The ß8 integrin EGF domains support a constitutive extended conformation, and the cytoplasmic domain impairs outside-in signaling.

Integrins are transmembrane proteins that transmit bi-directional signals across the cell membrane through global structural rearrangement among three different conformational states: bent, extended- closed, and extended-open conformations. However, the ß8 integrin is distinctive and may adopt only one conformation, that is, extended-closed conformation, with high affinity for ligands under physiological conditions, and may not transmit bi-directional signals like other integrin members. It is unclear how different ß8 domains affect its unique conformation and signaling. We swapped different domains of integrin ß3 with those of ß8 and investigated how they affected integrin ligand binding, global conformation, and outside-in signaling. We found that the ß8 epidermal growth factor (EGF) domains increased integrin ligand binding ability and contributed to its extended conformation. By comparison, the ß8 transmembrane and cytoplasmic domains had little effect on ligand binding or global conformation. The ß8 EGF and transmembrane domains did not affect integrin-mediated cell adhesion, cell spreading, focal adhesion formation, or colocalization of integrin with other proteins, but the cytoplasmic domain had a defective effect on outside-in signaling. Our results showed that different domains of ß8 play various roles on its unique conformation, ligand binding, and signaling, which are considered atypical among integrin members.

Effects of the association of the αv ß8 lower legs on integrin ligand binding.

Many integrins transmit signals through global conformational changes. However, it is unclear whether integrin αv ß8 adopts a similar mechanism during integrin activation and signaling on the cell surface. Here, we showed that disulfide-bonded mutants, which prevented integrin αv ß8 lower leg dissociation, bound ligands with similar level as the wild-type protein, suggesting that αv ß8 ligand binding did not require lower leg disassociation. We further showed that the N-glycosylation mutant at the interface between the ß I and hybrid domains did not affect ligand binding, suggesting that the αv ß8 open headpiece was not present on the cell surface. We proposed that αv ß8 integrin may adopt only one state, that is, the extended conformation with a closed headpiece. Our results showed that two lower legs retained heterodimeric interfaces, and this association might be important for stabilizing integrin in the extended conformation. Therefore, αv ß8 may not transmit bidirectional signals across the plasma membrane but instead may serve as an anchoring site with high affinity and high accessibility for extracellular ligands.

Atypical structure and function of integrin αV ß8.

Integrins are heterodimeric transmembrane proteins that play important roles in various biological processes. Most integrins serve as adhesion molecules and transmit bidirectional signaling across the cell membrane through global conformational changes from the bent closed to the extended open conformation. However, integrin ß8 is distinctive in structure and function. Its cytoplasmic domain lacks the conserved protein-binding sequence, which is important in transmitting inside-out signals, suggesting that integrin ß8 may have a different activation mechanism or lack such signaling. In addition, the ligand-binding or activating metal ion Mn2+ does not induce a global conformational change in integrin ß8 . It may have only one conformation, that is, an extended, closed conformation, but with high affinity for ligands under physiological conditions, and is, therefore, considered an atypical integrin member. The extended structure and high ligand-binding affinity of integrin αv ß8 make it ideal for encountering and binding ligands expressed on an opposing cell or in the extracellular matrix. In this review, we summarize the progress in integrin ß8 research with a focus on its distinctive function and structure among integrin members.

The interface between the EGF1 and EGF2 domains is critical in integrin affinity regulation.

It has been proposed that integrins adopt a bent, closed conformation with low ligand binding capability at resting state and switch into an extended, open conformation upon activation or interacting with extracellular matrix (ECM) ligand. In this study, we addressed how integrin conformational change at the ß genu affects ligand binding and signaling. We discovered that swapping of the ß3 epidermal growth factor-like (EGF) domain 1 and 2 with that of ß8 greatly promoted ligand binding in ß3 ß8 chimeras. Sequence alignment indicated that ß8 integrin uniquely lacks the interface between the EGF1 and 2. Disrupting this interface of the ß3 integrin increased integrin ligand binding. Furthermore, the interface is critical for integrin affinity regulation but not downstream outside-in signaling.

Structural basis of antifreeze activity of a bacterial multi-domain antifreeze protein.

Antifreeze proteins (AFPs) enhance the survival of organisms inhabiting cold environments by affecting the formation and/or structure of ice. We report the crystal structure of the first multi-domain AFP that has been characterized. The two ice binding domains are structurally similar. Each consists of an irregular ß-helix with a triangular cross-section and a long α-helix that runs parallel on one side of the ß-helix. Both domains are stabilized by hydrophobic interactions. A flat plane on the same face of each domain's ß-helix was identified as the ice binding site. Mutating any of the smaller residues on the ice binding site to bulkier ones decreased the antifreeze activity. The bulky side chain of Leu174 in domain A sterically hinders the binding of water molecules to the protein backbone, partially explaining why antifreeze activity by domain A is inferior to that of domain B. Our data provide a molecular basis for understanding differences in antifreeze activity between the two domains of this protein and general insight on how structural differences in the ice-binding sites affect the activity of AFPs.

Integrin αv ß8 Adopts a High Affinity State for Soluble Ligands Under Physiological Conditions.

It has been proposed that integrins adopt a low affinity conformation under physiological conditions. Integrin can either be activated through cytoplasm or by binding of cations such as Mn2+ to the head domain. The cytoplasmic activation pathway, that is, inside-out signaling has been regarded as the physiological pathway for integrin activation. Integrin ß8 is important for neuron vascular development. However, due to the highly divergent cytoplasmic domain, this integrin probably does not rely on inside-out signaling for affinity regulation. We therefore hypothesized that the ß8 integrin uniquely assumes a constitutively high affinity state under physiological conditions. We discovered that ß8 indeed exhibited high binding to soluble vitronectin in the presence of Ca2+ and the ligand binding could not be further enhanced by addition of Mn2+ . The lower ectodomain stalk of the integrin, which is comprised by the integrin epidermal growth factor-like (I-EGF) domains and ßTD domain, is critical for this high affinity conformation. In addition, we found that unlike other integrins, Mg2+ at low concentration inhibited ß8 ligand binding. Mutagenesis studies indicated that ß8 integrin possesses a unique cation binding site which might contribute to the ligand binding affinity. Our study showed that both the ß8 lower ectodomain stalk and the head domain play an important role in its high affinity state under physiological conditions. J. Cell. Biochem. 118: 2044-2052, 2017. © 2016 Wiley Periodicals, Inc.

Functional Analysis of a Bacterial Antifreeze Protein Indicates a Cooperative Effect between Its Two Ice-Binding Domains.

Antifreeze proteins make up a class of ice-binding proteins (IBPs) that are possessed and expressed by certain cold-adapted organisms to enhance their freezing tolerance. Here we report the biophysical and functional characterization of an IBP discovered in a bacterium recovered from a deep glacial ice core drilled at Vostok Station, Antarctica (IBPv). Our study showed that the recombinant protein rIBPv exhibited a thermal hysteresis of 2 °C at concentrations of >50 µM, effectively inhibited ice recrystallization, and enhanced bacterial viability during freeze-thaw cycling. Circular dichroism scans indicated that rIBPv mainly consists of ß strands, and its denaturing temperature was 53.5 °C. Multiple-sequence alignment of homologous IBPs predicted that IBPv contains two ice-binding domains, a feature unique among known IBPs. To examine functional differences between the IBPv domains, each domain was cloned, expressed, and purified. The second domain (domain B) expressed greater ice binding activity. Data from thermal hysteresis and gel filtration assays supported the idea that the two domains cooperate to achieve a higher ice binding effect by forming heterodimers. However, physical linkage of the domains was not required for this effect.

Integrin αIIbß3 transmembrane domain separation mediates bi-directional signaling across the plasma membrane.

Integrins play an essential role in hemostasis, thrombosis, and cell migration, and they transmit bidirectional signals. Transmembrane/cytoplasmic domains are hypothesized to associate in the resting integrins; whereas, ligand binding and intracellular activating signals induce transmembrane domain separation. However, how this conformational change affects integrin outside-in signaling and whether the α subunit cytoplasmic domain is important for this signaling remain elusive. Using Chinese Hamster Ovary (CHO) cells that stably expressed different integrin αIIbß3 constructs, we discovered that an αIIb cytoplasmic domain truncation led to integrin activation but not defective outside-in signaling. In contrast, preventing transmembrane domain separation abolished both inside-out and outside-in signaling regardless of removing the αIIb cytoplasmic tail. Truncation of the αIIb cytoplasmic tail did not obviously affect adhesion-induced outside-in signaling. Our research revealed that transmembrane domain separation is a downstream conformational change after the cytoplasmic domain dissociation in inside-out activation and indispensable for ligand-induced outside-in signaling. The result implicates that the ß TM helix rearrangement after dissociation is essential for integrin transmembrane signaling. Furthermore, we discovered that the PI3K/Akt pathway is not essential for cell spreading but spreading-induced Erk1/2 activation is PI3K dependent implicating requirement of the kinase for cell survival in outside-in signaling.

Recrystallization inhibition in ice due to ice binding protein activity detected by nuclear magnetic resonance.

Liquid water present in polycrystalline ice at the interstices between ice crystals results in a network of liquid-filled veins and nodes within a solid ice matrix, making ice a low porosity porous media. Here we used nuclear magnetic resonance (NMR) relaxation and time dependent self-diffusion measurements developed for porous media applications to monitor three dimensional changes to the vein network in ices with and without a bacterial ice binding protein (IBP). Shorter effective diffusion distances were detected as a function of increased irreversible ice binding activity, indicating inhibition of ice recrystallization and persistent small crystal structure. The modification of ice structure by the IBP demonstrates a potential mechanism for the microorganism to enhance survivability in ice. These results highlight the potential of NMR techniques in evaluation of the impact of IBPs on vein network structure and recrystallization processes; information useful for continued development of ice-interacting proteins for biotechnology applications.

Variation in one residue associated with the metal ion-dependent adhesion site regulates αIIbß3 integrin ligand binding affinity.

The Asp of the RGD motif of the ligand coordinates with the ß I domain metal ion dependent adhesion site (MIDAS) divalent cation, emphasizing the importance of the MIDAS in ligand binding. There appears to be two distinct groups of integrins that differ in their ligand binding affinity and adhesion ability. These differences may be due to a specific residue associated with the MIDAS, particularly the ß3 residue Ala(252) and corresponding Ala in the ß1 integrin compared to the analogous Asp residue in the ß2 and ß7 integrins. Interestingly, mutations in the adjacent to MIDAS (ADMIDAS) of integrins α4ß7 and αLß2 increased the binding and adhesion abilities compared to the wild-type, while the same mutations in the α2ß1, α5ß1, αVß3, and αIIbß3 integrins demonstrated decreased ligand binding and adhesion. We introduced a mutation in the αIIbß3 to convert this MIDAS associated Ala(252) to Asp. By combination of this mutant with mutations of one or two ADMIDAS residues, we studied the effects of this residue on ligand binding and adhesion. Then, we performed molecular dynamics simulations on the wild-type and mutant αIIbß3 integrin ß I domains, and investigated the dynamics of metal ion binding sites in different integrin-RGD complexes. We found that the tendency of calculated binding free energies was in excellent agreement with the experimental results, suggesting that the variation in this MIDAS associated residue accounts for the differences in ligand binding and adhesion among different integrins, and it accounts for the conflicting results of ADMIDAS mutations within different integrins. This study sheds more light on the role of the MIDAS associated residue pertaining to ligand binding and adhesion and suggests that this residue may play a pivotal role in integrin-mediated cell rolling and firm adhesion.

Integrin bi-directional signaling across the plasma membrane.

Integrins are heterodimeric cell adhesion molecules that are important in many biological functions, such as cell migration, proliferation, differentiation, and survival. They can transmit bi-directional signals across the plasma membrane. Inside-out activating signal from some cell surface receptors bound with soluble agonists triggers integrins conformational change leading to high affinity for extracellular ligands. Then binding of ligands to integrins results in outside-in signaling, leading to formation of focal adhesion complex at the integrin cytoplasmic tail and activation of downstream signal pathways. This bi-directional signaling is essential for rapid response of cell to surrounding environmental changes. During this process, the conformational change of integrin extracellular and transmembrane/cytoplasmic domains is particularly important. In this review, we will summarize recent progress in both inside-out and outside-in signaling with specific focus on the mechanism how integrins transmit bi-directional signals through transmembrane/cytoplasmic domains.

α(V)ß(3) integrin crystal structures and their functional implications.

Many questions about the significance of structural features of integrin α(V)ß(3) with respect to its mechanism of activation remain. We have determined and re-refined crystal structures of the α(V)ß(3) ectodomain linked to C-terminal coiled coils (α(V)ß(3)-AB) and four transmembrane (TM) residues in each subunit (α(V)ß(3)-1TM), respectively. The α(V) and ß(3) subunits with four and eight extracellular domains, respectively, are bent at knees between the integrin headpiece and lower legs, and the headpiece has the closed, low-affinity conformation. The structures differ in the occupancy of three metal-binding sites in the ßI domain. Occupancy appears to be related to the pH of crystallization, rather than to the physiologic regulation of ligand binding at the central, metal ion-dependent adhesion site. No electron density was observed for TM residues and much of the α(V) linker. α(V)ß(3)-AB and α(V)ß(3)-1TM demonstrate flexibility in the linker between their extracellular and TM domains, rather than the previously proposed rigid linkage. A previously postulated interface between the α(V) and ß(3) subunits at their knees was also not supported, because it lacks high-quality density, required rebuilding in α(V)ß(3)-1TM, and differed markedly between α(V)ß(3)-1TM and α(V)ß(3)-AB. Together with the variation in domain-domain orientation within their bent ectodomains between α(V)ß(3)-AB and α(V)ß(3)-1TM, these findings are compatible with the requirement for large structural changes, such as extension at the knees and headpiece opening, in conveying activation signals between the extracellular ligand-binding site and the cytoplasm.

Overview: structural biology of integrins.

Integrins are cell adhesion molecules that play important roles in many biological processes including hemostasis, immune responses, development, and cancer. Their adhesiveness is dynamically regulated through a process termed inside-out signaling. In addition, ligand binding transduces outside-in signals from the extracellular domain to the cytoplasm. Advances in the past several years have shed light on structural basis for integrin regulation and signaling, especially how the large-scale reorientations of the ectodomain are related to the inter-domain and intra-domain shape shifting that changes ligand-binding affinity. Experiments have also shown how the conformational changes of the ectodomain are linked to changes in the α- and ß-subunit transmembrane and cytoplasmic domains.

Mutagenesis studies of the ß I domain metal ion binding sites on integrin αVß3 ligand binding affinity.

Three divalent cation binding sites in the integrin ß I domain have been shown to regulate ligand binding and adhesion. However, the degree of ligand binding and adhesion varies among integrins. The αLß2 and α4ß7 integrins show an increase in ligand binding affinity and adhesion when one of their ADMIDAS (adjacent to MIDAS, or the metal ion-dependent adhesion site) residues is mutated. By contrast, the α2ß1, α5ß1, and αIIbß3 integrins show a decrease in binding affinity and adhesion when their ADMIDAS is mutated. Our study here indicated that integrin αVß3 had lower affinity when the ADMIDAS was mutated. By comparing the primary sequences of these integrin subunits, we propose that one residue associated with the MIDAS (ß3 Ala(252)) may account for these differences. In the ß1 integrin subunit, the corresponding residue is also Ala, whereas in both ß2 and ß7 integrin subunits, it is Asp. We mutated the ß3 residue Ala(252) to Asp and combined this mutant with mutations of one or two ADMIDAS residues. The mutant A252D showed reduced ligand binding affinity and adhesion. The ligand binding affinity and adhesion were increased when this A252D mutant was paired with mutations of one ADMIDAS residue. But when paired with mutations of two ADMIDAS residues the mutant nearly abolished ligand-binding ability, which was restored by the activating glycosylation mutation. Our study suggests that the variation of this residue contributes to the different ligand binding affinities and adhesion abilities among different integrin families.

Effects of the association between the α-subunit thigh and the ß-Subunit EGF2 domains on integrin activation and signaling.

Integrin bidirectional signaling is mediated by conformational change. It has been shown that the separation of the α- and ß-subunit transmembrane/cytoplasmic tails and the lower legs is required for transmitting integrin bidirectional signals across the plasma membrane. In this study, we address whether the separation of the αß knee is critical for integrin activation and outside-in signaling. By introducing three disulfide bonds to restrict dissociation of the α-subunit thigh domain and ß-subunit I-EGF2 domain, we found that two of them could completely abolish integrin inside-out activation, whereas the other could not. This disulfide-bonded mutant, in the context of the activation mutation of the cytoplasmic domain, had intermediate affinity for ligands and was able to mediate cell adhesion. Our data suggest that there exists rearrangement at the interface between the thigh domain and the I-EGF2 domain during integrin inside-out activation. None of the disulfide-bonded mutants could mediate cell spreading upon adhering to immobilized ligands, suggesting that dissociation of the integrin two knees is required for integrin outside-in signaling. Disrupting the interface by introducing a glycan chain into either subunit is sufficient for high affinity ligand binding and cell spreading.

Regulation of integrin αIIbß3 ligand binding and signaling by the metal ion binding sites in the ß I domain.

The ability of αIIbß3 to bind ligands and undergo outside-in signaling is regulated by three divalent cation binding sites in the ß I domain. Specifically, the metal ion-dependent adhesion site (MIDAS) and the synergistic metal binding site (SyMBS) are thought to be required for ligand binding due to their synergy between Ca(2+) and Mg(2+). The adjacent to MIDAS (ADMIDAS) is an important ligand binding regulatory site that also acts as a critical link between the ß I and hybrid domains for signaling. Mutations in this site have provided conflicting results for ligand binding and adhesion in different integrins. We have mutated the ß3 SyMBS and ADMIDAS. The SyMBS mutant abolished ligand binding and outside-in signaling, but when an activating glycosylation mutation in the αIIb Calf 2 domain was introduced, the ligand binding affinity and signaling were restored. Thus, the SyMBS is important but not absolutely required for integrin bidirectional signaling. The ADMIDAS mutants showed reduced ligand binding affinity and abolished outside-in signaling, and the activating glycosylation mutation could fully restore integrin signaling of the ADMIDAS mutant. We propose that the ADMIDAS ion stabilizes the low-affinity state when the integrin headpiece is in the closed conformation, whereas it stabilizes the high-affinity state when the headpiece is in the open conformation with the swung-out hybrid domain.

Tests of integrin transmembrane domain homo-oligomerization during integrin ligand binding and signaling.

Integrin transmembrane (TM) and/or cytoplasmic domains play a critical role in integrin bidirectional signaling. Although it has been shown that TM and/or cytoplasmic α and ß domains associate in the resting state and separation of these domains is required for both inside-out and outside-in signaling, the role of TM homomeric association remains elusive. Formation of TM homo-oligomers was observed in micelles and bacterial membranes previously, and it has been proposed that homomeric association is important for integrin activation and clustering. This study addresses whether integrin TM domains form homo-oligomers in mammalian cell membranes using cysteine scanning mutagenesis. Our results show that TM homomeric interaction does not occur before or after soluble ligand binding or during inside-out activation. In addition, even though the cysteine mutants and the heterodimeric disulfide-bounded mutant could form clusters after adhering to immobilized ligand, the integrin TM domains do not form homo-oligomers, suggesting that integrin TM homomeric association is not critical for integrin clustering or outside-in signaling. Therefore, integrin TM homo-oligomerization is not required for integrin activation, ligand binding, or signaling.

Dissociation of the α-subunit Calf-2 domain and the ß-subunit I-EGF4 domain in integrin activation and signaling.

Integrin conformational changes mediate integrin activation and signaling triggered by intracellular molecules or extracellular ligands. Even though it is known that αß transmembrane domain separation is required for integrin signaling, it is still not clear how this signal is transmitted from the transmembrane domain through two long extracellular legs to the ligand-binding headpiece. This study addresses whether the separation of the membrane-proximal extracellular αß legs is critical for integrin activation and outside-in signaling. Using a disulfide bond to restrict dissociation of the α-subunit Calf-2 domain and ß-subunit I-EGF4 domain, we were able to abolish integrin inside-out activation and outside-in signaling. In contrast, disrupting the interface by introducing a glycosylation site into either subunit activated integrins for ligand binding through a global conformational change. Our results suggest that the interface of the Calf-2 domain and the I-EGF4 domain is critical for integrin bidirectional signaling.

Structural basis of integrin transmembrane activation.

Integrins are cell adhesion receptors that transmit bidirectional signals across plasma membrane and are crucial for many biological functions. Recent structural studies of integrin transmembrane (TM) and cytoplasmic domains have shed light on their conformational changes during integrin activation. A structure of the resting state was solved based on Rosetta computational modeling and experimental data using intact integrins on mammalian cell surface. In this structure, the alpha(IIb) GXXXG motif and their beta(3) counterparts of the TM domains associate with ridge-in-groove packing, and the alpha(IIb) GFFKR motif and the beta(3) Lys-716 in the cytoplasmic segments play a critical role in the alpha/beta association. Comparing this structure with the NMR structures of the monomeric alpha(IIb) and beta(3) (represented as active conformations), the alpha subunit helix remains similar after dissociation whereas beta subunit helix is tilted by embedding additional 5-6 residues into the lipid bilayer. These conformational changes are critical for integrin activation and signaling across the plasma membrane. We thus propose a new model of integrin TM activation in which the recent NMR structure of the alpha(IIb)beta(3) TM/cytoplasmic complex represents an intermediate or transient state, and the electrostatic interaction in the cytoplasmic region is important for priming the initial alpha/beta association, but not absolutely necessary for the resting state.

The structure of a receptor with two associating transmembrane domains on the cell surface: integrin alphaIIbbeta3.

Structures of intact receptors with single-pass transmembrane domains are essential to understand how extracellular and cytoplasmic domains regulate association and signaling through transmembrane domains. A chemical and computational method to determine structures of the membrane regions of such receptors on the cell surface is developed here and validated with glycophorin A. An integrin heterodimer structure reveals association over most of the lengths of the alpha and beta transmembrane domains and shows that the principles governing association of hetero and homo transmembrane dimers differ. A turn at the Gly of the juxtamembrane GFFKR motif caps the alpha TM helix and brings the two Phe of GFFKR into the alpha/beta interface. A juxtamembrane Lys residue in beta also has an important role in the interface. The structure shows how transmembrane association/dissociation regulates integrin signaling. A joint ectodomain and membrane structure shows that substantial flexibility between the extracellular and TM domains is compatible with TM signaling.