Homomeric Interactions Research Articles

Detection of in vivo interactions between Arabidopsis class A-HSFs, using a novel BiFC fragment, and identification of novel class B-HSF interacting proteins

Class A heat shock factors (Hsfs) of Arabidopsis are known to function as transcriptional activators of stress genes. Genetic and functional analysis suggests that HsfA1a and HsfA1b are central regulators required in the early phase of the heat shock response, which have the capacity to functionally replace each other. In order to examine Hsf interaction in vivo, we conducted interaction assays using bimolecular fluorescence complementation (BiFC) on Arabidopsis protoplasts co-transformed with suitable Hsf-YFP fusion genes. BiFC assays were quantified with confocal laser scanning microscopy and flow cytometry, and confirmed with immunoprecipitation assays. For each Hsf we could not only demonstrate homomeric interactions but also detect heteromeric interaction between HsfA1a and HsfA1b. Truncated versions of these of Hsfs, containing deletions of the oligomerization domains (ODs), provided clear evidence that the ODs are required and sufficient for the HSF interaction in vivo. By contrast there was only homomeric but no heteromeric interaction detected between two different class B Hsf transcription factors (HsfB1 and HsfB2b) in a yeast two-hybrid assay. HsfB1/HsfB2b functions are not directly linked with the expression of conventional heat shock genes; class B Hsfs are devoid of the activation domain motif conserved in class A Hsfs. In order to identify other proteins interacting with HsfB1 and HsfB2b we performed yeast two-hybrid screenings of cDNA libraries. Three of the identified proteins were common to both screenings. This suggests that HsfB1 and HsfB2b may be involved in complex regulatory networks, which are linked to other stress responses and signaling processes.

Predicting Multicomponent Crystal Formation: The Interplay between Homomeric and Heteromeric Interactions

Current approaches to improving physicochemical properties without changing desirable therapeutic behavior of active pharmaceutical ingredients include formation of multicomponent crystals such as salts and cocrystals. We report a technique that can provide a priori prediction for multicomponent crystal formation based on intermolecular pair interactions characterized using pulsed gradient spin−echo nuclear magnetic resonance (PGSE NMR). The accuracy of our prediction technique in comparison to the well-adopted ΔpKa rule of thumb is tested against 25 molecular pairs including protonated amines, nitrogen-protonated heterocyclic bases, phenols, and carboxylic acids dissolved in six solvents. While the ΔpKa rule results in numerous contradicting exceptions, our technique robustly predicts multicomponent crystal formation. These results reveal that the application of PGSE NMR for determining the self-diffusivities of molecules and subsequently the strengths of intermolecular interactions has the potential to be developed into a standard and robust protocol for the study of multicomponent solution chemistry.

Maize AKINβγ dimerizes through the KIS/CBM domain and assembles into SnRK1 complexes

The SNF1/AMPK/SnRK1 complex is an intracellular energy sensor composed of three types of subunits: the SnRK1 kinase and two regulatory, non-catalytic subunits (designated β and γ). We have previously described an atypical plant γ-subunit, AKINβγ, which contains an N-terminal tail similar to the so-called KIS domain normally present in β-subunits. However, it is not known whether AKINβγ normally associates with endogenous SnRK1 complexes in vivo, nor how its unique domain structure might contribute to SnRK1 function. Here, we present evidence that maize AKINβγ is an integral component of active SnRK1 complexes in plant cells. Using complementary methodological approaches, we also show that AKINβγ associates through homomeric interactions mediated by both, the γ- and, unexpectedly, the KIS/CBM domain. Structured summary MINT- 7040005: AKIN (uniprotkb: B4FX20) and AKIN (uniprotkb: B4FX20) physically interact (MI: 0914) by chromatography technologies (MI: 0091) MINT- 7039992: AKIN (uniprotkb: B4FX20) and AKIN (uniprotkb: B4FX20) physically interact (MI: 0915) by bimolecular fluorescence complementation (MI: 0809) MINT- 7040024, MINT- 7040044, MINT- 7040067: AKIN (uniprotkb: B4FX20) and AKIN (uniprotkb: B4FX20) bind (MI: 0407) by pull down (MI: 0096) MINT- 7039978: SnRK1 (uniprotkb: Q8H1L5) and AKIN (uniprotkb: B4FX20) physically interact (MI: 0915) by bimolecular fluorescence complementation (MI: 0809)

An Orai1 Activating Minimal Fragment Of Stim1

In immune cells generation of sustained Ca2+ levels is mediated by the Ca2+ release activated Ca2+ (CRAC) current. Molecular key players in this process comprise the stromal interaction molecule (STIM1) that functions as a Ca2+ sensor in the endoplasmic reticulum and ORAI1 located in the plasma membrane. Depletion of ER Ca2+ store leads to STIM1 multimerization into discrete punctae that co-cluster with ORAI1 thereby triggering coupling to and activation of ORAI1 channels. The C-terminus of STIM1 is sufficient for the activation of ORAI1 currents independent of store depletion. Here we unmasked an ORAI activating minimal fragment (OAMF) within STIM1 C-terminus that exhibited enhanced interaction with ORAI1 and resulted in three-fold increased Ca2+ currents. STIM1-OAMF still showed the ability of a homomeric interaction similar to longer fragments as well as the full-length form of STIM1 C-terminus. In contrast, further deletion of a thirty amino acid region resulted in a substantial reduction of homomeric interaction concomitant to a loss of coupling to as well as activation of ORAI1. In aggregate, we have identified two key regions within STIM C-terminus that govern ORAI1 activation. (Supported by PhD-Program W1201 from the FWF)

Supramolecular synthesis of some molecular adducts of 4,4′-bipyridine N,N′-dioxide

Molecular adducts (1a–1e) of 4,4′-bipyridine N,N′-dioxide, 1, respectively with cyanuric acid, trithiocyanuric acid, 1,3,5-trihydroxybenzene (phloroglucinol), 1,3-dihydroxybenzene (resorcinol) and 1,2,4,5-benzenetetracarboxylic acid have been reported. The major interactions observed in the structures 1a–1e are N–H⋯O, N–H⋯S, O–H⋯O and C–H⋯O, in the form of homomeric and heteromeric patterns of the constituents, either as a single or cyclic hydrogen-bonded motifs. While in the adduct 1a, both homomeric and heteromeric units of both the constituents were observed, no heteromeric interactions were observed in 1b and 1c. In addition, in 1b, homomeric aggregation of molecules of 1 occurred in association with water molecules. However, while heteromeric interactions prevail between the constituents in 1d and 1e, only one of the co-crystallizing species gave homomeric interactions (4,4′-bipyridine N,N′-dioxide in 1d; 1,2,4,5-benzenetetracarboxylic acid in 1e). Further, in either type of the patterns, the cyclic motifs are formed as a pair-wise hydrogen bonds comprising of strong and weak hydrogen bonds (N–H⋯O/C–H⋯O or O–H⋯O/C–H⋯O). In three-dimensions, the ensembles of molecules yield planar sheets, ladders and pseudorotaxane type assemblies.

A Conserved Transmembrane Domain Interface Regulates Integrin Function

Integrins are a superfamily of transmembrane (TM) α/β heterodimers that mediate fundamental cellular adhesive functions. Platelet integrins, for example, mediate stable platelet adhesion to collagen and fibronectin and the formation of stable platelet aggregates. Integrins reside on cell surfaces in an equilibrium between inactive and active conformations. An essential feature of this equilibrium is interaction of the integrin α and β subunit TM domains. Thus, when integrins are inactive, the α and β TM domains are in proximity, but they separate when integrins assume an active conformation. Moreover, inducing TM domain separation alone is sufficient to cause integrin activation. Previously, we reported that the TM domains of the platelet integrin αIIbβ3 interact both heteromerically and homomerically and that the strength of their heteromeric interaction is necessarily weak to allow regulated TM domain separation. To address whether these observations can be extended to the other members of the integrin superfamily, we focused initially on αvβ3, α2β1 and α5β1, integrins present in platelets, using a dominant-negative ToxR-based assay. ToxR is a single-pass TM transcriptional factor from V. cholera that activates the cholera toxin (ctx) promoter when it dimerizes in the inner membrane of E. coli. By co-expressing wild-type ToxR with either wild-type ToxR or an R96K ToxR mutant that can dimerize but is unable to activate the ctx promoter, we can measure the homomeric and heteromeric interaction of each integrin TM domain. Using alanine and leucine scanning mutagenesis, we found that like αIIb, homo-oligomerization of other integrin α subunit TM domains is preferred over hetero-oligomerization, and that the relative strength of homo-oligomerization correlates with the presence of a canonical small residue-xxx-small residue motif followed one turn of the TM helix by a leucine (G, A, S-xxx-G-xxx-L). This motif also mediates the hetero-oligomerization of these TM domains with either β3 or β1. By contrast, a different motif (V-xxx-I-xxx-G) mediates the heteromeric interaction of both β3 and β1 with their complementary α subunits. Mutations that disrupt either the αIIb or β3 interaction motif induce constitutive αIIbβ3 activation. To determine if this is also the case for β1-containing integrins, we introduced disruptive interfacial mutations into the full-length integrins and expressed the mutants in either the β1-deficient Jurkat A1 cells or in HEK293 suspension cells. We found that the β1 mutations V716A, I720A and G724L caused a substantial increase in the static adhesion of A1 cells to laminin, fibronectin, the α4β1-specific peptide H1, as well as type I, II and type IV collagen, whereas mutation of the canonical G-xxx-G motif did not. On the other hand, an increase in binding to type I collagen and fibronectin was observed for mutations of the interfacial α2 residues S1009, G1013, and L1017 and the interfacial α5 residues A964, G968, and L972, respectively. Thus, our studies indicate that β1 and β3 integrins employ a novel, specific, and conserved reciprocating ‘large-small’ TM packing interface that interacts less strongly than the canonical small-residue-xxx-small residue motif. It is also noteworthy that this interface is present in all integrins except β4 and is overrepresented in databases of TM helix-helix interaction as well. Accordingly, it is likely that this type of interface evolved to mediate TM domain interactions that are capable of regulation.

Factors That Influence the Homo- and Hetero-Oligomerization of the Leukocyte Integrin Transmembrane Domains

Integrins are a large family of transmembrane α/β heterodimers that subserve various cell-cell, cell-matrix, and cell-pathogen interactions. Integrins are present on cell surfaces in a dynamic equilibrium between inactive and active conformations that can be regulated by interactions involving their transmembrane (TM) domains. Thus, in their inactive conformation, the α and β subunit TM domains interact, whereas the TM domains separate when integrins assume their active conformations. The role of TM domains in regulating integrin function has been extensively studied using the platelet integrin αIIbβ3 and has revealed that the αIIb and β3 subunit TM domains interact in both a heteromeric and homomeric fashion. These observations have led to a “push-pull” hypothesis for regulating integrin function: events that disrupt the heteromeric association of integrin TM domains stabilize the active integrin conformation and push the equilibrium toward the activated state, whereas interactions that require separation, or are more favorable when the TMs are separated, pull the equilibrium toward the activated state. Although the factors that influence the homo- and hetero-association of the platelet αIIb and β3 TM domains have been well studied, the “push-pull” hypothesis for regulating integrin function has yet to be applied to other integrins, specifically to the leukocyte integrins αMβ2 (MAC-1) and αLβ2 (LFA-1). To determine the importance of TM interactions in αMβ2 and αLβ2, we used modified ToxR-based assays, ToxRed and Dominant-Negative (DN)-ToxRed, to measure the specificity and strength of the homomeric and heteromeric associations of the αM, αL, and β2 TM domains in a bacterial membrane. ToxR is a single-pass TM transcriptional factor from V. cholera that activates the cholera toxin (ctx) promoter when it undergoes TM-driven dimerization in the inner membrane of E. coli. By co-expressing ToxR containing a β2 integrin TM domain with either wild-type ToxR or a R96K ToxR mutant that can dimerize but is unable to activate the ctx promoter, we can measure both the homomeric and heteromeric interaction of each TM domain. In contrast to the TM domains of β3-containing integrins that preferentially form homooligomers, the tendency of the αM, αL, and β2 TM domains to form homo- and heterooligomers is nearly the same. To identify residues that are important for the homo- and heterooligomerization of these TM domains, each was scanned with leucine replacements. A small residue motif that lies along one face of the αM TM helix, similar to that identified in the αIIb TM domain, was found to mediate αM TM domain interactions. Based on these observations, we designed CHAMP (Computed Helical Anti-Membrane Peptide) peptides to specifically target the αM TM interface. In the CHAMP method, the backbone conformation for an αM-binding peptide is selected based on known structural preferences for the motifs identified in the αM helix. The sequence for the binding peptide is then designed computationally using a side-chain repacking algorithm. Ten anti-αM CHAMP peptides were designed and tested for their ability to bind to the αM TM domain in the DN-ToxRed assay. All ten designed sequences interacted with the αM TM domain to an equivalent or better extent than the natural αM and β2 TM domain sequences. Thus, our studies indicate that like the TM domains of β3-containing integrins, the TM domains of the β2 integrins undergo both homomeric and heteromeric interactions, making use of canonical TM helix interaction motifs. Further, we have demonstrated the successful application of computational methods to design peptides that selectively target the αM TM domain, providing a novel method to address mechanistic questions about β2 integrin structure and function.

Structural Synergy and Molecular Crosstalk between Bacterial Helicase Loaders and Replication Initiators

The loading of oligomeric helicases onto replication origins marks an essential step in replisome assembly. In cells, dedicated AAA+ ATPases regulate loading, however, the mechanism by which these factors recruit and deposit helicases has remained unclear. To better understand this process, we determined the structure of the ATPase region of the bacterial helicase loader DnaC from Aquifex aeolicus to 2.7 A resolution. The structure shows that DnaC is a close paralog of the bacterial replication initiator, DnaA, and unexpectedly shares an ability to form a helical assembly similar to that of ATP-bound DnaA. Complementation and ssDNA-binding assays validate the importance of homomeric DnaC interactions, while pull-down experiments show that the DnaC and DnaA AAA+ domains interact in a nucleotide-dependent manner. These findings implicate DnaC as a molecular adaptor that uses ATP-activated DnaA as a docking site for regulating the recruitment and correct spatial deposition of the DnaB helicase onto origins.

ArPIKfyve Homomeric and Heteromeric Interactions Scaffold PIKfyve and Sac3 in a Complex to Promote PIKfyve Activity and Functionality

PtdIns(3,5)P2 (with PtdIns indicating phosphatidylinositol) is vital in the differentiation and development of multicellular organisms because knockout of the PtdIns(3,5)P2-synthesizing enzyme PIKfyve (phosphoinositide kinase for position 5 containing a FYVE finger domain) or its associated regulator ArPIKfyve is lethal. In previous work with endogenous proteins, we identified that Sac3, a phosphatase that turns over PtdIns(3,5)P2, associates with the PIKfyve–ArPIKfyve biosynthetic complex. However, whether the three proteins suffice for the organization/maintenance of this complex [referred to as the PAS (PIKfyve–ArPIKfyve–Sac3) complex], how they interact with one another, and what the functional relevance of this ternary association would be remained unresolved. Using co-immunoprecipitation analyses in transfected mammalian cells with increased or decreased levels of the three proteins, singly or in double versus triple combinations, herein we report that the triad is sufficient to form and maintain the PAS complex. ArPIKfyve is the principal organizer interacting with both Sac3 and PIKfyve, whereas Sac3 is permissive for maximal PIKfyve–ArPIKfyve association in the PAS complex. We further identified that ArPIKfyve scaffolds the PAS complex through homomeric interactions, mediated via its conserved C-terminal domain. Introduction of the C-terminal peptide fragment of the ArPIKfyve–ArPIKfyve contact sites effectively disassembled the PAS complex and reduced the in vitro PIKfyve lipid kinase activity. Exploring insulin-regulated GLUT4 translocation in 3T3L1 adipocytes as a functional readout, a process that is positively regulated by PIKfyve activity and ArPIKfyve levels, we determined that ectopic expression of the ArPIKfyve C-terminal peptide inhibits GLUT4 surface accumulation. Our data indicate that the PAS complex is organized to provide optimal PIKfyve functionality and is maintained via ArPIKfyve homomeric and heteromeric interactions.

Amino-terminal domain interactions of λ integrase on arm-type DNA

In contrast to the other tyrosine recombinase family members, integrase protein (Int) of bacteriophage λ has an additional amino-terminal domain that binds to “arm-type” DNA sequences distant from those involved in strand exchange. The homomeric interaction between neighboring amino-terminal domains of Int is contributed by R30-D71 salt-bridge in a non-equivalent manner on Holliday-junction intermediates. In this report, R30 and D71 residues were investigated in regard to Int’s cooperative binding to “arm-type” DNA and the attenuating function of “arm-type” DNA. The results suggest the electrostatic interaction between residues 30 and 71 is dependent on “arm-type” DNA and contributes the “selective” inhibition of catalytic activity of λ Int by “arm-type” DNA.

Characterization and Expression of a HumanKCTD1Gene Containing the BTB Domain, Which Mediates Transcriptional Repression and Homomeric Interactions

We identified potassium channel tetramerization domain-containing 1 (KCTD1) gene in a human brain cDNA library. Here, we report that the KCTD1 gene contains seven exons, encoding 257 amino acid residues with a predicted molecular mass of 29.4 kDa. Sequence alignments showed KCTD1 protein contains an N-terminal broad-complex, tramtrack, and bric-a-brac (BTB) domain. Northern blot analysis revealed that KCTD1 is expressed in the mammary gland, kidney, brain, and ovary compared to other tissues. Further, the subcellular localization results showed that KCTD1 is localized in the nuclei of HeLa and HBL100 cells. Reporter gene assays in HEK293FT and NIH3T3 cells further indicated that KCTD1 acts as a potent transcriptional repressor and inhibits the transcriptional activity via its BTB domain, though KCTD1 transcriptional repression is unaffected by the HDAC inhibitors, trichostatin A, and sodium butyrate. Finally, we found that the BTB domain of KCTD1 mediates homomeric protein-protein interactions by co-immunoprecipitation and GST pull-down assays. These data present the first characterization of human KCTD1 and suggest that KCTD1 is a nuclear protein that functions as a transcriptional repressor and mediates protein-protein interactions through a BTB domain.

Transmembrane helices that form two opposite homodimeric interactions: An asparagine scan study of αM and β2 integrins

Integrins are alpha/beta heterodimers, but recent in vitro and in vivo experiments also suggest an ability to associate through their transmembrane domains to form homomeric interactions. While the results of some in vitro experiments are consistent with an interaction mediated by a GxxxG-like motif, homo-oligomers observed after in vivo cross-linking are consistent with an almost opposite helix-helix interface. We have shown recently that both models of interaction are compatible with evolutionary conservation data, and we predicted that the alpha-helices in both models would have a similar rotational orientation. Herein, we have tested our prediction using in vitro asparagine scan of five consecutive residues along the GxxxG-like motif of the transmembrane domain of alpha and beta integrins, alphaM and beta2. We show that Asn-mediated dimerization occurs twice for every turn of the helix, consistent with two almost opposite forms of interaction as suggested previously for alphaIIb and beta3 transmembrane domains. The orientational parameters helix tilt and rotational orientation of each of these two Asn-stabilized dimers were measured by site-specific infrared dichroism (SSID) in model lipid bilayers and were found to be consistent with our predicted computational models. Our results highlight an intrinsic tendency for integrin transmembrane alpha-helices to form two opposite types of homomeric interaction in addition to their heteromeric interactions and suggest that integrins may form complex and specific networks at the transmembrane domain during function.

Subunit Dynamics in the IL‐17 Receptor Complex: Identification of a Pre‐ligand Assembly Domain (PLAD) and Ligand Binding site in IL‐17RA

IL‐17 is the defining cytokine of the “Th17” subset, and is implicated in the pathogenesis of numerous autoimmune diseases. The first IL‐17 binding protein, IL‐17RA, is the founder of a unique receptor superfamily, and there is considerable interest in targeting IL‐17 or its receptor for anti‐cytokine therapeutics. However, the composition, subunit dynamics and ligand contact sites of the IL‐17R complex remain poorly defined. We show by fluorescence resonance energy transfer (FRET) microscopy that IL‐17RA oligomerizes in the absence of ligand, suggesting that an unknown region mediates pre‐assembly. Using computational modeling, we identified 2 fibronectin‐like III (FN) domains connected by a non‐structured linker. Because FN domains are known to aggregate, we hypothesized that these were responsible for IL‐17RA self‐interactions. The membrane‐proximal FN domain (FN2) but not the membrane‐distal (FN1) domain was found to form homomeric interactions. Thus, FN2 constitutes a “pre‐ligand assembly domain” (PLAD). Interestingly, FN2 fused to the linker but not FN2 alone retained IL‐17‐dependent signaling activity, indicating that the FN2/linker domain contains the IL‐17 binding site. Therefore, while the PLAD is located entirely within FN2, effective ligand binding also involves contributions from the linker region and the FN1 domain.

The Highly Repetitive Region of the Helicobacter pylori CagY Protein Comprises Tandem Arrays of an α-Helical Repeat Module

The cag-pathogenicity-island-encoded type IV secretion system of Helicobacter pylori functions to translocate the effector protein CagA directly through the plasma membrane of gastric epithelial cells. Similar to other secretion systems, the Cag type IV secretion system elaborates a surface filament structure, which is unusually sheathed by the large cag-pathogenicity-island-encoded protein CagY. CagY is distinguished by unusual amino acid composition and extensive repetitive sequence organised into two defined repeat regions. The second and major repeat region (CagYrpt2) has a regular disposition of six repetitive motifs, which are subject to deletion and duplication, facilitating the generation of CagY size and phenotypic variants. In this study, we show CagYrpt2 to comprise two highly thermostable and acid-stable α-helical structural motifs, the most abundant of which (motif A) occurs in tandem arrays of one to six repeats terminally flanked by single copies of the second repeat (motif B). Isolated motifs demonstrate hetero- and homomeric interactions, suggesting a propensity for uniform assembly of discrete structural subunit motifs within the larger CagYrpt2 structure. Consistent with this, CagY proteins comprising substantially different repeat 2 motif organisations demonstrate equivalent CagA translocation competence, illustrating a remarkable structural and functional tolerance for precise deletion and duplication of motif subunits. We provide the first insight into the structural basis for CagYrpt2 assembly that accommodates both the variable motif sequence composition and the extensive contraction/expansion of repeat modules within the CagYrpt2 region.

Identification of Oligomerization Motifs in the β3 Transmembrane Domain.

The integrin heterodimer αIIbβ3 resides on the platelet surface in an equilibrium between inactive (low affinity) and active (high affinity) conformations. We have reported that the transmembrane (TM) domains of its αIIb and β3 subunits engage in specific heteromeric and homomeric interactions that define the αIIbβ3 activation state. Further, we have proposed a “push-pull” hypothesis to explain how αIIbβ3 activity is regulated. Thus, processes that disrupt the TM domain heterodimer stabilize the active αIIbβ3 conformation and “push” the low affinity/high affinity equilibrium toward the activated state. Conversely, interactions that either require separation of the TM domains or are more favorable when the TM domains are apart “pull” the equilibrium in the same direction. Moreover, as would be predicted by this hypothesis, we found that adding a soluble peptide corresponding to the β3 TM domain to suspensions of gel-filtered platelets induced signaling-independent, αIIbβ3-mediated platelet aggregation, confirming that disruption of the αIIb/β3 TM domain heterodimer activates αIIbβ3. To identify the motifs responsible for oligomerization of the β3 TM domain, we used the TOXCAT assay. In TOXCAT, a chimeric protein consisting of an N-terminal ToxR' DNA binding domain, a C-terminal maltose-binding protein domain, and an interposed β3 TM domain was expressed in the inner membrane of E. coli. TM domain-mediated dimerization of the chimeric protein then drove the transcriptional activation of a reporter gene chloramphenicol acetyl transferase (CAT). We replaced each residue in the β3 TM domain with either Leu, Ala, Val or Ile and measured the effect on CAT synthesis. The results were then used to calculate a perturbation index that reflects the mean fold-change in observed CAT activity for each of the mutants. When analyzed in this way, TOXCAT revealed that β3 helices associate homomerically with a 4 residue periodicity and that the interactive side of the helix lies along residues 700, 704, 708, and 712. Next, based on the TOXCAT results, we introduced selected mutations into the β3 TM domain of full-length αIIbβ3 and measured their effect on FITC-fibrinogen binding to αIIbβ3 expressed in CHO cells. We found that the disruptive mutations M701L, I704L, L705L, L712A, and L713A, located on the interactive side of the β3 helix, induced constitutive fibrinogen binding to αIIbβ3, whereas mutation of the intervening residues did not. Lastly, the TOXCAT and fibrinogen binding results were applied to a model of the β3 TM helix, revealing that the residues involved in oligomerization of the β3 TM domain were present as a “sticky” stripe along one face of the helix. Our results indicate that the face of the β3 helix that mediates its homomeric interaction also mediates its interaction with the αIIb TM domain; that mutations at this interface disrupt both homomeric and heteromeric interactions, and that these mutations strongly induce fibrinogen binding to αIIbβ3. Thus, our findings indicate that disruption of the α/β TM domain interface alone is sufficient to activate αIIbβ3 and that homomeric β3 interactions by themselves are not required to induce fibrinogen binding to αIIbβ3.

Cutting Edge: Identification of a Pre-Ligand Assembly Domain (PLAD) and Ligand Binding Site in the IL-17 Receptor

IL-17 is the hallmark cytokine of the newly described "Th17" lymphocyte population. The composition, subunit dynamics, and ligand contacts of the IL-17 receptor are poorly defined. We previously demonstrated that the IL-17RA subunit oligomerizes in the membrane without a ligand. In this study, computational modeling identified two fibronectin-III-like (FN) domains in IL-17RA connected by a nonstructured linker, which we predicted to mediate homotypic interactions. In yeast two-hybrid, the membrane-proximal FN domain (FN2), but not the membrane-distal domain (FN1), formed homomeric interactions. The ability of FN2 to drive ligand-independent multimerization was verified by coimmunoprecipitation and fluorescence resonance energy transfer microscopy. Thus, FN2 constitutes a "pre-ligand assembly domain" (PLAD). Further studies indicated that the FN2 linker domain contains the IL-17 binding site, which was never mapped. However, the FN1 domain is also required for high affinity interactions with IL-17. Therefore, although the PLAD is located entirely within FN2, effective ligand binding also involves contributions from the linker and FN1.

Cx29 and Cx32, two connexins expressed by myelinating glia, do not interact and are functionally distinct

In rodents, oligodendrocytes and myelinating Schwann cells express connexin32 (Cx32) and Cx29, which have different localizations in the two cell types. We show here that, in contrast to Cx32, Cx29 does not form gap junction plaques or functional gap junctions in transfected cells. Furthermore, when expressed together, Cx29 and Cx32 are not colocalized and do not coimmunoprecipitate. To determine the structural basis of their divergent behavior, we generated a series of chimeric Cx32-Cx29 proteins by exchanging their intracellular loops and/or their C-terminal cytoplasmic tails. Although some chimerae reach the cell membrane, others appear to be largely localized intracellularly; none form gap junction plaques or functional gap junctions. Substituting the C-terminus or the intracellular loop and the C-terminus of Cx32 with those of Cx29 does not disrupt their colocalization or coimmunoprecipitation with Cx32. Substituting the C-terminus of Cx29 with that of Cx32 does not disrupt the coimmunoprecipitation or the colocalization with Cx29, whereas substituting both the intracellular loop and the C-terminus of Cx32 with those of Cx29 diminishes the coimmunoprecipitation with Cx29. Conversely, the Cx32 chimera that contains the intracellular loop of Cx29 coimmunoprecipitates with Cx29, indicating that the intracellular loop participates in Cx29-Cx29 interactions. These data indicate that homomeric interactions of Cx29 and especially Cx32 largely require other domains: the N-terminus, transmembrane domains, and extracellular loops. Substituting the intracellular loop and/or tail of Cx32 with those of Cx29 appears to prevent Cx32 from forming functional gap junctions.

Direct Evidence for Specific Interactions of the Fibrinogen αC-Domains with the Central E Region and with Each Other

The carboxyl-terminal regions of the fibrinogen Aalpha chains (alphaC regions) form compact alphaC-domains tethered to the bulk of the molecule with flexible alphaC-connectors. It was hypothesized that in fibrinogen two alphaC-domains interact intramolecularly with each other and with the central E region preferentially through its N-termini of Bbeta chains and that removal of fibrinopeptides A and B upon fibrin assembly results in dissociation of the alphaC regions and their switch to intermolecular interactions. To test this hypothesis, we studied the interactions of the recombinant alphaC region (Aalpha221-610 fragment) and its subfragments, alphaC-connector (Aalpha221-391) and alphaC-domain (Aalpha392-610), between each other and with the recombinant (Bbeta1-66)2 and (beta15-66)2 fragments and NDSK corresponding to the fibrin(ogen) central E region, using laser tweezers-based force spectroscopy. The alphaC-domain, but not the alphaC-connector, bound to NDSK, which contains fibrinopeptides A and B, and less frequently to desA-NDSK and (Bbeta1-66)2 containing only fibrinopeptides B; it was poorly reactive with desAB-NDSK and (beta15-66)2 both lacking fibrinopeptide B. The interactions of the alphaC-domains with each other and with the alphaC-connector were also observed, although they were weaker and heterogeneous in strength. These results provide the first direct evidence for the interaction between the alphaC-domains and the central E region through fibrinopeptide B, in agreement with the hypothesis given above, and indicate that fibrinopeptide A is also involved. They also confirm the hypothesized homomeric interactions between the alphaC-domains and display their interaction with the alphaC-connectors, which may contribute to covalent cross-linking of alpha polymers in fibrin.

Organization and function of TRPC channelosomes

TRPC proteins constitute a family of conserved Ca2+-permeable cation channels which are activated in response to agonist-stimulated PIP2 hydrolysis. These channels were initially proposed to be components of the store-operated calcium entry channel (SOC). Subsequent studies have provided substantial evidence that some TRPCs contribute to SOC activity. TRPC proteins have also been shown to form agonist-stimulated calcium entry channels that are not store-operated but are likely regulated by PIP2 or diacylglycerol. Further, and consistent with the presently available data, selective homomeric or heteromeric interactions between TRPC monomers generate distinct agonist-stimulated cation permeable channels. We suggest that interaction between TRPC monomers, as well as the association of these channels with accessory proteins, determines their mode of regulation as well as their cellular localization and function. Currently identified accessory proteins include key Ca2+ signaling proteins as well as proteins involved in vesicle trafficking, cytoskeletal interactions, and scaffolding. Studies reported until now demonstrate that TRPC proteins are segregated into specific Ca2+ signaling complexes which can generate spatially and temporally controlled [Ca2+]i signals. Thus, the functional organization of TRPC channelosomes dictates not only their regulation by extracellular stimuli but also serves as a platform to coordinate specific downstream cellular functions that are regulated as a consequence of Ca2+ entry. This review will focus on the accessory proteins of TRPC channels and discuss the functional implications of TRPC channelosomes and their assembly in microdomains.

Solid-State Structures of 4-Carboxyphenylboronic Acid and Its Hydrates

Solid-state structures of 4-carboxyphenylboronic acid (1a) and its mono- and quarter-hydrates (1b and 1c, respectively) obtained by crystallization from different solvents, acetone, methanol, and 2-proponal, respectively, have been reported. In the anhydrous and monohydrate structures (1a and 1b), the H-atoms on the −B(OH)2 functional group exist in syn−syn conformation, but syn−anti conformation was found in the quarter-hydrate structure. While 1a and 1b crystallize through interactions between −B(OH)2 and −COOH groups (heteromeric), homomeric interactions of the functional groups (COOH···COOH and B(OH)2···B(OH)2) were observed in 1c. In addition, although similar interactions prevail in 1a and 1b, the latter crystallizes in a noncentrosymmetric space group Ccc2, registering itself as a unique structure with unusual catenation features with the aid of water molecules. However, 1a and 1c show many common features in the molecular arrangement in the crystal lattices with the formation of sheet structures.