Crystal Structure Of Alpha Research Articles

A novel Phe171Cys mutation in integrin αIIb causes Glanzmann thrombasthenia by abrogating αIIbβ3 complex formation

Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by lack of platelet aggregation induced by most agonists. The disease is caused by mutations in either alpha(IIb)[glycoprotein (GP) IIb] or beta(3) (GPIIIa) genes that lead to a lack or dysfunction of the integrin alpha(IIb)beta(3) which serves as a fibrinogen receptor. Mucocutaneous bleeding manifestations and platelet dysfunction consistent with GT were observed in three members of a Cypriot family: a 3-year-old proband, her father and her paternal uncle. To determine the molecular basis of GT in this family and to characterize possible biochemical and structural defects. Analysis of the patients' platelets by fluorescence-activated cell sorting demonstrated trace amounts of beta(3), no alpha(IIb) and no alpha(IIb)beta(3) on the membrane. Sequence analysis revealed a novel T607G transversion in exon 5 of the alpha(IIb) gene predicting a Phe171Cys alteration that created a PstI recognition site. All three patients were homozygous for the mutation, the mother and paternal grandparents of the proband were heterozygous, whereas 110 healthy subjects lacked this transversion. Chinese hamster ovary cells cotransfected with cDNAs of mutated alpha(IIb) and wild-type beta(3) failed to express alpha(IIb)beta(3) as shown by immunoprecipitation and immunohistochemistry experiments. Structural analysis of the alpha(IIb)beta(3) model, which was based on the crystal structure of alpha(v)beta(3), indicated that Phe171 plays an essential role in the interface between the beta-propeller domain of alpha(IIb) and the betaA domain of beta(3). A novel Phe171Cys mutation in the alpha(IIb) gene of patients with GT is associated with abrogation of alpha(IIb)beta(3) complex formation.

Molecular model of the alpha(IIb)beta(3) integrin.

A molecular model of the alpha(IIb)beta(3) integrin has been developed utilizing (i). the crystal structure of alpha(v)beta(3), (ii). homology model of the alpha(IIb) subdomain, and (iii). the docking of alpha(IIb)beta(3)/alpha(v)beta(3) dual and selective inhibitors into the putative binding sites of alpha(IIb)beta(3) and alpha(v)beta(3). Since the binding sites of these integrins are located at the interface between the two heads of the individual subunits, only the alpha(IIb)beta(3) head region is modeled. The 3D conformations of two loops in alpha(IIb), whose residues have been implicated in non-peptide ligand binding, could not be determined from homology with alpha(v) alone. Mutagenesis data and the modeling of small ligand binding contributed to the rational design of these loop conformations. The final energy minimized loop conformations exhibit permissible phi/psi angles and contribute to a binding site model of alpha(IIb)beta(3) that is consistent with both the known mutagenesis studies and in-house structure-activity relationships. The charged residues alpha(IIb):E117 and beta(3):R214 are found to dominate the ligand-protein binding interaction. The previously identified "exosite" is also identified as a hydrogen bond, hydrophobic or pi-pi interaction with Y190, similar to the recently proposed binding model of alpha(v)beta(3).

Electrochemical synthesis and structural and physical characterization of one- and two-electron-reduced forms of [SMo12O40]2-.

Isolation of a soluble [NHex4]+ salt has allowed a detailed electrochemical study of the anion alpha-[SMo12O40]2- to be undertaken. Four reversible one-electron-reduction processes are observed in CH2Cl2 solution. Controlled potential electrolysis led to isolation of tetraalkylammonium salts of the one-electron-reduced anion alpha-[SMo12O40]3- and the two-electron-reduced anion alpha-[SMo12O40].4- [SMo12O40]3- is stable to disproportionation in dry solvents (Kdis = 10(-7.4). EPR and magnetic susceptibility data indicate that [SMo12O40]3- is a simple paramagnet (S = 1/2) while [SMo12O40]4- is paramagnetic with the mu eff values decreasing at low temperatures. Solutions of the two-electron-reduced species are EPR silent, but microcrystalline powders show very weak signals. The crystal structure of alpha-[NBu4]3[SMo12O40] has been determined (triclinic P1; a = 13.840(3) A; b = 15.587(4) A; c = 19.370(3) A; alpha = 94.82(2) degrees; beta = 93.10(1) degrees; gamma = 91.05(2) degrees; Z = 2). There is disorder around the C2 axis of the central SO4(2-) tetrahedron. In the presence of aqueous HClO4 (0.045 M) in thf/H2O or MeCN/H2O (98/2 v/v), [SMo12O40]2- exhibits five two-electron-reduction processes. Under these conditions, [SMo12O40]3- protonates and disproportionates into [SMo12O40]2- and the (2e-, 2H+)-reduced anion [H2SMo12O40]2-.

Altered DNA recognition and bending by insertions in the alpha 2 tail of the yeast a1/alpha 2 homeodomain heterodimer.

The yeast MAT alpha 2 and MATa1 homeodomain proteins bind cooperatively as a heterodimer to sites upstream of haploid-specific genes, repressing their transcription. In the crystal structure of alpha 2 and a1 bound to DNA, each homeodomain makes independent base-specific contacts with the DNA and the two proteins contact each other through an extended tail region of alpha 2 that tethers the two homeodomains to one another. Because this extended region may be flexible, the ability of the heterodimer to discriminate among DNA sites with altered spacing between alpha 2 and a1 binding sites was examined. Spacing between the half sites was critical for specific DNA binding and transcriptional repression by the complex. However, amino acid insertions in the tail region of alpha 2 suppressed the effect of altering an a1/alpha 2 site by increasing the spacing between the half sites. Insertions in the tail also decreased DNA bending by a1/alpha 2. Thus tethering the two homeodomains contributes to DNA bending by a1/alpha 2, but the precise nature of the resulting bend is not essential for repression.

Hydrophobic cluster analysis (HCA) of the hormone-binding domain of receptor proteins

A new technique of protein sequence analysis, namely, Hydrophobic Cluster Analysis (HCA), has been used to align and compare the sequences of proteins belonging to the receptor superfamily (steroid, thyroid hormone and retinoic acid receptors) and serpin superfamily (corticosteroid binding globulin (CBG) and alpha 1-antitrypsin (alpha 1-AT]. By matching up clusters of hydrophobic amino-acids that oftenmost correspond to identifiable secondary structures (alpha-helices, beta-strands etc.), it has been possible to deduce the following information on the secondary structures of these proteins: CBG is structurally related to alpha 1-AT (HCA score greater than 80%), the structures of the hormone-binding domains of the steroid receptors that bind 3-keto-delta 4-steroids are closely interrelated (greater than 80%) but less closely related to that of the estrogen receptor (ER) (approximately 75%), vitamin D, retinoic acid and thyroid hormone receptors are structurally closely related (greater than or equal to 80%). Their secondary structures are, however, also related to that of the steroid receptors (approximately 70%), and a high degree of analogy exists between the structures of serpins and of the hormone-binding domains of members of the steroid superfamily (60-70%). HCA has clearly shown that a previous local sequence alignment of the estrogen receptor with other steroid receptors and cytochromes P450 has to be reconsidered. The published consensus steroid binding sequence previously identified in cytochromes is in fact 80 amino-acids upstream from its previously defined position. Other regions of contiguous sequence identity have also been identified which may be involved in the hydrophobic core of the protein or in steroid binding. Their positions have been indicated using the crystal structure of alpha 1-AT as a model.