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C O Pabo

Publications and source records attributed to C O Pabo.

At least 19 recordsLinked to original sources
PubMed1992

Refined 1.8 A crystal structure of the lambda repressor-operator complex.

The crystal structure of the lambda repressor-operator complex has been refined to an R-factor of 18.9% at 1.8 A resolution. This refinement, using data collected at low temperature, has revealed the structure of the N-terminal arm and shows that the interactions of repressor with the two halves of the pseudo-symmetric operator site are significantly different. The two halves of the complex are most similar near the outer edge of the operator site (in a region where the lambda and 434 repressors make similar contacts), but they become increasingly different toward the center of the operator. There are striking differences near the center of the site where it appears that the arm makes significant contacts to only one half of the DNA site. This suggested a new way of aligning the operator sites in phage lambda. The high resolution structure confirms many of the previously noted features of the complex, but also reveals a number of new protein-DNA contacts. It also gives a better view of the extensive H-bonding networks that couple contacts made by different residues and different regions of the protein, and reveals important new details about the helix-turn-helix (HTH) region, and the positions of many water molecules in the complex.

Bacteriophage lambda↗
PubMed1991

Crystal structure of a MAT alpha 2 homeodomain-operator complex suggests a general model for homeodomain-DNA interactions.

The MAT alpha 2 homeodomain regulates the expression of cell type-specific genes in yeast. We have determined the 2.7 A resolution crystal structure of the alpha 2 homeodomain bound to a biologically relevant DNA sequence. The DNA in this complex is contacted primarily by the third of three alpha-helices, with additional contacts coming from an N-terminal arm. Comparison of the yeast alpha 2 and the Drosophila engrailed homeodomain-DNA complexes shows that the protein fold is highly conserved, despite a 3-residue insertion in alpha 2 and only 27% sequence identity between the two homeodomains. Moreover, the orientation of the recognition helix on the DNA is also conserved. This docking arrangement is maintained by side chain contacts with the DNA--primarily the sugar-phosphate backbone--that are identical in alpha 2 and engrailed. Since these residues are conserved among all homeodomains, we propose that the contacts with the DNA are also conserved and suggest a general model for homeodomain-DNA interactions.

Base Sequence↗
PubMed1991

The DNA binding arm of lambda repressor: critical contacts from a flexible region.

Segments of protein that do not adopt a well-ordered conformation in the absence of DNA can still contribute to site-specific recognition of DNA. The first six residues (NH2-Ser1-Thr2-Lys3-Lys4-Lys5-Pro6-) of phage lambda repressor are flexible but are important for site-specific binding. Low-temperature x-ray crystallography and codondirected saturation mutagenesis were used to study the role of this segment. All of the functional sequences have the form [X]1-[X]2-[Lys or Arg]3-[Lys]4-[Lys or Arg]5-[X]6. A high-resolution (1.8 angstrom) crystal structure shows that Lys3 and Lys4 each make multiple hydrogen bonds with guanines and that Lys5 interacts with the phosphate backbone. The symmetry of the complex breaks down near the center of the site, and these results suggest a revision in the traditional alignment of the six lambda operator sites.

Amino Acid Sequence↗
PubMed1991

Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A.

The zinc finger DNA-binding motif occurs in many proteins that regulate eukaryotic gene expression. The crystal structure of a complex containing the three zinc fingers from Zif268 (a mouse immediate early protein) and a consensus DNA-binding site has been determined at 2.1 angstroms resolution and refined to a crystallographic R factor of 18.2 percent. In this complex, the zinc fingers bind in the major groove of B-DNA and wrap part way around the double helix. Each finger has a similar relation to the DNA and makes its primary contacts in a three-base pair subsite. Residues from the amino-terminal portion of an alpha helix contact the bases, and most of the contracts are made with the guanine-rich strand of the DNA. This structure provides a framework for understanding how zinc fingers recognize DNA and suggests that this motif may provide a useful basis for the design of novel DNA-binding proteins.

Amino Acid Sequence↗
PubMed1991

Crystallization and preliminary X-ray diffraction studies of a MAT alpha 2-DNA complex.

Crystals have been obtained of the DNA-binding domain of the yeast MAT alpha 2 repressor bound to a 21 base-pair DNA site. The crystals are grown from polyethylene glycol and CaCl2 and form in space group P2(1) with a = 60.1 A, b = 39.4 A, c = 68.7 A and beta = 98 degrees. They diffract to 2.9 A resolution and contain one protein-DNA complex in the crystallographic asymmetric unit.

Base Sequence↗
PubMed1990

Crystal structure of an engrailed homeodomain-DNA complex at 2.8 A resolution: a framework for understanding homeodomain-DNA interactions.

The crystal structure of a complex containing the engrailed homeodomain and a duplex DNA site has been determined at 2.8 A resolution and refined to a crystallographic R factor of 24.4%. In this complex, two separate regions of the 61 amino acid polypeptide contact a TAAT subsite. An N-terminal arm fits into the minor groove, and the side chains of Arg-3 and Arg-5 make contacts near the 5' end of this "core consensus" binding site. An alpha helix fits into the major groove, and the side chains of IIe-47 and Asn-51 contact base pairs near the 3' end of the TAAT site. This "recognition helix" is part of a structurally conserved helix-turn-helix unit, but these helices are longer than the corresponding helices in the lambda repressor, and the relationship between the helix-turn-helix unit and the DNA is significantly different.

Amino Acid Sequence↗
PubMed1990

Crystallization and preliminary X-ray diffraction studies of the engrailed homeodomain and of an engrailed homeodomain/DNA complex.

The homeodomain from the engrailed protein of Drosophila has been crystallized from ammonium phosphate at pH 6.8. The crystals form in space group P6(1)22 (or P6(5)22), with cell dimensions a = b = 44.8 A and c = 118.2 A. These crystals diffract to 1.8 A resolution. A complex containing the engrailed homeodomain and a duplex DNA site also has been crystallized. The cocrystals form in space group C2 with a = 131.2 A, b = 45.5 A, c = 72.9 A and beta = 119.0 degrees. These crystals diffract to 2.6 A resolution.

Animals↗
PubMed1990

Conserved residues make similar contacts in two repressor-operator complexes.

Comparison of a lambda repressor-operator complex and a 434 repressor-operator complex reveals that three conserved residues in the helix-turn-helix (HTH) region make similar contacts in each of the crystallographically determined structures. These conserved residues and their interactions with phosphodiester oxygens help establish a frame of reference within which other HTH residues make contacts that are critical for site-specific recognition. Such "positioning contacts" may be important conserved features within families of HTH proteins. In contrast, the structural comparisons appear to rule out any simple "recognition code" at the level of detailed side chain-base pair interactions.

Amino Acid Sequence↗
PubMed1990

Identification of protein folds: matching hydrophobicity patterns of sequence sets with solvent accessibility patterns of known structures.

Hydrophobic side chains often are buried in the interior of a protein, and evolutionarily related proteins usually maintain the hydrophobic character of buried positions. In this paper we show that a pattern of hydrophobicity values derived from a set of related protein sequences is well correlated with the linear pattern of side-chain solvent accessibility values, calculated from a known protein structure representative of the sequences. In several cases, information from aligned sequences can be used to select the correct tertiary fold from a large data base of protein structures.

Algorithms↗
PubMed1988

Cellular uptake of the tat protein from human immunodeficiency virus.

While developing an assay to measure the activity of the tat protein from human immunodeficiency virus 1 (HIV-1), we discovered that the purified protein could be taken up by cells growing in tissue culture and subsequently trans-activate the viral promoter. Trans-activation is dramatically increased by a variety of lysosomotrophic agents. For example, trans-activation can be detected at tat concentrations as low as 1 nM in the presence of chloroquine. Experiments using radioactive protein show that tat becomes localized to the nucleus after uptake and suggest that chloroquine protects tat from proteolytic degradation. These results raise the possibility that, under some conditions, tat might act as a viral growth factor to stimulate viral replication in latently infected cells or alter expression of cellular genes.

Animals↗
PubMed1988

Structure of the lambda complex at 2.5 A resolution: details of the repressor-operator interactions.

The crystal structure of a complex containing the DNA-binding domain of lambda repressor and a lambda operator site was determined at 2.5 A resolution and refined to a crystallographic R factor of 24.2 percent. The complex is stabilized by an extensive network of hydrogen bonds between the protein and the sugar-phosphate backbone. Several side chains form hydrogen bonds with sites in the major groove, and hydrophobic contacts also contribute to the specificity of binding. The overall arrangement of the complex is quite similar to that predicted from earlier modeling studies, which fit the protein dimer against linear B-form DNA. However, the cocrystal structure reveals important side chain-side chain interactions that were not predicted from the modeling or from previous genetic and biochemical studies.

Base Composition↗
PubMed1988

Combining thermostable mutations increases the stability of lambda repressor.

We have combined three mutations previously shown to stabilize lambda repressor against thermal denaturation. Two of these mutations are in helix 3, where Gly-46 and Gly-48 have been replaced by alanines [Hecht, M. H., et al. (1986) Proteins: Struct., Funct., Genet. 1, 43-46]. The other mutation, which replaces Tyr-88 with cysteine, allows the protein to form an intersubunit disulfide bond [Sauer, R. T., et al. (1986) Biochemistry 25, 5992-5998]. Calorimetric measurements show that the two alanine substitutions stabilize repressor by about 8 degrees C, that the disulfide bond stabilizes repressor by about 8 degrees C, and that the triple mutant is 16 degrees C more stable than wild-type repressor.

Amino Acid Sequence↗
PubMed1988

Tat protein from human immunodeficiency virus forms a metal-linked dimer.

Tat, the transactivating protein from HIV, forms a metal-linked dimer with metal ions bridging cysteine-rich regions from each monomer. This novel arrangement is distinct from the "zinc finger" domain observed in other eukaryotic regulatory proteins. Ultraviolet absorption spectra show that Tat binds two Zn2+ or two Cd2+ ions per monomer, and electrophoresis of the Tat-metal complexes demonstrates that the protein forms metal-linked dimers. Partial proteolysis and circular dichroism spectra suggest that metal binding has its primary effects in the cysteine-rich region and relatively little effect on the folding of other regions. These results suggest new directions for biological studies and new approaches to drug design.

Cadmium↗
PubMed1988

Dimerization of the tat protein from human immunodeficiency virus: a cysteine-rich peptide mimics the normal metal-linked dimer interface.

We have synthesized an 18-amino acid peptide that contains the cysteine-rich region of the tat protein from human immunodeficiency virus. Previous experiments in vitro with the intact tat protein have shown that these cysteines serve as metal ligands, causing tat to form metal-linked dimers. Ultraviolet absorption spectra show that the synthetic peptide (tat21-38) binds two Cd2+ or two Zn2+ ions per peptide monomer, and some changes in the circular dichroism spectra are seen as the metals bind. The peptide-metal complexes are completely resistant to proteolytic digestion, and mass spectrometry demonstrates that this peptide forms metal-linked dimers. The peptide can also combine with the intact tat protein to form metal-linked heterodimers. If these heterodimers are unable to trans-activate viral transcription, tat21-38 could be a lead compound for designing drugs to treat acquired immunodeficiency syndrome.

Amino Acid Sequence↗
PubMed1987

Dimerization of the operator binding domain of phage lambda repressor.

Dimerization of lambda repressor is required for its binding to operator DNA. As part of a continuing study of the structural basis of the coupling between dimer formation and operator binding, we have undertaken 1H NMR and gel filtration studies of the dimerization of the N-terminal domain of lambda repressor. Five protein fragments have been studied: three are wild-type fragments of different length (1-102, 1-92, and 1-90), and two are fragments bearing single amino acid substitutions in residues involved in the dimer interface (1-102, Tyr-88----Cys; 1-92, Ile-84----Ser). The tertiary structure of each species is essentially the same, as monitored by the 1H NMR resonances of internal aromatic groups. However, significant differences are observed in their dimerization properties. 1H NMR resonances of aromatic residues that are involved in the dimer contact allow the monomer-dimer equilibrium to be monitored in solution. The structure of the wild-type dimer contact appears to be similar to that deduced from X-ray crystallography and involves the hydrophobic packing of symmetry-related helices (helix 5) from each monomer. Removal of two contact residues, Val-91 and Ser-92, by limited proteolysis disrupts this interaction and also prevents crystallization. The Ile-84----Ser substitution also disrupts this interaction, which accounts for the severely reduced operator affinity of this mutant protein.

Amino Acids↗