Biomedical subjects

D Mahadevan

Publications and source records attributed to D Mahadevan.

At least 19 recordsLinked to original sources

PubMed1999

The extracellular regions of PSMA and the transferrin receptor contain an aminopeptidase domain: implications for drug design.

The Aeromonas proteolytica aminopeptidase (AMP), Pseudomonas sp. (RS-16) carboxypeptidase G2 (CPG2), and Streptomyces griseus aminopeptidase (SGAP) are zinc dependent proteolytic enzymes with cocatalytic zinc ion centers and a conserved aminopeptidase fold. A BLAST search with the sequence of the solved AMP structure indicated that a similar domain could be found in prostate-specific membrane antigen (PSMA) and the transferrin receptor (TfR). When the PSMA or TfR sequence was input into the THREADER program, the top structural matches were SGAP and AMP confirming that these are structurally conserved domains. Optimal sequence alignment of PSMA and TfR using the known three-dimensional structures of AMP, CPG2, and SGAP shows that the critical amino acids involved in forming the catalytic pocket are conserved in PSMA but absent in the TfR. The specificity pocket in AMP is formed from four aromatic side chains and the equivalent region in CPG2/PSMA has a changed sequence pattern. Since CPG2 and PSMA are folate hydrolases, the changed specificity pocket leaves space to accommodate the large pteroate moiety of folic acid. In contrast, no enzyme function has been ascribed to the TfR.

Aeromonas↗

PubMed1998

Identification of a Frizzled-like cysteine rich domain in the extracellular region of developmental receptor tyrosine kinases.

In Drosophila, members of the Frizzled family of tissue-polarity genes encode proteins that appear to function as cell-surface receptors for Wnts. The Frizzled genes belong to the seven transmembrane class of receptors (7TMR) and have on their extracellular region a cysteine-rich domain that has been implicated as the Wnt binding domain. This region has a characteristic spacing of ten cysteines, which has also been identified in FrzB (a secreted antagonist of Wnt signaling) and Smoothened (another 7TMR, which is involved in suppression of the hedgehog pathway). We have identified, using BLAST, sequence similarity between the cysteine-rich domain of Frizzled and several receptor tyrosine kinases, which have roles in development. These include the muscle-specific receptor tyrosine kinase (MuSK), the neuronal specific kinase (NSK2), and ROR1 and ROR2. At present, the ligands for these developmental tyrosine kinases are unknown. Our results suggest that Wnt-like ligands may bind to these developmental tyrosine kinases.

Amino Acid Sequence↗

PubMed1998

Identification of a Frizzled-like cysteine rich domain in the extracellular region of developmental receptor tyrosine kinases.

In Drosophila, members of the Frizzled family of tissue-polarity genes encode proteins that appear to function as cell-surface receptors for Wnts. The Frizzled genes belong to the seven transmembrane class of receptors (7TMR) and have on their extracellular region a cysteine-rich domain that has been implicated as the Wnt binding domain. This region has a characteristic spacing of ten cysteines, which has also been identified in FrzB (a secreted antagonist of Wnt signaling) and Smoothened (another 7TMR, which is involved in the hedgehog signalling pathway). We have identified, using BLAST, sequence similarity between the cysteine-rich domain of Frizzled and several receptor tyrosine kinases, which have roles in development. These include the muscle-specific receptor tyrosine kinase (MuSK), the neuronal specific kinase (NSK2), and ROR1 and ROR2. At present, the ligands for these developmental tyrosine kinases are unknown. Our results suggest that Wnt-like ligands may bind to these developmental tyrosine kinases

Amino Acid Sequence↗

PubMed1996

Functional interactions between isolated SH2 domains and insulin/Ras signaling pathways of Xenopus oocytes: opposite effects of the carboxy- and amino-terminal SH2 domains of p85 PI 3-kinase.

Purified amino-terminal Src homology 2 (SH2) domains of GAP, PLCgamma1 and the p85alpha subunit of PI 3-kinase, as well as the carboxy-terminal SH2 domain of the latter protein and the unique SH2 domain of Grb2, were injected into full grown, stage VI Xenopus laevis oocytes. None of the injected domains showed any effect when injected alone, nor did they affect the rate of GVBD induced by progesterone, an adenylate cyclase-dependent process. On the other hand, the unique Grb2 SH2 domain and all N-terminal SH2 domains injected inhibited to various degrees the rate of insulin-induced GVBD, a tyrosine kinase dependent pathway. Interestingly, and in contrast to the behavior shown by the N-terminal domain of the same molecule, the C-terminal SH2 domain of p85 did not inhibit, but slightly accelerated the rate of GVBD induced by insulin. Furthermore, whereas the Grb SH2 domain and all N-terminal SH2 domains tested failed to co-operate with normal Ras protein to induce GVBD, the C-terminal SH2 domain of p85alpha exhibited significant synergy when coinjected with normal Ras protein, indicating that the C- and N-terminal SH2 domains of p85alpha exert opposite (positive and negative, respectively) regulatory roles in the control of oocyte insulin/Ras signaling pathways. Our results demonstrate that the purified, isolated SH2 domains retain structural and functional specificity and that Xenopus oocytes constitute an useful biological system to analyse their functional role in tyrosine kinase signaling pathways.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Isolated Sos1 PH domain exhibits germinal vesicle breakdown-inducing activity in Xenopus oocytes.

Purified, bacterially expressed PH domains of Sos1, IRS-1, betaARK, and PLCdelta1 were analyzed functionally by means of microinjection into full grown, stage VI Xenopus laevis oocytes. Whereas the PH domains from IRS-1, betaARK, or PLCdelta1 did not show any effect in the oocytes, injection of the purified Sos1 PH domain resulted in induction of significant rates of germinal vesicle breakdown and meiotic maturation. Furthermore, the Sos1 PH domain exhibited also significant synergy with insulin or coinjected normal Ras protein in induction of germinal vesicle breakdown, although it did not affect the rate of progesterone-induced maturation. These results suggest that purified, isolated PH domains retain, at least in part, their functional specificity and that Xenopus oocytes may constitute a useful biological system to analyze the functional role of the Sos1 PH domain in Ras signaling pathways.

Amino Acid Sequence↗

PubMed1996

Identification of a domain within the carboxyl-terminal region of the beta platelet-derived growth factor (PDGF) receptor that mediates the high transforming activity of PDGF.

We have reported previously that a chimeric platelet-derived growth factor receptor (PDGFR) possessing the ligand binding domain of the alpha PDGFR and the intracellular domain of the beta PDGFR (alpha 340 beta 342 R) was markedly more efficient than the wild type alpha PDGFR (alpha RWT) in its ability to enhance PDGF-A transforming activity in NIH/3T3 fibroblasts. To determine the region within the cytoplasmic domain of beta PDGFR that confers this higher transforming activity, we generated several additional alpha/beta PDGFR chimerae. When a chimeric PDGFR possessing the first 933 amino-terminal amino acids from the alpha PDGFR and the final 165 amino acids from the carboxyl-terminal of the beta PDGFR (alpha 933 beta 942 R) was cotransfected with the PDGF-A gene into NIH/3T3 cells, it showed a similar high efficiency to enhance PDGF-A chain transforming activity as alpha 340 beta 342 R. However, when chimeric PDGFRs in which either the kinase insert domain (alpha beta RKI) or the last 79 amino acids from the carboxyl-terminal end of the beta PDGFR (alpha 1024 beta 1028 R) were substituted into alpha PDGFR sequences were cotransfected with PDGF-A, they showed similar low efficiencies in enhancing transforming activity as the alpha RWT. These results predicted that the 86 amino acids following the tyrosine kinase 2 domain of beta PDGFR (amino acid residues 942-1027) were responsible for the higher transforming activity of beta PDGFR. To confirm this finding, we next constructed a chimera in which amino acid residues 942-1028 of the beta PDGFR (alpha beta 942-1028R) were substituted for those in the alpha PDGFR. Cotransfection experiments indicated that alpha beta 942-1028R increased transforming activity of PDGF-A to similar extent as the alpha 933 beta 942R, or alpha 340 beta 342R. Therefore, our findings define a critical domain within the noncatalytic region of beta PDGFR intracellular domain that confers the higher focus forming activity mediated by the beta PDGFR.

3T3 Cells↗

PubMed1996

The PH domain of Ras-GAP is sufficient for in vitro binding to beta gamma subunits of heterotrimeric G proteins.

1. The noncatalytic domain of Ras-GAP can affect signaling through G protein-coupled receptors by a poorly understood mechanism. 2. In this study, fusion proteins containing elements of the noncatalytic domain of ras-GAP were examined for their ability to bind beta gamma subunits of heterotrimeric G proteins and phosphotyrosine-containing polypeptides. 3. Our results demonstrate that purified beta gamma dimers associated with bacterially expressed GAP proteins and that this association does not require SH2 or SH3 domains but is dependent on the presence of the GAP pleckstrin-homology (PH) domain. In contrast, only the SH2 domains are necessary for binding to tyrosine phosphorylated proteins. 4. These findings raise the possibility that heterotrimeric G proteins might affect functioning of ras-like proteins through beta gamma subunits acting on their regulatory molecules.

Binding Sites↗

PubMed1995

Structural role of extracellular domain 1 of alpha-platelet-derived growth factor (PDGF) receptor for PDGF-AA and PDGF-BB binding.

The purpose of this study was to bacterially express, purify, and refold combinations of the extracellular immunoglobulin (Ig)-like domains (2-3, 1-3, and 1-5) of the human alpha-platelet-derived growth factor receptor (alpha PDGFR) to characterize molecular interactions with its ligand, platelet-derived growth factor (PDGF). The far UV circular dichroism spectroscopy of the alpha-PDGFR extracellular domains (ECDs) revealed a predominantly beta-sheet protein, with a structure consistent with folded Ig-like domains. The addition of PDGF-BB to these ECD types changed the conformation of all three types with a decrease in mean residue ellipticity in the following rank order: 1-5 = 1-3 > 2-3. In striking contrast, addition of PDGF-AA to these ECD types markedly changed the conformation of ECD 2-3, by an increased mean residue ellipticity but no changes were observed for ECDs 1-3 and 1-5. PDGF-AA bound to the immobilized ECD types 2-3, 1-3, and 1-5 at concentrations of 20, 11, and 7.5 nM, respectively. In contrast, PDGF-BB bound the ECD types 2-3, 1-3, and 1-5 at concentrations of 3, 3, and 2.2 nM, respectively. Scatchard analysis of binding studies using labeled ECDs indicated that PDGF-BB bound ECD 1-3 and ECD 2-3 with KD values of 74 and 72 nM, respectively. While, PDGF-AA bound ECD 1-3 and ECD 2-3 with KD values of 33 and 87 nM, respectively. Therefore, our results indicated that the loss of ECD 1 impaired the binding affinity of alpha PDGFR ECD 1-3 toward PDGF-AA without having a similar effect on PDGF-BB binding. Together all of our data suggest that ECD 1 is differentially required for proper orientation of PDGF-AA but not PDGF-BB binding determinant within ECDs 2 and 3.

Becaplermin↗

PubMed1995

Structural studies on the PH domains of Db1, Sos1, IRS-1, and beta ARK1 and their differential binding to G beta gamma subunits.

Pleckstrin homology (PH) domains are approximately 110 amino acid residues in length and are structurally conserved in a number of intracellular signaling proteins. A role for these domains has been postulated for beta ARK, which binds to G beta gamma subunits. We have quantified the binding of individual (His)6-tag PH domains of human Db1, human Sos1, rat IRS-1, human beta ARK, and human beta ARK with an extra 33-residue C-terminal extension (beta ARK + C) to G beta gamma subunits. Our in vitro binding studies show that all of the PH domains (apart from Sos1), bind G beta gamma subunits in a dose-dependent manner, but beta ARK + C binds 4 times as much G beta gamma at saturation as the others. The IRS-1 PH domain has a similar half-maximal concentration of G beta gamma binding (18 nM) to beta ARK + C (30 nM), suggesting that the IRS-1 PH domain has sufficient determinants for G beta gamma binding. The beta ARK PH domain alone has a half-maximal value of 45 nM but a drastically reduced extent of G beta gamma binding, suggesting that both the PH domain and the C-terminal 33 residues are necessary for maximal binding. Db1 has a half-maximum concentration of G beta gamma binding of 45 nM and a maximal extent of binding similar to that of beta ARK, but it is difficult to demonstrate saturable binding of G beta gamma to Sos1. Since it was previously predicted that the C-terminal PH domain of Pleckstrin [Tyers, M., et al. (1988) Nature 333, 470-473] contains a potential calcium binding site, we have tested the different PH domains for calcium binding. Only the PH domain of Db1 bound 45Ca2+ with a Kd of 10 microM. CD spectroscopy of the purified recombinant PH domains indicated that they are predominantly beta-sheet structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

A divalent metal ion binding site in the kinase insert domain of the alpha-platelet-derived growth factor receptor regulates its association with SH2 domains.

To investigate the effects of metal ion binding to the alpha-PDGFR kinase insert domain, a PCR product representing amino acid residues 691-795 (104 amino acids) was bacterially expressed and purified. Secondary structure prediction and circular dichroism spectroscopy indicated this domain to be a mixed alpha + beta protein with a large coil/turn contribution. This 16 kDa, soluble, nonphosphorylated domain bound to 45Ca2+ and 65Zn2+ through a common shared site. Of the unlabeled divalent and trivalent metal ions tested, Ho3+ = Zn2+ > Ni2+ > Ca2+ = Mn2+ > Mg2+, Ba2+ in competing for 45Ca2+ binding to this domain. In the presence of Ca2+ ions, the conformation of the KI domain changed significantly, and this changed conformation was resistant to subtilisin proteolysis. However, in the presence of Zn2+ ions, the conformation of the KI domain changed only slightly. Nevertheless, Zn2+ ions were more effective in rendering the KI domain resistant to proteolysis as compared to that shown by Ca2+ ions. In vitro binding studies using purified baculovirus-expressed alpha-PDGFR showed a marked increase in binding the p85 N-SH2 domain in the presence of Ca2+ or Zn2+ ions (KD = 0.5 microM), suggesting that metal ion binding enhances association of the p85 N-SH2 domain with the receptor. To confirm this, association of the alpha-PDGFR with the p85 N-SH2 domain was tested in the presence of the KI domain. The nonphosphorylated KI domain was effective in competing with the alpha-PDGFR for the binding of the p85 N-SH2 domain. This effect was more pronounced in the presence of Ca2+ ions. Microinjection of this domain into Xenopus oocytes delayed maturation in the presence of insulin but not progesterone. This suggests that the KI domain has a correctly folded three-dimensional structure compatible with biological activity. Together these findings indicate that the recombinant alpha-PDGFR KI domain binds the p85 N-SH2 domain and this binding is modulated by the presence of a novel divalent metal ion binding site within its structure.

Amino Acid Sequence↗

PubMed1995

Beta PDGFR-IgG chimera demonstrates that human beta PDGFR Ig-like domains 1 to 3 are sufficient for high affinity PDGF BB binding.

To localize human beta PDGFR binding determinants, we constructed a fusion protein comprising beta PDGFR Ig-like domains 1 to 3 and an IgG1 Fc domain (beta PDGFR-HFc). beta PDGFR-HFc was expressed as a 200 kDa dimeric molecule and contained Fc epitopes as demonstrated by anti-mouse Fc antibody recognition. Scatchard analysis revealed that PDGF BB possessed a dissociation constant of 1.5 nM for beta PDGFR-HFc. Thus, beta PDGFR Ig-like domains 1 to 3 are sufficient for high affinity PDGF BB binding. We exploited this fusion protein technology to identify and characterize beta PDGFR antagonists using a sensitive beta PDGFR immunosorbent assay. In this assay, beta PDGFR-HFc half-maximally bound to PDGF BB with an affinity of around 150 pM. Suramin, as well as bacterially expressed and refolded human alpha PDGFR domains 1-3, inhibited beta PDGFR-HFc binding to PDGF BB half-maximally at 25 microM and 10 nM respectively. Therefore, alpha PDGFR D1-3, like beta PDGFR D1-3, are sufficient for high affinity PDGF BB binding. Furthermore, the beta PDGFR-HFc immunosorbent assay will be useful to identify beta PDGFR antagonists as well as to study alpha and beta PDGFR substitution mutants which further map receptor binding determinants.

3T3 Cells↗

PubMed1994

Structural coincidence of alpha PDGFR epitopes binding to platelet-derived growth factor-AA and a potent neutralizing monoclonal antibody.

We have generated two groups of deletion mutants of the alpha PDGFR and one group of chimeras between alpha PDGFR and beta PDGFR to further investigate the structural requirements of the alpha PDGFR for binding to platelet-derived growth factor (PDGF)-AA and to a monoclonal antibody against alpha PDGFR (designated as mAb-alpha R1). The mAb-alpha R1 has recently been reported to block high affinity binding of PDGF-AA to alpha PDGFR. The first group of mutants were carboxyl-terminal deletion mutants encoding the first two immunoglobulin (Ig)-like domains (alpha R1-216), the first four Ig-like domains (alpha R1-415), or all five Ig-like domains (alpha R1-530) of the alpha PDGFR. Since these mutants lacked transmembrane domains, their expression in NIH/3T3 cells resulted in secretion of the truncated alpha PDGFRs. Using conditioned medium from NIH/3T3 transfectants, we showed that mAb-alpha R1 was able to immunoprecipitate each of these secreted form of alpha PDGFRs, suggesting that the epitope recognized by mAb-alpha R1 is located within Ig-like domains 1 and 2 of the alpha PDGFR. Furthermore, PDGF-AA exhibited detectable binding to alpha R1-415 or alpha R1-530 but failed to interact with alpha R1-216, suggesting that the first two Ig-like domains of the alpha PDGFR are not sufficient for PDGF-AA binding. The second group of alpha PDGFR mutants were internal deletion mutants lacking Ig-like loop 1 (alpha R delta 49-100), Ig-like loop 2 (alpha R delta 150-189), Ig-like loop 3 (alpha R delta 235-290), or part of Ig-like loops 4 and 5 (alpha R delta 375-450). The internal deletion mutants were transfected into 32D cells which lack both alpha PDGFR and beta PDGFR. PDGF-AA bound with high affinity to 32D cells expressing alpha R delta 375-450 but not to 32D cells expressing the other three internal deletion mutants, suggesting that the region required for PDGF-AA binding should be within the first three Ig-like domains of the alpha PDGFR. In addition, mAb-alpha R1 bound to 32D cells expressing alpha R delta 235-290 but failed to bind 32D cells transfected with alpha R delta 49-100 or alpha R delta 150-189.(ABSTRACT TRUNCATED AT 400 WORDS)

3T3 Cells↗

PubMed1994

Comparison of calcium-dependent conformational changes in the N-terminal SH2 domains of p85 and GAP defines distinct properties for SH2 domains.

Src-homology region 2 (SH2) domains are stretches of about 100 amino acids which are found to be structurally conserved in a number of signaling molecules. These regions have been shown to bind with high affinity to phosphotyrosine residues within activated receptor tyrosine kinases. Here we report the bacterial expression and purification of individual N-terminal SH2 (NSH2) domains of phosphatidylinositol 3-kinase (PI-3K) binding subunit (p85) and Ras GTPase activating protein (GAP) in amounts suitable for structure-function studies. The p85NSH2 domain stains dark purple and absorbs around 620-640 nm with Stains-all, a dye known to bind to calcium binding proteins. This effect was not observed for the GAPNSH2 domain. Circular dichroism analysis of the N-terminal SH2 domain of these proteins shows that p85NSH2, but not GAPNSH2, undergoes a significant dose-dependent change in conformation in the presence of increasing calcium concentrations. Moreover, the conformational change of p85NSH2 induced by calcium could be replicated by addition of a phosphorylated hexapeptide (DYpMDMK) representing the alpha-PDGFR binding site for p85. Limited proteolysis studies showed a significant calcium-dependent increase in protection of p85NSH2 but not GAPNSH2 from degradation by subtilisin. Our results further indicate that holmium, a trivalent lanthanide ion, which has been previously shown to substitute for calcium, could also protect the p85NSH2 domain from proteolysis even at 10-fold lower concentrations. In vitro binding studies using purified preparations of activated alpha-PDGFR show that calcium did not affect the binding of GAPNSH2 domains to activated alpha-PDGFR.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Biological function of PDGF-induced PI-3 kinase activity: its role in alpha PDGF receptor-mediated mitogenic signaling.

The tyrosine phosphorylation sites in the human alpha PDGF receptor (alpha PDGFR) required for association with PI-3 kinase have been identified as tyrosines 731 and 742. Mutation of either tyrosine substantially reduced PDGF-induced PI-3 kinase activity but did not impair the receptor-mediated mitogenic response. We sought to determine whether PDGF-induced PI-3 kinase activity could be further ablated so as to exclude a low threshold requirement for PDGFR signal transduction. Thus, we mutated both tyrosine 731 and 742 and expressed the double mutant (Y731F/Y742F) in 32D hematopoietic cells. In such transfectants, PDGF induced no detectable receptor-associated or anti-P-Tyr recoverable PI-3 kinase activity. Under the same conditions, neither mobility shift of raf-1 nor tyrosine phosphorylation of either PLC gamma or MAP kinase was impaired. 32D transfectants expressing the double mutant showed wild-type alpha PDGFR levels of mitogenic and chemotactic responses to PDGF. To examine the effect of the double mutation in cells that normally respond to PDGF, we generated chimeras in which the cytoplasmic domains of wild-type alpha PDGFR, Y731F, and Y731F/Y742F were linked to the extracellular domain of colony-stimulating factor-1 (CSF-1) receptor (fms). After introduction of the chimeric receptors into mouse NIH/3T3 fibroblasts, the ability of CSF-1 to stimulate growth of these transfectants was examined. Our data show that all these chimeric receptors exhibited similar abilities to mediate CSF-1-stimulated cell growth. These findings lead us to conclude that PDGF-induced PI-3 kinase activity is not required for PDGF-stimulated mitogenic pathway in both NIH/3T3 fibroblasts and 32D hematopoietic cells.

3T3 Cells↗

PubMed1991

Structure of oligomeric beta B2-crystallin: an application of the T2 translation function to an asymmetric unit containing two dimers.

The molecular structure of the main subunit of the beta-crystallins, components of the vertebrate eye lens, has recently been solved by molecular replacement at 2.1 A resolution [Bax, Lapatto, Nalini, Driessen, Lindley, Mahadevan, Blundell & Slingsby (1990). Nature (London), 347, 776-780]. The protein, beta B2, is a dimer in solution, but a tetramer in the crystal with one subunit in the asymmetric unit of space group I222. Using the crystallographic dimer from this I-centred form the structure of a C222 crystal form of the beta B2 protein with four subunits in the asymmetric unit has now been solved by molecular replacement at 3.3 A. The solution involved the use of a new translation function for non-crystallographic symmetry, based on the T2 function of Crowther & Blow [Acta Cryst. (1967), 23, 544-548].

Crystallins↗

PubMed1991

Molecular model-building of amylin and alpha-calcitonin gene-related polypeptide hormones using a combination of knowledge sources.

Amylin is the major component of the amyloid found in the pancreases of noninsulin-dependent diabetics (type 2 diabetes). It is a 37 amino acid polypeptide and has been shown to have 46% sequence identity with the neuropeptide alpha-calcitonin gene-related peptide (alpha-CGRP). Both amylin and alpha-CGRP are known to be potent inhibitors of glycogen synthesis in stripped rat soleus muscle. Secondary structure prediction and tertiary structure model-building show the two polypeptides to have an alpha-helix/beta-strand motif similar to that observed in the insulin B-chain. The results have been supported by CD spectroscopy, although there is no sequence similarity between insulin and amylin/alpha-CGRP. Aggregation states have been predicted based on the dimeric and hexameric arrangements seen in porcine insulin. Rat and hamster amylin have a changed sequence motif in the beta-strand which results in lack of amyloid formation and type 2 diabetes. This, we propose, is caused by disruption of hydrogen bonding which prevents the formation of the dimer.

Algorithms↗

PubMed1990

X-ray analysis of beta B2-crystallin and evolution of oligomeric lens proteins.

The beta, gamma-crystallins form a class of homologous proteins in the eye lens. Each gamma-crystallin comprises four topologically equivalent, Greek key motifs; pairs of motifs are organized around a local dyad to give domains and two similar domains are in turn related by a further local dyad. Sequence comparisons and model building predicted that hetero-oligomeric beta-crystallins also had internally quadruplicated subunits, but with extensions at the N and C termini, indicating that beta, gamma-crystallins evolved in two duplication steps from an ancestral protein folded as a Greek key. We report here the X-ray analysis at 2.1 A resolution of beta B2-crystallin homodimer which shows that the connecting peptide is extended and the two domains separated in a way quite unlike gamma-crystallin. Domain interactions analogous to those within monomeric gamma-crystallin are intermolecular and related by a crystallographic dyad in the beta B2-crystallin dimer. This shows how oligomers can evolve by conserving an interface rather than connectivity. A further interaction between dimers suggests a model for more complex aggregates of beta-crystallin in the lens.

Amino Acid Sequence↗

PubMed1990

Prediction of domain organisation and secondary structure of thyroid peroxidase, a human autoantigen involved in destructive thyroiditis.

Organ specific autoimmune diseases are relatively common immunological disorders in man which include thyroid autoimmune disease, insulin-dependent diabetes mellitus and myasthenia gravis. The target autoantigens in some of these diseases have recently been characterised. In thyroid autoimmune disease this includes the key enzyme, thyroid peroxidase (TPO), which is involved in the generation of thyroid hormone. Structural knowledge about autoantigens such as thyroid peroxidase will allow a greater understanding of the interaction between autoantigens and the aberrant immune response, and facilitate the development of strategies for antigen-specific therapeutic manipulation. We report here a prediction of the secondary structure of thyroid peroxidase, together with the results of circular dichroic spectroscopy of a homologous purified enzyme. A combination of 3 secondary structure prediction programs has been used, following multiple sequence alignment, and TPO has been found to consist mainly of alpha-helical conformation, with little beta-sheet present. This structure prediction, together with knowledge of the exon-intron boundaries allows a model for the domain organisation of the TPO molecule to be proposed.

Amino Acid Sequence↗