The C-terminal FT domain decreased Env cleavage and syncytium-forming ability by approximately three-fold; alteration of the FT trimerization interface restored Env cleavage and syncytium formation to near-wild-type levels.ConclusionThe modified transmembrane region was not conducive to trimerization of soluble Envs. However, for HIV-1 Env ectodomains that are minimally modified, membrane-anchored Envs exhibit the most native structures and can be stabilized by appropriately positioned FT domains.

Structural and Functional Comparisons of Retroviral Envelope Protein C-Terminal Domains: Still Much to Learn

Retroviruses are a family of viruses that cause a broad range of pathologies in animals and humans, from the apparently harmless, long-term genomic insertion of endogenous retroviruses, to tumors induced by the oncogenic retroviruses and acquired immunodeficiency syndrome (AIDS) resulting from human immunodeficiency virus infection. Disease can be the result of diverse mechanisms, including tumorigenesis induced by viral oncogenes or immune destruction, leading to the gradual loss of CD4 T-cells. Of the virally encoded proteins common to all retroviruses, the envelope (Env) displays perhaps the most diverse functionality. Env is primarily responsible for binding the cellular receptor and for effecting the fusion process, with these functions mediated by protein domains localized to the exterior of the virus. The remaining C-terminal domain may have the most variable functionality of all retroviral proteins. The C-terminal domains from three prototypical retroviruses are discussed, focusing on the different structures and functions, which include fusion activation, tumorigenesis and viral assembly and lifecycle influences. Despite these genetic and functional differences, however, the C-terminal domains of these viruses share a common feature in the modulation of Env ectodomain conformation. Despite their differences, perhaps each system still has information to share with the others.

EM Structure of the Ectodomain of Integrin CD11b/CD18 and Localization of Its Ligand-Binding Site Relative to the Plasma Membrane

One-half of the integrin α-subunit Propeller domains contain and extra vWFA domain (αA domain), which mediates integrin binding to extracellular physiologic ligands via its metal-ion-dependent adhesion site (MIDAS). We used electron microscopy to determine the 3D structure of the αA-containing ectodomain of the leukocyte integrin CD11b/CD18 (αMβ2) in its inactive state. A well defined density for αA was observed within a bent ectodomain conformation, while the structure of the ectodomain in complex with the Fab fragment of mAb107, which binds at the MIDAS face of CD11b and stabilizes the inactive state, further revealed that αA is restricted to a relatively small range of orientations relative to the Propeller domain. Using Fab 107 as probe in fluorescent lifetime imaging microscopy (FLIM) revealed that αA is positioned relatively far from the membrane surface in the inactive state, and a systematic orientation search revealed that the MIDAS face would be accessible to extracellular ligand in the inactive state of the full-length cellular integrin. These studies are the first to define the 3D EM structure of an αA-containing integrin ectodomain and to position the ligand-binding face of αA domain in relation to the plasma membrane, providing new insights into current models of integrin activation.

Effects of Stabilization of the gp41 Cytoplasmic Domain on Fusion Activity and Infectivity of SIVmac239

We investigated the effects of introducing specific sequences that are predicted to affect trimer stability into the CT domain of the SIV Env protein. Two constructs, 3HBai and 3HBaa, with additional GCN4-related sequences in the CT domain (45 aa) had enhanced infectivity, and differed in their fusion activity and trimer stability. Another construct, 3HBii, exhibited a very stable trimeric structure. Pseudotyped virions containing 3HBii retained infectivity despite the lack of syncytia formation. In contrast, 3HBai and 3HBaa, which caused extensive syncytia formation, had a less stable trimeric structure. We observed an inverse correlation between trimer stability and fusion activity but no correlation between syncytia formation activity and infectivity. Quantitative cell-cell fusion assays, analysis of Env incorporation, measurement of ectodomain conformation by CD4 binding, and CCR5 blocking assays indicated differential effects on fusion activity and infectivity of the viruses with Env CT modifications. Differences in interaction with CD4 were not affected by trimer stability and were not related to fusion activity or infectivity. The results indicate that changes in the stability of the CT domain can have significant effects on functional activities of the Env external domain and can impact viral biological properties.

Structural Model and trans-Interaction of the Entire Ectodomain of the Olfactory Cell Adhesion Molecule

The ectodomain of olfactory cell adhesion molecule (OCAM/NCAM2/RNCAM) consists of five immunoglobulin (Ig) domains (IgI-V), followed by two fibronectin-type 3 (Fn3) domains (Fn3I-II). A complete structural model of the entire ectodomain of human OCAM has been assembled from crystal structures of six recombinant proteins corresponding to different regions of the ectodomain. The model is the longest experimentally based composite structural model of an entire IgCAM ectodomain. It displays an essentially linear arrangement of IgI-V, followed by bends between IgV and Fn3I and between Fn3I and Fn3II. Proteins containing IgI-IgII domains formed stable homodimers in solution and in crystals. Dimerization could be disrupted invitro by mutations in the dimer interface region. In conjunction with the bent ectodomain conformation, which can position IgI-V parallel with the cell surface, the IgI-IgII dimerization enables OCAM-mediated trans-interactions with an intercellular distance of about 20nm, which is consistent with that observed in synapses.

Modification of the L1-CAM carboxy-terminus in pancreatic adenocarcinoma cells

The neural cell adhesion molecule L1 has recently been shown to be expressed in pancreatic adenocarcinoma (PDAC) cells. In this report, we demonstrate that L1 is expressed by moderately- to poorly-differentiated PDAC cells in situ, and that L1 expression is a predictor of poor patient survival. In vitro, reduced reactivity of an anti-L1 carboxy-terminus-specific antibody was observed in the more poorly differentiated fast-growing (FG) variant of the COLO357 population, versus its well-differentiated slow-growing (SG) counterpart, even though they express equivalent total L1. The carboxy-terminus of L1 mediates binding to the MAP kinase-regulating protein RanBPM and mutation of T1247/S1248 within this region attenuates the expression of malignancy associated proteins and L1-induced tumorigenicity in mice. Therefore, we reasoned that the differential epitope exposure observed might be indicative of modifications responsible for regulating these events. However, epitope mapping demonstrated that the major determinant of binding was actually N1251; mutation of T1247 and S1248, alone or together, had little effect on C20 binding. Moreover, cluster assays using CD25 ectodomain/L1 cytoplasmic domain chimeras demonstrated the N1251-dependent, RanBPM-independent stimulation of erk phosphorylation in these cells. Reactivity of this antibody also reflects the differential exposure of extracellular epitopes in these COLO357 sublines, consistent with the previous demonstration of L1 ectodomain conformation modulation by intracellular modifications. These data further support a central role for L1 in PDAC, and define a specific role for carboxy-terminal residues including N1251 in the regulation of L1 activity in PDAC cells.

α9β1 Integrin in melanoma cells can signal different adhesion states for migration and anchorage

Cell surface integrins are the primary receptors for cell migration on extracellular matrix, and exist in several activation states regulated in part by ectodomain conformation. The α9 integrin subunit, which pairs only with β1, has specific roles in the immune system and may regulate cell migration. Melanoma cells express abundant α9β1 integrin, and its role in cell migration was assessed. Ligands derived from Tenascin-C and ADAM12 supported α9β1 integrin-mediated cell attachment and GTP-Rac dependent migration, but not focal adhesion formation. Manganese ions induced α9β1 integrin- and Rho kinase-dependent focal adhesion and stress fibre formation, suggesting that the activation status of α9β1 integrin was altered. The effect of manganese ions in promoting focal adhesion formation was reproduced by β1 integrin activating antibody. The α9β1 integrin translocated to focal adhesions, where active β1 integrin was also detected by conformation-specific antibodies. Focal adhesion assembly was commensurate with reduced cell migration. Endogenous α9β1 integrin-mediated adhesion was sensitive to the PP1 chemical inhibitor and an inhibitor of endosomal vesicle recycling, but not inhibitors of protein kinase C or the small GTPase Rho. Our results demonstrated that although α9β1 integrin can induce and localise to focal adhesions in a high activation state, its intermediate activity state normally supports cell adhesion consistent with migration.

Urokinase-type Plasminogen Activator Receptor Induces Conformational Changes in the Integrin αMβ2 Headpiece and Reorientation of Its Transmembrane Domains

The glycosylphosphatidylinositol-linked urokinase-type plasminogen activator receptor (uPAR) interacts with the heterodimer cell adhesion molecules integrins to modulate cell adhesion and migration. Devoid of a cytoplasmic domain, uPAR triggers intracellular signaling via its associated molecules that contain cytoplasmic domains. Interestingly, uPAR changes the ectodomain conformation of one of its partner molecules, integrin alpha(5)beta(1), and elicits cytoplasmic signaling. The separation or reorientation of integrin transmembrane domains and cytoplasmic tails are required for integrin outside-in signaling. However, there is a lack of direct evidence showing these conformational changes of an integrin that interacts with uPAR. In this investigation we used reporter monoclonal antibodies and fluorescence resonance energy transfer analyses to show conformational changes in the alpha(M)beta(2) headpiece and reorientation of its transmembrane domains when alpha(M)beta(2) interacts with uPAR.

Polybasic KKR Motif in the Cytoplasmic Tail of Nipah Virus Fusion Protein Modulates Membrane Fusion by Inside-Out Signaling

The cytoplasmic tails of the envelope proteins from multiple viruses are known to contain determinants that affect their fusogenic capacities. Here we report that specific residues in the cytoplasmic tail of the Nipah virus fusion protein (NiV-F) modulate its fusogenic activity. Truncation of the cytoplasmic tail of NiV-F greatly inhibited cell-cell fusion. Deletion and alanine scan analysis identified a tribasic KKR motif in the membrane-adjacent region as important for modulating cell-cell fusion. The K1A mutation increased fusion 5.5-fold, while the K2A and R3A mutations decreased fusion 3- to 5-fold. These results were corroborated in a reverse-pseudotyped viral entry assay, where receptor-pseudotyped reporter virus was used to infect cells expressing wild-type or mutant NiV envelope glycoproteins. Differential monoclonal antibody binding data indicated that hyper- or hypofusogenic mutations in the KKR motif affected the ectodomain conformation of NiV-F, which in turn resulted in faster or slower six-helix bundle formation, respectively. However, we also present evidence that the hypofusogenic phenotypes of the K2A and R3A mutants were effected via distinct mechanisms. Interestingly, the K2A mutant was also markedly excluded from lipid rafts, where approximately 20% of wild-type F and the other mutants can be found. Finally, we found a strong negative correlation between the relative fusogenic capacities of these cytoplasmic-tail mutants and the avidities of NiV-F and NiV-G interactions (P = 0.007, r(2) = 0.82). In toto, our data suggest that inside-out signaling by specific residues in the cytoplasmic tail of NiV-F can modulate its fusogenicity by multiple distinct mechanisms.

Activation of a paramyxovirus fusion protein is modulated by inside-out signaling from the cytoplasmic tail.

Many viruses have evolved fusion-mediating glycoproteins that couple the energy released from irreversible protein refolding to the work of membrane fusion. The viral fusion proteins require a triggering event to undergo a cascade of tightly regulated conformational changes. Different isolates of the paramyxovirus SV5 fusion (F) protein have either a short (20-residue) or long (42-residue) cytoplasmic tail (CT), and a long CT suppresses fusion activity in a sequence-specific manner. Addition of a domain to the F protein CT, which has the propensity to form a three-helix bundle, stabilizes the F protein and increases the energy required for fusion activation. Quantitative cell-cell fusion assays and measurement of ectodomain conformation by monoclonal antibody reactivity indicate that this suppression of fusion by the long CT or addition of a three-helix bundle occurs at a step preceding initial membrane merger. The data suggest that F protein activation involves CT signaling to the ectodomain.

Defining the boundaries of the testis angiotensin I-converting enzyme ectodomain

Numerous cytokines, receptors, and ectoenzymes, including angiotensin I-converting enzyme (ACE), are shed from the cell surface by limited proteolysis at the juxtamembrane stalk region. The membrane-proximal C domain of ACE has been implicated in sheddase–substrate recognition. We mapped the functional boundaries of the testis ACE ectodomain (identical to the C domain of somatic ACE) by progressive deletions from the N- and C-termini and analysing the effects on catalytic activity, stability, and shedding in transfected cells. We found that deletions extending beyond Leu37 at the N-terminus and Trp616 at the C-terminus abolished catalytic activity and shedding, either by disturbing the ectodomain conformation or by inhibiting maturation and surface expression. Based on these data and on sequence alignments, we propose that the boundaries of the ACE ectodomain are Asp40 at the N-terminus and Gly615 at the C-terminus.

Environmental control of collateral branching of mitral cells

The ectodomain of olfactory cell adhesion molecule (OCAM/NCAM2/RNCAM) consists of five immunoglobulin (Ig) domains (IgI–V), followed by two fibronectin-type 3 (Fn3) domains (Fn3I–II). A complete structural model of the entire ectodomain of human OCAM has been assembled from crystal structures of six recombinant proteins corresponding to different regions of the ectodomain. The model is the longest experimentally based composite structural model of an entire IgCAM ectodomain. It displays an essentially linear arrangement of IgI–V, followed by bends between IgV and Fn3I and between Fn3I and Fn3II. Proteins containing IgI–IgII domains formed stable homodimers in solution and in crystals. Dimerization could be disrupted in vitro by mutations in the dimer interface region. In conjunction with the bent ectodomain conformation, which can position IgI–V parallel with the cell surface, the IgI–IgII dimerization enables OCAM-mediated trans-interactions with an intercellular distance of about 20 nm, which is consistent with that observed in synapses.