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PubMed2005

Ionization of His 55 at the dimer interface of dynein light-chain LC8 is coupled to dimer dissociation.

LC8 is a highly conserved light-chain subunit of cytoplasmic dynein that interacts with a wide variety of cellular proteins and is presumed to play a fundamental role in dynein assembly and cargo recruitment and in the assembly of protein complexes unrelated to dynein. LC8 is a dimer at physiological pH but dissociates to a folded monomer at pH < 4.8. We have suggested that acid-induced dimer dissociation is due to protonation of His 55, which is stacked against His 55' and completely buried in the dimer interface. In this work, we show that the pH-induced dissociation is reversible and indeed governed by the ionization state of His 55. Mutagenesis of His 55 to Lys results in a monomer in the pH range of 3-8, while the mutation to Ala results in a dimer in the same pH range. Mutations that disrupt intermolecular hydrogen bonds between Tyr 65 and Lys 44' and His 55 and Thr 67' do not change the association state of the dimer. Titration curves for His 55 and the two other histidines, His 72 and 68, were determined by (13)C-(1)H NMR for H55K and for WT-LC8 in the monomeric and dimeric states. The pK(a) values of His 72 and His 68 are 6 in the WT dimer and 6.2-6.5 in monomeric H55K, while the pK(a) of His 55 is about 4.5 in the WT dimer. These results indicate that deprotonation of His 55 is linked to dimer formation and that mutation of His 55 to a small neutral residue or to a positively charged residue uncouples the protonation and dissociation processes.

Animals↗

PubMed2006

Selection between pinching-type and supramolecular polymer-type complexes by alpha-cyclodextrin-beta-cyclodextrin hetero-dimer and hetero-cinnamamide guest dimers.

Novel supramolecular complexes have been prepared from an alpha-cyclodextrin-beta-cyclodextrin hetero-dimer (alpha-CD-beta-CD hetero-dimer) and hetero-cinnamamide guest dimers, G-t-Boc and G-NH2, having adamantyl groups in aqueous solutions. On addition of the competitive guest, the supramolecular structure formed by a mixture of the alpha-CD-beta-CD hetero-dimer and G-t-Boc was found to be different from that of a mixture of the alpha-CD-beta-CD hetero-dimer and G-NH2 by the 1H NMR spectroscopy, the ROESY NMR spectroscopy, and the circular dichroism spectroscopy. The size of the supramolecular complex from the mixture of the alpha-CD-beta-CD hetero-dimer and G-NH2 is larger than that from the mixture of the alpha-CD-beta-CD hetero-dimer and G-t-Boc, which was proved by the pulse field gradient spin-echo NMR and the atomic force microscopy. These results suggest that the mixture of the alpha-CD-beta-CD hetero-dimer and G-t-Boc formed a pinching-type complex, and the mixture of the alpha-CD-beta-CD hetero-dimer and G-NH2 formed a supramolecular polymer-type complex.

Cinnamates↗

PubMed2000

The HIV-1(Lai) RNA dimerization. Thermodynamic parameters associated with the transition from the kissing complex to the extended dimer.

Retroviruses contain dimeric RNA consisting of two identical copies of the genomic RNA. The interaction between these two RNA molecules occurs near their 5' ends. A region upstream from the splice donor comprising an auto-complementary sequence has been identified as being responsible for the initiation of the formation of dimeric HIV-1(Lai) RNA. This region (SL1), part of the PSI encapsidation domain, can adopt a stem-loop structure. It has already been shown that this stem-loop structure can initiate the formation of two distinct dimers differing in their thermostability: a loop-loop dimer or 'kissing complex' and an extended dimer. We report here a study using UV and 1D NMR spectroscopy of the dimerization of a short oligoribonucleotide (23 nucleotides) spanning nucleotides 248-270 of the HIV-1(Lai) SL1 sequence, in order to derive the thermodynamic parameters associated with the transition from the loop-loop complex to the extended dimer. The temperature dependence of the UV absorbency shows an hypochromicity for this transition with a small enthalpy change equal to - 29.4 +/- 5 kcal x mol-1, together with a concentration independent transition which implies a monomolecular reaction. On the other hand, our NMR results don't indicate a dissociation of the GCGCGC sequence engaged in the loop-loop interaction during the rearrangement of the loop-loop complex into the extended dimer. Our data suggest that the loop-loop interaction is maintained during the temperature dependent conformational change while the intramolecular base-pairing of the stems is disrupted and then reconstituted to form an intermolecular base-pairing leading to an extended dimer.

Base Sequence↗

PubMed2001

The dimer initiation site hairpin mediates dimerization of the human immunodeficiency virus, type 2 RNA genome.

The untranslated leader of retroviral RNA genomes encodes multiple structural signals that are critical for virus replication. In the human immunodeficiency virus, type 1 (HIV-1) leader, a hairpin structure with a palindrome-containing loop is termed the dimer initiation site (DIS), because it triggers in vitro RNA dimerization through base pairing of the loop-exposed palindromes (kissing loops). Controversy remains regarding the region responsible for HIV-2 RNA dimerization. Different studies have suggested the involvement of the transactivation region, the primer binding site, and a hairpin structure that is the equivalent of the HIV-1 DIS hairpin. We have performed a detailed mutational analysis of the HIV-2 leader RNA, and we also used antisense oligonucleotides to probe the regions involved in dimerization. Our results unequivocally demonstrate that the DIS hairpin is the main determinant for HIV-2 RNA dimerization. The 6-mer palindrome sequence in the DIS loop is essential for dimer formation. Although the sequence can be replaced by other 6-mer palindromes, motifs that form more than two A/U base pairs do not dimerize efficiently. The inability to form stable kissing-loop complexes precludes formation of dimers with more extended base pairing. Structure probing of the DIS hairpin in the context of the complete HIV-2 leader RNA suggests a 5-base pair elongation of the DIS stem as it is proposed in current RNA secondary structure models. This structure is supported by phylogenetic analysis of leader RNA sequences from different viral isolates, indicating that RNA genome dimerization occurs by a similar mechanism for all members of the human and simian immunodeficiency viruses.

Base Sequence↗

PubMed2003

Dimerization of cGMP-dependent protein kinase Ibeta is mediated by an extensive amino-terminal leucine zipper motif, and dimerization modulates enzyme function.

All mammalian cGMP-dependent protein kinases (PKGs) are dimeric. Dimerization of PKGs involves sequences located near the amino termini, which contain a conserved, extended leucine zipper motif. In PKG Ibeta this includes eight Leu/Ile heptad repeats, and in the present study, deletion and site-directed mutagenesis have been used to systematically delete these repeats or substitute individual Leu/Ile. The enzymatic properties and quaternary structures of these purified PKG mutants have been determined. All had specific enzyme activities comparable to wild type PKG. Simultaneous substitution of alanine at four or more of the Leu/Ile heptad repeats ((L3A/L10A/L17A/I24A), (L31A/I38A/L45A/I52A), (L17A/I24A/L31A/I38A/L45A/I52A), and (L3A/L10A/L45A/I52A)) of the motif produces a monomeric PKG Ibeta. Mutation of two Leu/Ile heptad repeats can produce either a dimeric (L3A/L10A) or monomeric (L17A/I24A and L31A/I38A) PKG. Point mutation of Leu-17 or Ile-24 (L17A or I24A) does not disrupt dimerization. These results suggest that all eight Leu/Ile heptad repeats are involved in dimerization of PKG Ibeta. Six of the eight repeats are sufficient to mediate dimerization, but substitutions at some positions (Leu-17, Ile-24, Leu-31, and Ile-38) appear to have greater impact than others on dimerization. The Ka of cGMP for activation of monomeric mutants (PKG Ibeta (delta1-52) and PKG Ibeta L17A/I24A/L31A/I38A/L45A/I52A) is 2- to 3-fold greater than that for wild type dimeric PKG Ibeta, and there is a corresponding 2- to 3-fold increase in cGMP-dissociation rate of the high affinity cGMP-binding site (site A) of these monomers. These results indicate that dimerization increases sensitivity for cGMP activation of the enzyme.

Amino Acid Motifs↗

PubMed2004

Dissociation of human copper-zinc superoxide dismutase dimers using chaotrope and reductant. Insights into the molecular basis for dimer stability.

The dissociation of apo- and metal-bound human copper-zinc superoxide dismutase (SOD1) dimers induced by the chaotrope guanidine hydrochloride (GdnHCl) or the reductant Tris(2-carboxyethyl)phosphine (TCEP) has been analyzed using analytical ultracentrifugation. Global fitting of sedimentation equilibrium data under native solution conditions (without GdnHCl or TCEP) demonstrate that both the apo- and metal-bound forms of SOD1 are stable dimers. Sedimentation velocity experiments show that apo-SOD1 dimers dissociate cooperatively over the range 0.5-1.0 M GdnHCl. In contrast, metal-bound SOD1 dimers possess a more compact shape and dissociate at significantly higher GdnHCl concentrations (2.0-3.0 M). Reduction of the intrasubunit disulfide bond within each SOD1 subunit by 5-10 mM TCEP promotes dissociation of apo-SOD1 dimers, whereas the metal-bound enzyme remains a stable dimer under these conditions. The Cys-57 --> Ser mutant of SOD1, a protein incapable of forming the intrasubunit disulfide bond, sediments as a monomer in the absence of metal ions and as a dimer when metals are bound. Taken together, these data indicate that the stability imparted to the human SOD1 dimer by metal binding and the formation of the intrasubunit disulfide bond are mediated by independent molecular mechanisms. By combining the sedimentation data with previous crystallographic results, a molecular explanation is provided for the existence of different SOD1 macromolecular shapes and multiple SOD1 dimeric species with different stabilities.

Chemical Phenomena↗

PubMed1998

Mutations in MHC class II dimer of dimers contact residues: effects on antigen presentation.

The recent solutions of the MHC class II crystal structure reveal dimerization of the alphabeta heterodimers. These dimer of dimers structures may also exist either on resting cells or after engagement by TCR, and may be involved in B cell signaling and up-regulation of co-stimulatory molecules such as B7 which facilitate T cell activation. By combining crystallographic data on HLA-DR1 with the sequence of murine I-Ak and refining the resulting structure through energy minimization calculations, we have predicted the contact amino acids expected to stabilize the I-Ak dimer of dimers structure. As in HLA-DR1, three salt bridges in I-Ak (D alpha62-Hbeta112, H alpha181-E beta163, E alpha183-Hbeta113) appear to provide the main interaction. Guided by this structural data, we prepared 45 B cell transfectants representing 20 different class II mutation phenotypes in the contact region containing these salt bridges. We examined their abilities to activate three T cell hybrids. Antigen-specific h4Ly50.5 cells were not greatly affected by changes in the dimer of dimer contact residues. In contrast, autoreactive C8.A3 T cells were very sensitive to changes in this region but presentation of class II of many mutation phenotypes could be rescued by treatments that up-regulate B7-1. The alloreactive hybridoma 2H40.2.5 was less sensitive to changes in the contact residues. A simple model was developed that summarizes the effects of the mutations for the T cells tested. Mutations at D alpha162, E alpha183, H alpha181 and Rbeta106 had the largest negative impact, while D alpha166, E alpha185, Hbeta112, Hbeta113 and E beta163 were less disruptive. Results are consistent with mutations interfering with class II interaction with another molecule which might or might not be another class II heterodimer. However, the larger negative impact of alpha chain mutations in salt bridge pairs suggests that these sites also help maintain some essential conformation of the alpha chain apart from any possible impact on dimer of dimers stability.

Antibodies, Monoclonal↗

PubMed2003

Insights into dimerization and four-helix bundle formation found by dissection of the dimer interface of the GrpE protein from Escherichia coli.

The GrpE heat shock protein from Escherichia coli has a homodimeric structure. The dimer interface encompasses two long alpha-helices at the NH(2)-terminal end from each monomer (forming a "tail"), which lead into a small four-helix bundle from which each monomer contributes two short sequential alpha-helices in an antiparallel topological arrangement. We have created a number of different deletion mutants of GrpE that have portions of the dimer interface to investigate requirements for dimerization and to study four-helix bundle formation. Using chemical crosslinking and analytical ultracentrifugation techniques to probe for multimeric states, we find that a mutant containing only the long alpha-helical tail portion (GrpE1-88) is unable to form a dimer, most likely due to a decrease in alpha-helical content as determined by circular dichroism spectroscopy, thus one reason for a dimeric structure for the GrpE protein is to support the tail region. Mutants containing both of the short alpha-helices (GrpE1-138 and GrpE88-197) are able to form a dimer and presumably the four-helix bundle at the dimer interface. These two mutants have equilibrium constants for the monomer-dimer equilibrium that are very similar to the full-length protein suggesting that the tail region does not contribute significantly to the stability of the dimer. Interestingly, one mutant that contains just one of the short alpha-helices (GrpE1-112) exists as a tetrameric species, which presumably is forming a four-helix bundle structure. A proposed model is discussed for this mutant and its relevance for factors influencing four-helix bundle formation.

Bacterial Proteins↗

PubMed2006

The two-state dimer receptor model: a general model for receptor dimers.

Nonlinear Scatchard plots are often found for agonist binding to G-protein-coupled receptors. Because there is clear evidence of receptor dimerization, these nonlinear Scatchard plots can reflect cooperativity on agonist binding to the two binding sites in the dimer. According to this, the "two-state dimer receptor model" has been recently derived. In this article, the performance of the model has been analyzed in fitting data of agonist binding to A(1) adenosine receptors, which are an example of receptor displaying concave downward Scatchard plots. Analysis of agonist/antagonist competition data for dopamine D(1) receptors using the two-state dimer receptor model has also been performed. Although fitting to the two-state dimer receptor model was similar to the fitting to the "two-independent-site receptor model", the former is simpler, and a discrimination test selects the two-state dimer receptor model as the best. This model was also very robust in fitting data of estrogen binding to the estrogen receptor, for which Scatchard plots are concave upward. On the one hand, the model would predict the already demonstrated existence of estrogen receptor dimers. On the other hand, the model would predict that concave upward Scatchard plots reflect positive cooperativity, which can be neither predicted nor explained by assuming the existence of two different affinity states. In summary, the two-state dimer receptor model is good for fitting data of binding to dimeric receptors displaying either linear, concave upward, or concave downward Scatchard plots.

Adenosine A1 Receptor Agonists↗

PubMed1998

Activation of Neu (ErbB-2) mediated by disulfide bond-induced dimerization reveals a receptor tyrosine kinase dimer interface.

Receptor dimerization is a crucial intermediate step in activation of signaling by receptor tyrosine kinases (RTKs). However, dimerization of the RTK Neu (also designated ErbB-2, HER-2, and p185(neu)), while necessary, is not sufficient for signaling. Earlier work in our laboratory had shown that introduction of an ectopic cysteine into the Neu juxtamembrane domain induces Neu dimerization but not signaling. Since Neu signaling does require dimerization, we hypothesized that there are additional constraints that govern signaling ability. With the importance of the interreceptor cross-phosphorylation reaction, a likely constraint was the relative geometry of receptors within the dimer. We have tested this possibility by constructing a consecutive series of cysteine substitutions in the Neu juxtamembrane domain in order to force dimerization along a series of interreceptor faces. Within the group that dimerized constitutively, a subset had transforming activity. The substitutions in this subset all mapped to the same face of a predicted alpha helix, the most likely conformation for the intramembrane domain. Furthermore, this face of interaction aligns with the projected Neu* V664E substitution and with a predicted amphipathic interface in the Neu juxtamembrane domain. We propose that these results identify an RTK dimer interface and that dimerization of this RTK induces an extended contact between juxtamembrane and intramembrane alpha helices.

3T3 Cells↗

PubMed2006

Validation of a new D-dimer microparticle enzyme immunoassay (AxSYM D-Dimer) in patients with suspected pulmonary embolism (PE).

OBJECTIVES: The aim of the study was to evaluate a new automated assay for D-dimer testing (AxSYM D-Dimer) based on microparticle enzyme-immunoassay technology by comparing it with three well established D-dimer assays. PATIENTS AND METHODS: The performance of the new assay was evaluated in 280 plasma samples that were collected prospectively from out-patients included in a management study evaluating a decision based algorithm. RESULTS: 58/280 patients (21%) had PE diagnosed by CT. Median values of AxSYM D-dimer in patients with PE were 3689 ng/mL (range 775-9000). Comparison analysis displayed excellent agreement with VIDAS (kappa=0.84) and Asserachrom (kappa=0.81) D-dimer assays. A strong correlation was found between AxSYM and the VIDAS (r=0.96) and Asserachrom (r=0.89) D-dimer assays. The highest cut-off value for AxSYM that yielded a sensitivity of 100% was 765 ng/mL with a specificity of 50%. At the cut-off level <500 ng/mL, the sensitivity and specificity of AxSYM D-dimer were 100% and 34%; VIDAS 100% and 42%; Asserachrom 100% and 40%; and STALiatest 100% and 37%, respectively. AxSYM D-dimer was negative in 75 patients (33.8%). None of these had PE at the initial work-up or VTE during the 3-month follow-up. CONCLUSIONS: AxSYM D-dimer seems to be safe and effective in ruling out PE in out-patients. The cut-off level can be set at 500 to 750 ng/mL, at which the assay displays a performance that is comparable to that of the ELISA based assays. However, further studies are needed to confirm the safety of the assay and to determine the most optimal cut-off level in patients with venous thromboembolism.

Adolescent↗

PubMed1996

A kissing complex together with a stable dimer is involved in the HIV-1Lai RNA dimerization process in vitro.

Retroviruses contain a dimeric RNA consisting of two identical molecules of genomic RNA. The interaction between the two monomers is thought to occur near their 5'ends. We previously identified a region upstream from the splice donor site, comprising an autocomplementary sequence, responsible for the formation of dimeric HIV-1Lai RNA [Muriaux, D., Girard, P.-M., Bonnet-Mathonire, B., & Paoletti, J.(1995) J. Biol. Chem. 270,8209-8216]. This region appeared to be confined within a putative stem-loop structure. Here we report an in vitro model under conditions of low inioc strength. Two dimers of RNA 77-402 were identified as a function of temperature, and a significant difference was found in their thermostability. Dimer D55, formed at 55 degrees Celsius, is more stable than dimer D37, formed at 37 degrees C. RNase probing experiments confirm the involvement of a stem-loop structure in the dimerization process. In the monomer, the free G257-CGCGC262 sequence forms a loop in the 240-280 region of RNA 77-402, whereas this sequence is engaged in base pairing when D55 and D37 dimers are formed. Our results show that the loop-loop interaction of the autocomplementary G257CGCGC262 sequence, though hydrogen bonding, is responsible for the formation of dimer D37 and strongly suggest that D37 is a "kissing" complex. In contrast, in dimer D55, all the nucleotides of the two hairpin stems, 243-254/264-277, are involved in a complete interstrand interaction.

Base Sequence↗

PubMed1990

Stability and activity of human immunodeficiency virus protease: comparison of the natural dimer with a homologous, single-chain tethered dimer.

A single-chain tethered dimer of human immunodeficiency virus protease (HIV-PR) was produced by expression of a synthetic gene in Escherichia coli. The tethered dimer, which consists of two 99-amino acid HIV-PR subunits linked together by a pentapeptide, was isolated from inclusion bodies and refolded as an active protease with enzymatic properties very similar to those of the natural dimer at pH 5.5. In addition to demonstrating that the tethered dimer is active, we have shown that the tethered dimer is more stable than the natural HIV-PR dimer at pH 7.0. This is attributed to dissociation of the natural HIV-PR dimer, for which a surprisingly high dissociation constant, 5 X 10(-8) M was measured. Furthermore, the tethered dimer offers an opportunity to produce asymmetric dimer mutants and thereby determine the effect of changes in one of the two subunits on protease activity. In one such mutant, a single active-site aspartic residue was changed to a glycine residue. This protein was inactive, consistent with a requirement for an aspartic residue from each subunit to constitute an active site of HIV-PR.

Amino Acid Sequence↗

PubMed1996

Tetramethylrhodamine dimer formation as a spectroscopic probe of the conformation of Escherichia coli ribosomal protein L7/L12 dimers.

The fluorescent probe tetramethylrhodamine iodoacetamide was attached to cysteine residues substituted at various specific locations in full-length and deletion variants of the homodimeric Escherichia coli ribosomal protein L7/L12. Ground-state tetramethylrhodamine dimers form between the two subunits of L7/L12 depending upon the location of the probe. The formation of tetramethylrhodamine dimers caused the appearance of a new absorption band at 518 nm that was used to estimate the extent of interaction of the probes in the different protein variants. Intersubunit tetramethylrhodamine dimers form when tetramethylrhodamine acetamide is attached to two different sites in the N-terminal domain of the L7/L12 dimer (residues 12 or 33), but not when attached to sites in the C-terminal domain (residues 63, 89, or 99). The tetramethylrhodamine dimers do form at sites in the C-terminal domain in L7/L12 variants that contain deletions of 11 or 18 residues within the putative flexible hinge that separates the N- and C-terminal domains. The tetramethylrhodamine dimers disappear rapidly (within 5 s) upon addition of excess unlabeled wild-type L7/L12. It appears that singly labeled L7/L12 dimers are formed by exchange with wild-type dimers. Binding of L7/L12:tetramethylrhodamine cysteine 33 or cysteine 12 dimers either to L7/L12-depleted ribosomal core particles, or to ribosomal protein L10 alone, results in disappearance of the 518-nm absorption band. This result implies a conformational change in the N-terminal domain of L7/L12 upon its binding to the ribosome, or to L10.

Binding Sites↗

PubMed1998

D-dimer testing as an adjunct to ultrasonography in patients with clinically suspected deep vein thrombosis: prospective cohort study. The Multicentre Italian D-dimer Ultrasound Study Investigators Group.

OBJECTIVE: To investigate the efficacy of using a rapid plasma D-dimer test as an adjunct to compression ultrasound for diagnosing clinically suspected deep vein thrombosis. DESIGN: D-dimer concentrations were determined in all patients with a normal ultrasonogram at presentation. Repeat ultrasonography was performed 1 week later only in patients with abnormal D-dimer test results. MAIN OUTCOME AND MEASURES: Patients with normal ultrasonograms were not treated with anticoagulants and were followed for 3 months for thromboembolic complications. SETTING: University research and affiliated centres. SUBJECTS: 946 patients with clinically suspected deep vein thrombosis. RESULTS: Ultrasonograms were abnormal at presentation in 260 (27.5%) patients. Of the remaining 686 patients tested for D-dimer, 88 (12.8%) had abnormal concentrations. During follow up venous thromboembolic complications occurred in one of the 598 patients who were not treated with anticoagulants and who had an initial normal ultrasonogram and D-dimer concentration, whereas thromboembolic complications occurred in two of the 83 untreated patients who had abnormal D-dimer concentrations but a normal repeat ultrasonogram. The cumulative incidence of venous thromboembolic complications during follow up was 0.4% (95% confidence interval 0% to 0.9%). The rapid plasma D-dimer test used as an adjunct to compression ultrasonography resulted in a reduction in the mean number of repeat ultrasound examinations and additional hospital visits from 0.7 to 0.1 per patient. CONCLUSIONS: Testing for D-dimer as an adjunct to a normal baseline ultrasound examination decreased the number of subsequent ultrasound examinations considerably without any increased risk of venous thromboembolic complications in patients not receiving anticoagulants. The use of ultrasound and testing for D-dimer enabled treatment decisions to be made at the time of presentation in most patients.

Biomarkers↗

PubMed1998

Carboxyl-terminal domain dimer interface mutant 434 repressors have altered dimerization and DNA binding specificities.

Strong dimerization of the repressor, mediated by the carboxyl (C)-terminal domain, is a prerequisite for forming a specific complex with DNA and cooperative DNA binding to form tetramers. We have generated a computer model of the C-terminal domain of the 434 repressor based on the crystal structure of the homologous UmuD' protein. This model predicts that residues in the primary sequence between 93 and 168 contribute to the dimer interface. We changed several amino acid residues located in this region. Gel filtration and crosslinking assays were used to characterize the strength and specificity of dimerization of the purified repressor C-terminal domain dimer interface mutants. These results indicate that amino acid residues K121, H139, D161 and N163 contribute to the strength and/or specificity of dimerization. The relative affinity of the bacteriophage 434 repressor for 434 operators is determined, in part, by the repressor's ability to detect sequence-dependent structural alterations in the non-contacted region at the center of an operator site. We find that the relative ability of C-terminal domain dimer interface mutant repressors to dimerize does not necessarily predict their relative abilities to bind DNA, and that these proteins are deficient in detecting non-contacted base-dependent differences in operator strength. Our results show that the structure of the DNA in complex with these mutant proteins differs from that found in wild-type repressor-operator complexes, even though the sites of these mutations lie in a separate domain from that which contacts the DNA. These observations demonstrate that the structural integrity of the C-terminal domain dimer interface is required to appropriately orient the DNA binding information contained within the DNA-contacting N-terminal domain.

Amino Acid Sequence↗

PubMed1996

Rhamnogalacturonan-II, a pectic polysaccharide in the walls of growing plant cell, forms a dimer that is covalently cross-linked by a borate ester. In vitro conditions for the formation and hydrolysis of the dimer.

Rhamnogalacturonan II (RG-II) is a structurally complex pectic polysaccharide present in the walls of growing plant cells. We now report that RG-II, released by endopolygalacturonase treatment of the walls of suspension-cultured sycamore cells and etiolated pea stems, exists mainly as a dimer that is cross-linked by a borate ester. The borate ester is completely hydrolyzed at room temperature within 30 min at pH 1, partially hydrolyzed between pH 2 and 4, and stable above pH 4. The dimer is formed in vitro between pH 2.4 and 6. 2 by treating monomeric RG-II (0.5 mM) with boric acid (1.2 mM); the dimer formed after 24 h at pH 3.4 and 5.0 accounts for approximately 30 and approximately 5%, respectively, of the RG-II. In contrast, the dimer accounts for approximately 80 and approximately 54% of the RG-II when the monomer is treated for 24 h at pH 3.4 and 5.0, respectively, with boric acid and 0.5 m Sr2+, Pb2+, or Ba2+. The amount of dimer formed at pH 3.4 or 5.0 is not increased by addition of 0.5 mM Ca2+, Cd2+, Cu2+, Mg2+, Ni2+, and Zn2+. Steric considerations appear to regulate dimer formation since those divalent cations that enhance dimer formation have an ionic radius >1.1 A. Our data suggest that the borate ester is located on C-2 and C-3 of two of the four 3'-linked apiosyl residues of dimeric RG-II. Our results, taken together with the results of two previous studies (Kobayashi, M., Matoh, T., and Azuma, J.-I. (1996) Plant Physiol. 110, 1017-1020; Ishii, T., and Matsunaga, T. (1996) Carbohydr. Res. 284, 1-9) provide substantial evidence that this plant cell wall pectic polysaccharide is covalently cross-linked.

Barium↗

PubMed2003

Constitutive activation and uncoupling of the atrial natriuretic peptide receptor by mutations at the dimer interface. Role of the dimer structure in signalling.

The crystal packing of the extracellular hormone binding domain of the atrial natriuretic peptide (ANP) receptor contains two possible dimer pairs, the head-to-head (hh) and tail-to-tail (tt) dimer pairs associated through the membrane-distal and membrane-proximal subdomains, respectively. The tt-dimer structure has been proposed previously (van den Akker, F., Zhang, X., Miyagi, M., Huo, X., Misono, K. S., and Yee, V. C. (2000) Nature 406, 101-104). However, no direct evidence is available to identify the physiological dimer form. Here we report site-directed mutagenesis studies of residues at the two alternative dimer interfaces in the full-length receptor expressed on COS cells. The Trp74 to Arg mutation (W74R) or D71R at the hh-dimer interface caused partial constitutive guanylate cyclase activation, whereas mutation F96D or H99D caused receptor uncoupling. In contrast, mutation Y196D or L225D at the tt-interface had no such effect. His99 modification at the hh-dimer interface by ethoxyformic anhydride abolished ANP binding. These results suggest that the hh-dimer represents the physiological structure. Recently, we determined the crystal structure of ANPR complexed with ANP and proposed a hormone-induced rotation mechanism mediating transmembrane signaling (H. Ogawa, Y. Qiu, C. M. Ogata, and K. S. Misono, submitted for publication). The observed effects of mutations are consistent with the ANP-induced structural change identified from the crystal structures with and without ANP and support the proposed rotation mechanism for ANP receptor signaling.

Animals↗