Structure Of Cytoplasmic Domain Research Articles

Identification and mechanistic analysis of an inhibitor of the CorC Mg2+ transporter

SummaryThe CorC/CNNM family of Na+-dependent Mg2+ transporters is ubiquitously conserved from bacteria to humans. CorC, the bacterial CorC/CNNM family of proteins, is involved in resistance to antibiotic exposure and in the survival of pathogenic microorganisms in their host environment. The CorC/CNNM family proteins possess a cytoplasmic region containing the regulatory ATP-binding site. CorC and CNNM have attracted interest as therapeutic targets, whereas inhibitors targeting the ATP-binding site have not been identified. Here, we performed a virtual screening of CorC by targeting its ATP-binding site, identified a compound named IGN95a with inhibitory effects on ATP binding and Mg2+ export, and determined the cytoplasmic domain structure in complex with IGN95a. Furthermore, a chemical cross-linking experiment indicated that with ATP bound to the cytoplasmic domain, the conformational equilibrium of CorC was shifted more toward the inward-facing state of the transmembrane domain. In contrast, IGN95a did not induce such a shift.

A novel pathogenic variant in the FZD6 gene causes recessive nail dysplasia in a large Iranian kindred

BackgroundNail disorder nonsyndromic congenital (NDNC) is a very rare clinically and genetically heterogeneous disease inherited both in recessive or dominant modes. FZD6 is a component of Wnt-FZD signaling pathway in which recessive loss-of-function variants in the corresponding genes could lead to nail anomalies. ObjectiveA large multiplex family with NDNC was referred for genetic counselling. Thorough genetic evaluation was performed. MethodsPCR-Sanger sequencing was carried out for the coding exons and exon-intron boundaries of the FZD6 gene. Co-segregation analysis, in silico evaluation and computational protein modeling was accomplished. ResultsA homozygous 1bp deletion variant, c.1859delC (p.Ser620Cysfs*75), leading to a truncating protein was found in the patient. Parents were heterozygous for the variant. The variant was found to be co-segreagting with the phenotype in the family. Computational analysis and protein modeling revealed its pathogenic consequence by disturbing the cytoplasmic domain structure and signaling through loss of phosphorylation residues. The variant met the criteria of being pathogenic according to the ACMG guideline. ConclusionsThis is the first report of the genetic diagnosis of NDNC in Iran. We also report a novel pathogenic variant. The study of the FZD6 gene is recommended as the first step in the diagnostic routing of the autosomal recessive NDNC patients with enlarged nails.

A Common Signal Patch Drives AP-1 Protein-dependent Golgi Export of Inwardly Rectifying Potassium Channels

Nearly all members of the inwardly rectifying potassium (Kir) channel family share a cytoplasmic domain structure that serves as an unusual AP-1 clathrin adaptor-dependent Golgi export signal in one Kir channel, Kir2.1 (KCNJ2), raising the question whether Kir channels share a common Golgi export mechanism. Here we explore this idea, focusing on two structurally and functionally divergent Kir family members, Kir2.3 (KCNJ4) and Kir4.1/5.1 (KCNJ10/16), which have ∼50% amino identity. We found that Golgi export of both channels is blocked upon siRNA-mediated knockdown of the AP-1 γ subunit, as predicted for the common AP-1-dependent trafficking process. A comprehensive mutagenic analysis, guided by homology mapping in atomic resolution models of Kir2.1, Kir2.3, and Kir4.1/5.1, identified a common structure that serves as a recognition site for AP-1 binding and governs Golgi export. Larger than realized from previous studies with Kir2.1, the signal is created by a patch of residues distributed at the confluence of cytoplasmic N and C termini. The signal involves a stretch of hydrophobic residues from the C-terminal region that form a hydrophobic cleft, an adjacent cluster of basic residues within the N terminus, and a potential network of salt bridges that join the N- and C-terminal poles together. Because patch formation and AP-1 binding are dependent on proper folding of the cytoplasmic domains, the signal provides a common quality control mechanism at the Golgi for Kir channels. These findings identify a new proteostatic mechanism that couples protein folding of channels to forward trafficking in the secretory pathway.

Functional Implications of Domain Organization Within Prokaryotic Rhomboid Proteases.

Intramembrane proteases are membrane embedded enzymes that cleave transmembrane substrates. This interesting class of enzyme and its water mediated substrate cleavage mechanism occurring within the hydrophobic lipid bilayer has drawn the attention of researchers. Rhomboids are a family of ubiquitous serine intramembrane proteases. Bacterial forms of rhomboid proteases are mainly composed of six transmembrane helices that are preceded by a soluble N-terminal domain. Several crystal structures of the membrane domain of the E. coli rhomboid protease ecGlpG have been solved. Independently, the ecGlpG N-terminal cytoplasmic domain structure was solved using both NMR and protein crystallography. Despite these structures, we still do not know the structure of the full-length protein, nor do we know the functional role of these domains in the cell. This chapter will review the structural and functional roles of the different domains associated with prokaryotic rhomboid proteases. Lastly, we will address questions remaining in the field.

Time-resolved FRET reveals the structural mechanism of SERCA–PLB regulation

We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to characterize the interaction between phospholamban (PLB) and the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA) under conditions that relieve SERCA inhibition. Unphosphorylated PLB inhibits SERCA in cardiac SR, but inhibition is relieved by either micromolar Ca2+ or PLB phosphorylation. In both cases, it has been proposed that inhibition is relieved by dissociation of the complex. To test this hypothesis, we attached fluorophores to the cytoplasmic domains of SERCA and PLB, and reconstituted them functionally in lipid bilayers. TR-FRET, which permitted simultaneous measurement of SERCA–PLB binding and structure, was measured as a function of PLB phosphorylation and [Ca2+]. In all cases, two structural states of the SERCA–PLB complex were resolved, probably corresponding to the previously described T and R structural states of the PLB cytoplasmic domain. Phosphorylation of PLB at S16 completely relieved inhibition, partially dissociated the SERCA–PLB complex, and shifted the T/R equilibrium within the bound complex toward the R state. Since the PLB concentration in cardiac SR is at least 10times that in our FRET measurements, we calculate that most of SERCA contains bound phosphorylated PLB in cardiac SR, even after complete phosphorylation. 4μM Ca2+ completely relieved inhibition but did not induce a detectable change in SERCA–PLB binding or cytoplasmic domain structure, suggesting a mechanism involving structural changes in SERCA’s transmembrane domain. We conclude that Ca2+ and PLB phosphorylation relieve SERCA–PLB inhibition by distinct mechanisms, but both are achieved primarily by structural changes within the SERCA–PLB complex, not by dissociation of that complex.

Structure of the cytoplasmic domain of Yersinia pestis YscD, an essential component of the type III secretion system.

The Yersinia pestis YscD protein is an essential component of the type III secretion system. YscD consists of an N-terminal cytoplasmic domain (residues 1-121), a transmembrane linker (122-142) and a large periplasmic domain (143-419). Both the cytoplasmic and the periplasmic domains are required for the assembly of the type III secretion system. Here, the structure of the YscD cytoplasmic domain solved by SAD phasing is presented. Although the three-dimensional structure is similar to those of forkhead-associated (FHA) domains, comparison with the structures of canonical FHA domains revealed that the cytoplasmic domain of YscD lacks the conserved residues that are required for binding phosphothreonine and is therefore unlikely to function as a true FHA domain.

The Receptor–CheW Binding Interface in Bacterial Chemotaxis

The basic structural unit of the signaling complex in bacterial chemotaxis consists of the chemotaxis kinase CheA, the coupling protein CheW, and chemoreceptors. These complexes play an important role in regulating the kinase activity of CheA and in turn controlling the rotational bias of the flagellar motor. Although individual three-dimensional structures of CheA, CheW, and chemoreceptors have been determined, the interaction between chemoreceptor and CheW is still unclear. We used nuclear magnetic resonance to characterize the interaction modes of chemoreceptor and CheW from Thermotoga maritima. We find that chemoreceptor binding surface is located near the highly conserved tip region of the N-terminal helix of the receptor, whereas the binding interface of CheW is placed between the β-strand 8 of domain 1 and the β-strands 1 and 3 of domain 2. The receptor–CheW complex shares a similar binding interface to that found in the “trimer-of-dimers” oligomer interface seen in the crystal structure of cytoplasmic domains of chemoreceptors from Escherichia coli. Based on the association constants inferred from fast exchange chemical shifts associated with receptor–CheW titrations, we estimate that CheW binds about four times tighter to its first binding site of the receptor dimer than to its second binding site. This apparent anticooperativity in binding may reflect the close proximity of the two CheW binding surfaces near the receptor tip or further, complicating the events at this highly conserved region of the receptor. This work describes the first direct observation of the interaction between chemoreceptor and CheW.

The Dependence Receptor UNC5H2/B Triggers Apoptosis via PP2A-Mediated Dephosphorylation of DAP Kinase

The UNC5H dependence receptors promote apoptosis in the absence of their ligand, netrin-1, and this is important for neuronal and vascular development and for limitation of cancer progression. UNC5H2 (also called UNC5B) triggers cell death through the activation of the serine-threonine protein kinase DAPk. While performing a siRNA screen to identify genes implicated in UNC5H-induced apoptosis, we identified the structural subunit PR65β of the holoenzyme protein phosphatase 2A (PP2A). We show that UNC5H2/B recruits a protein complex that includes PR65β and DAPk and retains PP2A activity. PP2A activity is required for UNC5H2/B-induced apoptosis, since it activates DAPk by triggering its dephosphorylation. Moreover, netrin-1 binding to UNC5H2/B prevents this effect through interaction of the PP2A inhibitor CIP2A to UNC5H2/B. Thus we show here that, in the absence of netrin-1, recruitment of PP2A to UNC5H2/B allows the activation of DAPk via a PP2A-mediated dephosphorylation and that this mechanism is involved in angiogenesis regulation.

Structure of the Cytoplasmic Segment of Histidine Kinase Receptor QseC: A Key Player in Bacterial Virulence

QseC is a histidine kinase (HK) receptor involved in quorum sensing, a mechanism by which bacteria respond to fluctuations in cell population. We conducted a structural study of the cytoplasmic domain of QseC (QseC-CD) using X-ray crystallography. The 2.5 Å structure of the apo-enzyme revealed that the kinase domain of QseC retains the overall fold of the typical HK kinase domain. The construct that we used is inactive in the autokinase reaction and its inactivity is most likely caused by its atypical dimerization interface, as compared to that observed in the T.maritima HK cytoplasmic domain structure. Restoration of the activity may require that the entire dimerization domain be present in the protein construct. QseC, which plays an important role in bacterial pathogenesis, is a promising drug target and the structure of QseC-CD provides a platform for developing more potent inhibitors of pathogen virulence.

Protein-Protein and Protein-Lipid Interactions Modulate αIIbβ3 Inside-out Signaling.

Abstract 152Integrins are ubiquitously expressed α/β heterodimers that mediate cell-cell and cell-extracellular matrix interactions. The platelet integrin αIIbβ3 binds soluble fibrinogen following platelet activation, an event necessary for the formation of platelet aggregates. Integrins reside on plasma membranes in a highly regulated and dynamic equilibrium between inactive resting states and active ligand binding conformations. An essential feature of this equilibrium is the association and dissociation of integrin transmembrane (TM) and cytoplasmic domains. Thus, when integrins are inactive, the TM and cytoplasmic domains of their α and β subunits are in proximity; the domains separate when integrins assume their active conformations. Agonist-induced activation of αIIbβ3 in platelets (inside-out activation) requires binding of the FERM domain of the cytoskeletal protein talin to highly conserved membrane proximal and distal sequences in the β3 subunit cytoplasmic domain. FERM domain binding to the β3 cytoplasmic domain likely disrupts the αIIbβ3 TM domain-cytoplasmic domain heterodimer. As the β3 cytoplasmic domain exists in close apposition to the inner leaflet of the plasma membrane and the talin FERM domain is known to interact with the negatively-charged phospholipids that are common in eukaryotic membranes, we have investigated the effects of the membrane environment on β3 and talin structure and the talin-β;3 protein-protein interaction that regulates αIIbβ3 activation. A water soluble form of the β3 cytoplasmic domain was expressed, purified, and chemically conjugated to phospholipid bilayers, mimicking the native environment of the αIIb and β3 cytoplasmic domains as they exit the membrane. Using circular dichroism (CD) spectroscopy, the β3 cytoplasmic domain was found to have a significant increase in secondary structure and overall helicity when attached to the bilayer. Similarly, using hydrogen-deuterium exchange (HDX) mass spectrometry, we found that there is an overall decrease in amide backbone flexibility, particularly in the membrane proximal talin-interacting region. We also found significant changes in talin secondary structure in detergents by CD and by limited proteolysis mapping, supporting dramatic conformational changes in talin upon membrane binding. Using isothermal titration calorimetry (ITC) and integrin-conjugated lipid bilayers, we investigated the effects of lipid composition on talin-bilayer and talin-β3 cytoplasmic domain interactions. We found that the talin FERM domain likely has two binding sites for the membrane-conjugated β3 cytoplasmic domain, one in the canonical F3 subdomain and a second that depends on the F2 subdomain. Moreover, the talin head domain does not bind to β3 on neutral bilayers and requires a significant excess of phosphatidylserine (PS) relative to β3 to allow saturation. Interestingly phosphatidylinositol (4, 5)-bisphosphate containing bilayers have significantly higher affinity and mitigate the need for excess PS. Our results point toward a model in which β3 cytoplasmic domain structure is dependent on the lipid environment and its interaction with talin. Thus, αIIbβ3 activation is significantly regulated by lipid which may play a role in constraining αIIbβ3 activation by talin in the crowded platelet intracellular environment. Disclosures:No relevant conflicts of interest to declare.

Autoinhibition of UNC5b Revealed by the Cytoplasmic Domain Structure of the Receptor

The cytoplasmic domains of UNC5 are responsible for its netrin-mediated signaling events in axonal migrations, blood vessel patterning, and apoptosis, although the molecular mechanisms governing these processes are unknown. To provide a foundation for the elucidation of the UNC5-mediated signaling mechanism, we determined the crystal structure of the cytoplasmic portion of UNC5b. We found that it contains three distinctly folded domains, namely ZU5, UPA, and death domain (DD). These three domains form a structural supramodule, with ZU5 binding to both UPA and DD, thereby locking the ZU5-UPA-DD supramodule in a closed conformation and suppressing its biological activities. Release of the closed conformation of the ZU5-UPA-DD supramodule leads to the activation of the receptor in the promotion of apoptosis and blood vessel patterning. Finally, we provide evidence showing that the supramodular nature of UNC5 ZU5-UPA-DD is likely to be shared by the ankyrin and PIDD families of scaffold proteins.

The three-dimensional structure of the cytoplasmic domains of EpsF from the type 2 secretion system of Vibrio cholerae

The type 2 secretion system (T2SS), a multi-protein machinery that spans both the inner and the outer membranes of Gram-negative bacteria, is used for the secretion of several critically important proteins across the outer membrane. Here we report the crystal structure of the N-terminal cytoplasmic domain of EpsF, an inner membrane spanning T2SS protein from Vibrio cholerae. This domain consists of a bundle of six anti-parallel helices and adopts a fold that has not been described before. The long C-terminal helix alpha6 protrudes from the body of the domain and most likely continues as the first transmembrane helix of EpsF. Two N-terminal EpsF domains form a tight dimer with a conserved interface, suggesting that the observed dimer occurs in the T2SS of many bacteria. Two calcium binding sites are present in the dimer interface with ligands provided for each site by both subunits. Based on this new structure, sequence comparisons of EpsF homologs and localization studies of GFP fused with EpsF, we propose that the second cytoplasmic domain of EpsF adopts a similar fold as the first cytoplasmic domain and that full-length EpsF, and its T2SS homologs, have a three-transmembrane helix topology.

Solution Structure of the Cytoplasmic Domain of Syndecan-3 by Two-dimensional NMR Spectroscopy

Syndecan-3 is a cell-surface heparan sulfate proteoglycan, which performs a variety of functions during cell adhension process. It is also a coreceptor for growth factor, mediating cell-cell and cell-matrix interaction. Syndecan-3 contains a cytoplasmic domain potentially associated with the cytoskeleton. Syndecan-3 is specifically expressed in neuron cell and has related to neuron cell differentiation and development of actin filament in cell migration. Syndecans each have a unique, central, and variable (V) region in their cytoplasmic domains. And that region of syndecan-3 may modulate the interactions of the conserved C1 regions of the cytoplasmic domains by tyrosine phosphorylation. Cytoplasmic domain of syndecan-3 has been synthesized for NMR structural studies. The solution structure of syndecan-3 cytoplasmic domain has been determined by two-dimensional NMR spectroscopy and simulated-annealing calculation. The cytoplasmic domain of the syndecan proteins has a tendency to form a dimmer conformation with a central cavity, however, that of syndecan-3 demonstrated a monomer conformation with a flexible region near C-terminus. The structural information might add knowledge about the structure-function relationships among syndecan proteins.

The Structure of the Cytoplasmic Domain of EpsL, An Inner Membrane Component of the Type II Secretion System of Vibrio cholerae: An Unusual Member of the Actin-like ATPase Superfamily

The type II secretion system (T2SS) is used by several Gram-negative bacteria for the secretion of hydrolytic enzymes and virulence factors across the outer membrane. In these secretion systems, a complex of 12-15 so-called "Gsp proteins" spans from a regulatory ATPase in the cytoplasm, via several signal or energy transducing proteins in the inner membrane and the pseudopilins in the periplasm, to the actual pore in the outer membrane. The human pathogen Vibrio cholerae employs such an assembly, called the Eps system, for the export of its major virulence factor, cholera toxin, from its periplasm into the lumen of the gastro-intestinal tract of the host. Here, we report the atomic structure of the major cytoplasmic domain of the inner membrane-spanning EpsL protein from V. cholerae. EpsL is the binding partner of the regulatory ATPase EpsE as well as of EpsM and pseudopilins, and is therefore a critical link between the cytoplasmic and the periplasmic part of the Eps-system. The 2.7A resolution structure was determined by a combination of Se-Met multiple anomalous dispersion (MAD) and multiple isomorphous replacement with anomalous scattering (MIRAS) phasing methods. The 28kDa cytoplasmic domain of EpsL (cyto-EpsL) consists of three beta-sheet-rich domains. With domains I and III similar to the RNaseH-fold, cyto-EpsL unexpectedly shows structural homology with the superfamily of actin-like ATPases. cyto-EpsL, however, is an unusual member of this superfamily as it misses the canonical actin domains 1B and 2B, which are common yet variable in this superfamily. Moreover, cyto-EpsL has an additional domain II, which has the topology of an SHS2-fold module. Within the superfamily this fold module has been observed only for domain 1C of the cell division protein FtsA, in which it mediates protein-protein interactions. This domain II displays great flexibility and contributes to a pronounced negatively charged canyon on the surface of cyto-EpsL. Functional data as well as structural homology and sequence conservation suggest that domain II interacts with EpsE, the major cytoplasmic binding partner of EpsL.

Stabilization of the activity of ATP-sensitive potassium channels by ion pairs formed between adjacent Kir6.2 subunits.

ATP-sensitive potassium (KATP) channels are formed by the coassembly of four Kir6.2 subunits and four sulfonylurea receptor subunits (SUR). The cytoplasmic domains of Kir6.2 mediate channel gating by ATP, which closes the channel, and membrane phosphoinositides, which stabilize the open channel. Little is known, however, about the tertiary or quaternary structures of the domains that are responsible for these interactions. Here, we report that an ion pair between glutamate 229 and arginine 314 in the intracellular COOH terminus of Kir6.2 is critical for maintaining channel activity. Mutation of either residue to alanine induces inactivation, whereas charge reversal at positions 229 and 314 (E229R/R314E) abolishes inactivation and restores the wild-type channel phenotype. The close proximity of these two residues is demonstrated by disulfide bond formation between cysteine residues introduced at the two positions (E229C/R314C); disulfide bond formation abolishes inactivation and stabilizes the current. Using Kir6.2 tandem dimer constructs, we provide evidence that the ion pair likely forms by residues from two adjacent Kir6.2 subunits. We propose that the E229/R314 intersubunit ion pairs may contribute to a structural framework that facilitates the ability of other positively charged residues to interact with membrane phosphoinositides. Glutamate and arginine residues are found at homologous positions in many inward rectifier subunits, including the G-protein–activated inwardly rectifying potassium channel (GIRK), whose cytoplasmic domain structure has recently been solved. In the GIRK structure, the E229- and R314-corresponding residues are oriented in opposite directions in a single subunit such that in the tetramer model, the E229 equivalent residue from one subunit is in close proximity of the R314 equivalent residue from the adjacent subunit. The structure lends support to our findings in Kir6.2, and raises the possibility that a homologous ion pair may be involved in the gating of GIRKs.

Probing the dark state tertiary structure in the cytoplasmic domain of rhodopsin: proximities between amino acids deduced from spontaneous disulfide bond formation between Cys316 and engineered cysteines in cytoplasmic loop 1.

A dark state tertiary structure in the cytoplasmic domain of rhodopsin is presumed to be the key to the restriction of binding of transducin and rhodopsin kinase to rhodopsin. Upon light-activation, this tertiary structure undergoes a conformational change to form a new structure, which is recognized by the above proteins and signal transduction is initiated. In this and the following paper in this issue [Cai, K., Klein-Seetharaman, J., Altenbach, C., Hubbell, W. L., and Khorana, H. G. (2001) Biochemistry 40, 12479-12485], we probe the dark state cytoplasmic domain structure in rhodopsin by investigating proximity between amino acids in different regions of the cytoplasmic face. The approach uses engineered pairs of cysteines at predetermined positions, which are tested for spontaneous formation of disulfide bonds between them, indicative of proximity between the original amino acids. Focusing here on proximity between the native cysteine at position 316 and engineered cysteines at amino acid positions 55-75 in the cytoplasmic sequence connecting helices I-II, disulfide bond formation was studied under strictly defined conditions and plotted as a function of the position of the variable cysteines. An absolute maximum was observed for position 65 with two additional relative maxima for cysteines at positions 61 and 68. The observed disulfide bond formation rates correlate well with proximity of these residues found in the crystal structure of rhodopsin in the dark. Modeling of the engineered cysteines in the crystal structure indicates that small but significant motions are required for productive disulfide bond formation. During these motions, secondary structure elements are retained as indicated by the lack of disulfide bond formation in cysteines that do not face toward Cys316 in the crystal structure model. Such motions may be important in light-induced conformational changes.

Definition of epitopes for monoclonal antibodies developed against purified sodium channel protein: implications for channel structure.

To test sodium channel structural models, we defined the epitopes for nineteen independently cloned monoclonal antibodies previously generated against purified, detergent-solubilized, adult rat skeletal muscle sodium channel protein using channel proteolysis, synthetic peptides, and fusion proteins. All identified epitopes were continuous and unique to the skeletal muscle subtype alpha-subunit. Of the nineteen independent clones, seventeen had epitopes located either in the origin of the amino-terminus or in the interdomain 2-3 region while only two antibodies had epitopes located in the mid-portion of the interdomain 1-2 region. No immunogenic regions were identified on the alpha-subunit's extracellular regions, interdomain 3-4 segment, or carboxyl-terminus or on channel beta-subunits. While immune tolerance may explain the lack of immunogenicity of extracellular regions, the lack of immunogenicity of most of the channel's cytoplasmic mass may be due to segment inaccessibility from organization of these regions as globular domains, to insertion of parts of these regions into the membrane phase, or to interaction with other protein elements. The definition of monoclonal antibody epitopes allows us to reinterpret previously reported monoclonal antibody competition studies, providing independent support for our model of sodium channel cytoplasmic domain structure. In addition, these data suggest additional testable hypotheses concerning the interactions of the sodium channel amino- and carboxyl-termini with each other as well as with other protein elements.

Requirements for the cytoplasmic domain of the alphaPS1, alphaPS2 and betaPS integrin subunits during Drosophila development.

The integrins are a family of transmembrane heterodimeric proteins that mediate adhesive interactions and participate in signaling across the plasma membrane. In this study we examine the functional significance of the cytoplasmic domains of the alphaPS1, alphaPS2 and betaPS subunits of the Drosophila Position Specific (PS) integrin family by analyzing the relationship between cytoplasmic domain structure and function in the context of a developing organism. By examining the ability of ssPS molecules lacking the cytoplasmic domain to rescue embryonic abnormalities associated with PS integrin loss, we find that although many embryonic events require the betaPS cytoplasmic domain, this portion of the molecule is not required for at least two processes requiring PS integrins: formation of midgut constrictions and maintaining germband integrity. Furthermore, our studies demonstrate that mutant proteins affecting four highly conserved amino acid residues in the cytoplasmic tail function with different efficiencies during embryonic development, suggesting that interaction of PS integrins with cytoplasmic ligands is developmentally modulated during embryogenesis. We have also examined the ability of alphaPS1 and alphaPS2 to function without their cytoplasmic domains. By analyzing the ability of transgenes producing truncated alphaPS molecules to rescue abnormalities associated with integrin loss, we find that the cytoplasmic tail of alphaPS2 is essential for both embryonic and postembryonic processes, while this portion of alphaPS1 is not required for function in the wing and in the retina. Furthermore, temperature-shift experiments suggest roles for the alphaPS2 cytoplasmic domain in signaling events occurring in the developing wing.

Solution structure of the cytoplasmic domain of the human immunodeficiency virus type 1 encoded virus protein U (Vpu)

The HIV-1-specific Vpu protein is an 81 amino acid class I integral membrane phosphoprotein that induces degradation of the virus receptor CD4 in the endoplasmic reticulum and enhances the release of virus particles from infected cells. Vpu is of amphipathic nature and consists of a hydrophobic N-terminal membrane anchor proximal to a polar C-terminal cytoplasmic domain. In our recent work, focussed on the structural analysis of the cytoplasmic tail, we established an alpha-helix-flexible-alpha-helix-turn model. Now we present the experimental solution structure of the Vpu cytoplasmic domain which has been elucidated in aqueous 50% trifluoroethanol solution by 2D 1H NMR spectroscopy, and restrained molecular dynamics and energy minimization calculations. Under these conditions the peptide, Vpu32-81, is predominantly monomeric and adopts a well defined helix-interconnection-helix-turn conformation, in which the four regions are bounded by residues 37-51, 52-56, 57-72 and 73-78. The presence of the cis isomer of Pro-75 manifests itself as a doubling of cross peaks of neighbouring residues in the 2D spectra. A related variant peptide, Vpum32-81, in which the Vpu-phosphoacceptor sites Ser52 and Ser56 were exchanged for Asn, adopts a very similar structure and, taken together, provides evidence that the second helix and the turn form a comparatively rigid region. Both helices are amphipathic in character, but show different charge distributions. In general the cytoplasmic region is N-terminally positively charged, passes through a region of alternating charges in helix 1 and then becomes negatively charged. The flexibility of the interconnection permits orientational freedom of the two helices. The motif found here is the first experimentally refined solution structure of the cytoplasmic domain of Vpu, and it is conceivable that these alpha-helices are important for a previously defined physical interaction with an alpha-helical Vpu-responsive element located within the cytoplasmic tail of CD4.

Structure of the third cytoplasmic loop of bovine rhodopsin.

The three-dimensional high-resolution structure of rhodopsin is unknown, as is the case for almost all integral membrane patients. As part of an alternative approach to determine of membrane protein structure, we are pursuing the structure of cytoplasmic domains of this G-protein receptor. A peptide, rhoIII, with the sequence of the third cytoplasmic loop of bovine rhodopsin was synthesized. This soluble peptide was biologically active, inhibiting the light-stimulated activation of the rod cell phosphodiesterase by rhodopsin in rod outer segment disks. Therefore rhoIII likely contains structural elements characteristic of native rhodopsin. The structure of rhoIII was determined by H nuclear magnetic resonance. A defined structure was obtained for about 70% of rhoIII. A model of a turn-helix-turn motif could then be proposed for the third cytoplasmic loop of rhodopsin, which suggested a molecular switch for activation of the G-protein by the receptor.