Sequence-selective carbohydrate-DNA interaction: dimeric and monomeric forms of the calicheamicin oligosaccharide interfere with transcription factor function.

The synthetic oligosaccharide moiety of the antibiotic calicheamicin and the head-to-head dimer of this oligosaccharide are known to bind to the minor groove of DNA in a sequence-selective manner preferring distinct target sequences. We tested these carbohydrates for their ability to interfere with transcription factor function. The oligosaccharides inhibit binding of transcription factors to DNA in a sequence-selective manner, probably by inducing a conformational change in DNA structure. They also interfere with transcription by polymerase II in vitro. The effective concentrations of the oligosaccharides for inhibition of transcription factor binding and for transcriptional inhibition are in the micromolar range. The dimer is a significantly more active inhibitor than is the monomer.

Identification, purification, and molecular cloning of autonomously replicating sequence-binding protein 1 from fission yeast Schizosaccharomyces pombe.

Autonomously replicating sequence (ARS) elements of the fission yeast Schizosaccharomyces pombe contain multiple imperfect copies of the consensus sequence reported by Maundrell et al. [Maundrell K., Hutchison, A. & Shall, S. (1988) EMBO J. 7, 2203-2209]. When cell free extracts of S. pombe were incubated with a dimer or tetramer of an oligonucleotide containing the ARS consensus sequence, several complexes were detected using a gel mobility-shift assay. The proteins forming these complexes also bind ars3002, which is the most active origin in the ura4 region of chromosome III of S. pombe. One protein, partly responsible for the binding activity observed with crude extracts, was purified to near homogeneity. It is a 60-kDa protein and was named ARS-binding protein 1 (Abp1). Abp1 preferentially binds to multiple sites in ARS 3002 and to the DNA polymer poly[d(A.T)]. The cloning and sequence of the gene coding for Abp1 revealed that it encodes a protein of 59.8 kDa (522 amino acids). Abp1 has significant homology (25% identity, 50% similarity) to the N-terminal region (approximately 300 amino acids) of the human and mouse centromere DNA-binding protein CENP-B. Because centromeres of S. pombe contain a high density of ARS elements, Abp1 may play a role connecting DNA replication and chromosome segregation.

Inducible nitric oxide synthase: identification of amino acid residues essential for dimerization and binding of tetrahydrobiopterin.

Nitric oxide synthases (NOSs) require tetrahydrobiopterin (BH4) for dimerization and NO production. Mutation analysis of mouse inducible NOS (iNOS; NOS2) identified Gly-450 and Ala-453 as critical for NO production, dimer formation, and BH4 binding. Substitutions at five neighboring positions were tolerated, and normal binding of heme, calmodulin, and NADPH militated against major distortions affecting the NH2-terminal portion, midzone, or COOH terminus of the inactive mutants. Direct involvement of residues 450 and 453 in the binding of BH4 is supported by the striking homology of residues 448-480 to a region extensively shared by the three BH4-utilizing aromatic amino acid hydroxylases and is consistent with the conservation of these residues among all 10 reported NOS sequences, including mammalian NOSs 1, 2, and 3, as well as avian and insect NOSs. Altered binding of BH4 and/or L-arginine may explain how the addition of a single methyl group to the side chain of residue 450 or the addition of three methylenes to residue 453 can each abolish an enzymatic activity that reflects the concerted function of 1143 other residues.

Effects of receptor dimerization on the interaction between the class I major histocompatibility complex-related Fc receptor and IgG.

The neonatal Fc receptor (FcRn) transports maternal IgG from ingested milk in the gut to the bloodstream of newborn mammals. An FcRn dimer was observed in crystals of the receptor alone and of an FcRn-Fc complex, but its biological relevance was unknown. Here we use surface plasmon resonance-based biosensor assays to assess the role of FcRn dimerization in IgG binding. We find high-affinity IgG binding when FcRn is immobilized on a biosensor chip in an orientation facilitating dimerization but not when its orientation disrupts dimerization. This result supports a model in which IgG-induced dimerization of FcRn is relevant for signaling the cell to initiate endocytosis of the IgG-FcRn complex.

Crystal structure of the cell cycle-regulatory protein suc1 reveals a beta-hinge conformational switch.

The Schizosaccharomyces pombe cell cycle-regulatory protein suc1, named as the suppressor of cdc2 temperature-sensitive mutations, is essential for cell cycle progression. To understand suc1 structure-function relationships and to help resolve conflicting interpretations of suc1 function based on genetic studies of suc1 and its functional homologs in both lower and higher eukaryotes, we have determined the crystal structure of the beta-interchanged suc1 dimer. Each domain consists of three alpha-helices and a four-stranded beta-sheet, completed by the interchange of terminal beta-strands between the two subunits. This beta-interchanged suc1 dimer, when compared with the beta-hairpin single-domain folds of suc1, reveals a beta-hinge motif formed by the conserved amino acid sequence HVPEPH. This beta-hinge mediates the subunit conformation and assembly of suc1: closing produces the intrasubunit beta-hairpin and single-domain fold, whereas opening leads to the intersubunit beta-strand interchange and interlocked dimer assembly reported here. This conformational switch markedly changes the surface accessibility of sequence-conserved residues available for recognition of cyclin-dependent kinase, suggesting a structural mechanism for beta-hinge-mediated regulation of suc1 biological function. Thus, suc1 belongs to the family of domain-swapping proteins, consisting of intertwined and dimeric protein structures in which the dual assembly modes regulate their function.

Tertiary structure of an amyloid immunoglobulin light chain protein: a proposed model for amyloid fibril formation.

An immunoglobulin light chain protein was isolated from the urine of an individual (BRE) with systemic amyloidosis. Complete amino acid sequence of the variable region of the light chain (VL) protein established it as a kappa I, which when compared with other kappa I amyloid associated proteins had unique residues, including Ile-34, Leu-40, and Tyr-71. To study the tertiary structure, BRE VL was expressed in Escherichia coli by using a PCR product amplified from the patient BRE's bone marrow DNA. The PCR product was ligated into pCZ11, a thermal-inducible replication vector. Recombinant BRE VL was isolated, purified to homogeneity, and crystallized by using ammonium sulfate as the precipitant. Two crystal forms were obtained. In crystal form I the BRE VL kappa domain crystallizes as a dimer with unit cell constants isomorphous to previously published kappa protein structures. Comparison with a nonamyloid VL kappa domain from patient REI, identified significant differences in position of residues in the hypervariable segments plus variations in framework region (FR) segments 40-46 (FR2) and 66-67 (FR3). In addition, positional differences can be seen along the two types of local diads, corresponding to the monomer-monomer and dimer-dimer interfaces. From the packing diagram, a model for the amyloid light chain (AL) fibril is proposed based on a pseudohexagonal spiral structure with a rise of approximately the width of two dimers per 360 degree turn. This spiral structure could be consistent with the dimensions of amyloid fibrils as determined by electron microscopy.

Retinoid X receptor alpha forms tetramers in solution.

Protein-protein interactions allow the retinoid X receptor (RXR) to bind to cognate DNA as a homo- or a heterodimer and to participate in mediating the effects of a variety of hormones on gene transcription. Here we report a systematic study of the oligomeric state of RXR in the absence of a DNA template. We have used electrophoresis under nondenaturing conditions and chemical crosslinking to show that in solution, RXR alpha forms homodimers as well as homotetramers. The dissociation constants governing dimer and tetramer formation were estimated by fluorescence anisotropy studies. The results indicate that RXR tetramers are formed with a high affinity and that at protein concentrations higher than about 70 nM, tetramers will constitute the predominant species. Tetramer formation may provide an additional level of the regulation of gene transcription mediated by RXRs.

A variant of lambda repressor with an altered pattern of cooperative binding to DNA sites.

The bacteriophage lambda repressor binds cooperatively to pairs of adjacent sites in the lambda chromosome, one repressor dimer binding to each site. The repressor's amino domain (that which mediates DNA binding) is connected to its carboxyl domain (that which mediates dimerization and the interaction between dimers) by a protease-sensitive linker region. We have generated a variant lambda repressor that lacks this linker region. We show that dimers of the variant protein are deficient in cooperative binding to sites at certain, but not all, distances. The linker region thus extends the range over which carboxyl domains of DNA-bound dimers can interact. In particular, the linker is required for cooperative binding to a pair of sites as found in the lambda chromosome, and thus is essential for the repressor's physiological function.

Repacking protein cores with backbone freedom: structure prediction for coiled coils.

Progress in homology modeling and protein design has generated considerable interest in methods for predicting side-chain packing in the hydrophobic cores of proteins. Present techniques are not practically useful, however, because they are unable to model protein main-chain flexibility. Parameterization of backbone motions may represent a general and efficient method to incorporate backbone relaxation into such fixed main-chain models. To test this notion, we introduce a method for treating explicitly the backbone motions of alpha-helical bundles based on an algebraic parameterization proposed by Francis Crick in 1953 [Crick, F. H. C. (1953) Acta Crystallogr. 6, 685-689]. Given only the core amino acid sequence, a simple calculation can rapidly reproduce the crystallographic main-chain and core side-chain structures of three coiled coils (one dimer, one trimer, and one tetramer) to within 0.6-A root-mean-square deviations. The speed of the predictive method [approximately 3 min per rotamer choice on a Silicon Graphics (Mountain View, CA) 4D/35 computer] permits it to be used as a design tool.

The CuA center of cytochrome-c oxidase: electronic structure and spectra of models compared to the properties of CuA domains.

The electronic structure and spectrum of several models of the binuclear metal site in soluble CuA domains of cytochrome-c oxidase have been calculated by the use of an extended version of the complete neglect of differential overlap/spectroscopic method. The experimental spectra have two strong transitions of nearly equal intensity around 500 nm and a near-IR transition close to 800 nm. The model that best reproduces these features consists of a dimer of two blue (type 1) copper centers, in which each Cu atom replaces the missing imidazole on the other Cu atom. Thus, both Cu atoms have one cysteine sulfur atom and one imidazole nitrogen atom as ligands, and there are no bridging ligands but a direct Cu-Cu bond. According to the calculations, the two strong bands in the visible region originate from exciton coupling of the dipoles of the two copper monomers, and the near-IR band is a charge-transfer transition between the two Cu atoms. The known amino acid sequence has been used to construct a molecular model of the CuA site by the use of a template and energy minimization. In this model, the two ligand cysteine residues are in one turn of an alpha-helix, whereas one ligand histidine is in a loop following this helix and the other one is in a beta-strand.

One sequence, two folds: a metastable structure of CD2.

When expressed as part of a glutathione S-transferase fusion protein the NH2-terminal domain of the lymphocyte cell adhesion molecule CD2 is shown to adopt two different folds. The immunoglobulin superfamily structure of the major (85%) monomeric component has previously been determined by both x-ray crystallography and NMR spectroscopy. We now describe the structure of a second, dimeric, form present in about 15% of recombinant CD2 molecules. After denaturation and refolding in the absence of the fusion partner, dimeric CD2 is converted to monomer, illustrating that the dimeric form represents a metastable folded state. The crystal structure of this dimeric form, refined to 2.0-A resolution, reveals two domains with overall similarity to the IgSF fold found in the monomer. However, in the dimer each domain is formed by the intercalation of two polypeptide chains. Hence each domain represents a distinct folding unit that can assemble in two different ways. In the dimer the two domains fold around a hydrophilic interface believed to mimic the cell adhesion interaction at the cell surface, and the formation of dimer can be regulated by mutating single residues at this interface. This unusual misfolded form of the protein, which appears to result from inter- rather than intramolecular interactions being favored by an intermediate structure formed during the folding process, illustrates that evolution of protein oligomers is possible from the sequence for a single protein domain.

Identification of four acidic amino acids that constitute the catalytic center of the RuvC Holliday junction resolvase.

Escherichia coli RuvC protein is a specific endonuclease that resolves Holliday junctions during homologous recombination. Since the endonucleolytic activity of RuvC requires a divalent cation and since 3 or 4 acidic residues constitute the catalytic centers of several nucleases that require a divalent cation for the catalytic activity, we examined whether any of the acidic residues of RuvC were required for the nucleolytic activity. By site-directed mutagenesis, we constructed a series of ruvC mutant genes with similar amino acid replacements in 1 of the 13 acidic residues. Among them, the mutant genes with an alteration at Asp-7, Glu-66, Asp-138, or Asp-141 could not complement UV sensitivity of a ruvC deletion strain, and the multicopy mutant genes showed a dominant negative phenotype when introduced into a wild-type strain. The products of these mutant genes were purified and their biochemical properties were studied. All of them retained the ability to form a dimer and to bind specifically to a synthetic Holliday junction. However, they showed no, or extremely reduced, endonuclease activity specific for the junction. These 4 acidic residues, which are dispersed in the primary sequence, are located in close proximity at the bottom of the putative DNA binding cleft in the three-dimensional structure. From these results, we propose that these 4 acidic residues constitute the catalytic center for the Holliday junction resolvase and that some of them play a role in coordinating a divalent metal ion in the active center.

An antiestrogen: a phosphotyrosyl peptide that blocks dimerization of the human estrogen receptor.

We have previously identified tyrosine-537 as a constitutively phosphorylated site on the human estrogen receptor (hER). A 12-amino acid phosphotyrosyl peptide containing a selected sequence surrounding tyrosine-537 was used to investigate the function of phosphotyrosine-537. The phosphotyrosyl peptide completely blocked the binding of the hER to an estrogen response element (ERE) in a gel mobility shift assay. Neither the nonphosphorylated tyrosyl peptide nor an unrelated phosphotyrosyl peptide previously shown to inhibit the signal transducers and activators of transcription factor (STAT) blocked binding of the hER to the ERE. The hER phosphotyrosyl peptide was shown by molecular sizing chromatography to dissociate the hER dimer into monomers. The hER specifically bound the 32P-labeled phosphotyrosyl peptide, indicating that the inhibition of ERE binding was caused by the phosphotyrosyl peptide binding directly to the hER and blocking dimerization. These data suggest that the phosphorylation of tyrosine-537 is a necessary step for the formation of the hER dimer. In addition, we propose that the dimerization of the hER occurs by a previously unrecognized Src homology 2 domain (SH2)-like phosphotyrosyl coupling mechanism. Consequently, the phosphotyrosyl peptide represents a class of antagonists that inhibits estrogen action by a mechanism other than interacting with the receptor's hormone binding site.

Transition metal ion activation of DNA binding by the diphtheria tox repressor requires the formation of stable homodimers.

The diphtheria tox repressor (DtxR) is a transition metal ion-dependent regulatory element that controls the expression of diphtheria toxin and several genes involved in the synthesis of siderophores in Corynebacterium diphtheriae. In the presence of transition metal ions apo-DtxR becomes activated and specifically binds to its target DNA sequences. We demonstrate by glutaraldehyde cross-linking that monomeric apo-DtxR is in weak equilibrium with a dimeric form and that upon addition of activating metal ions to the reaction mixture a dimeric complex is stabilized. Addition of the DNA-binding-defective mutant apo-DtxR(delta 1-47) to apo-DtxR in the absence of transition metal ions inhibits conversion of the apo-repressor to its activated DNA-binding form. We also show that the binding of Ni2+ to both apo-DtxR and apo-DtxR(delta 1-47) is cooperative and that upon ion binding there is a conformational change in the environment of the indole ring moiety of Trp-104. For the wild-type repressor the consequences of this conformational change include a shift in equilibrium toward dimer formation and activation of target DNA binding by the repressor. We conclude that the formation of DtxR homodimers is mediated through a protein-protein interaction domain that is also activated on metal ion binding.

Function of the p86 subunit of eukaryotic initiation factor (iso)4F as a microtubule-associated protein in plant cells.

The isozyme form of eukaryotic initiation factor 4F [eIF-(iso)4F] from wheat germ is composed of a p28 subunit that binds the 7-methylguanine cap of mRNA and a p86 subunit having unknown function. The p86 subunit was found to have limited sequence similarity to a kinesin-like protein encoded by the katA gene of Arabidopsis thaliana. Native wheat germ eIF-(iso)4F and bacterially expressed p86 subunit and p86-p28 complex bound to taxol-stabilized maize microtubules (MTs) in vitro. Binding saturation occurred at 1 mol of p86 per 5-6 mol of polymerized tubulin dimer, demonstrating a substoichiometric interaction of p86 with MTs. No evidence was found for a direct interaction of the p28 subunit with MTs. Unlike kinesin, cosedimentation of eIF-(iso)4F with MTs was neither reduced by MgATP nor enhanced by adenosine 5'-[gamma-imido]triphosphate. Both p86 subunit and p86-p28 complex induced the bundling of MTs in vitro. The p86 subunit was immunolocalized to the cytosol in root maize cells and existed in three forms: fine particles, coarse particles, and linear patches. Many coarse particles and linear patches were colocalized or closely associated with cortical MT bundles in interphase cells. The results indicate that the p86 subunit of eIF-(iso)4F is a MT-associated protein that may simultaneously link the translational machinery to the cytoskeleton and regulate MT disposition in plant cells.