Structure of the dna binding cleft of the gene 5 protein from bacteriophage fd

The structure of the gene 5 DNA unwinding protein from bacteriophage fd has been solved to 2.3-Å resolution by X-ray diffraction techniques. The molecule contains an extensive cleft region that we have identified as the DNA binding site on the basis of the residues that comprise its surface. The interior of the groove has a rather large number of basic amino acid residues that serve to draw the polynucleotide backbone into the cleft. Arrayed along the external edges of the groove are a number of aromatic amino acid side groups that are in position to stack upon the bases of the DNA and fix it in place. The structure and binding mechanism as we visualize it appear to be fully consistent with evidence provided by physical-chemical studies of the protein in solution.     

Structure at 2.3 A resolution of the gene 5 product of bacteriophage fd: a DNA unwinding protein.

The structure of the gene 5 DNA unwinding protein from bacteriophage fd has been determined by X-ray diffraction analysis of single crystals to 2.3 Å resolution using six isomorphous heavy-atom derivatives. The essentially globular monomer appears to consist of three secondary structural elements, a radically twisted three-stranded antiparallel β sheet and two distinct anti-parallel β loops, which are joined by short segments of extended polypeptide chain. The molecule contains no α-helix. A long groove, or arch, 30 Å in length is formed by the underside of the twisted β sheet and one of the two β ribbons. We believe this groove to be the DNA binding region, and this is supported by the assignment of residues on its surface implicated in binding by solution studies. These residues include several aromatic amino acids which may intercalate or stack upon the bases of the DNA. Two monomers are maintained as a dimer by the very close interaction of symmetry related β ribbons about the molecular dyad. About six residues at the amino and carboxyl terminus are in extended conformation and both seem to exhibit some degree of disorder. The amimo-terminal methionine is the locus for binding the platinum heavy-atom derivatives and tyrosine 26 for attachment of the major iodine substituent.     

Preliminary molecular replacement results for a crystalline gene 5 protein–deoxyoligonucleotide complex

Complexes of the gene 5 protein from bacteriophage fd with a variety of oligodeoxynucleotides, ranging in length from two to eight and comprised of several different sequences, have been formed and crystallized for X-ray diffraction analysis. The crystallographic parameters of four different unit cells, all of which are based on hexagonal packing arrangements, indicate that the fundamental unit of the complex is composed of six gene 5 protein dimers. We believe this aggregate has 622 point group symmetry and is a ring formed by end-to-end closure of a linear array of six dimers. From our results we have proposed a double-helix model for the gene 5 protein–DNA complex in which the protein forms a spindle or core around which the DNA is spooled. Currently 5.0-Å X-ray diffraction data from one of the crystalline complexes is being analyzed by molecular replacement techniques to obtain a direct image of the protein–nucleic acid complex.     

Three-dimensional structure of the ribonuclease T1 2'-GMP complex at 1.9-A resolution

The complex formed between the enzyme ribonuclease T1 (EC 3.1.27.3) and its specific inhibitor 2'-guanylic acid (2'-GMP) has been refined to R = 0.180 using x-ray diffraction data to 1.9-A resolution. The protein molecule displays a compact fold; a 4.5 turn alpha-helix packed over an antiparallel beta-pleated sheet shields most of the hydrophobic interior of the protein against the solvent. The extended pleated sheet structure of ribonuclease T1 is composed of three long and four short strands building up a two-stranded minor beta-sheet near the amino terminus and a five-stranded major sheet in the interior of the protein molecule. In the complex with ribonuclease T1, the inhibitor 2'-guanylic acid adopts the syn-conformation and C2'-endo sugar pucker. Binding of the nucleotide is mainly achieved through amino acid residues 38-46 of the protein. The catalytically active amino acid residues of ribonuclease T1 (His40, Glu58, Arg77, and His92) are located within the major beta-sheet which, as evident from the analysis of atomic temperature factors, provides an environment of minimal local mobility. The geometry of the active site is consistent with a mechanism for phosphodiester hydrolysis where, in the transesterification step, His40 and/or Glu58 act as a general base toward the ribose 2'-hydroxyl group and His92, as a general acid, donates a proton to the leaving 5'-hydroxyl group.     

Recognition of a TG mismatch: the crystal structure of very short patch repair endonuclease in complex with a DNA duplex

The crystal structure of very short patch repair (Vsr) endonuclease, in complex with Mg2+ and with duplex DNA containing a TG mismatch, has been determined at 2.3 A resolution. In E. coli, the enzyme recognizes a TG mismatched base pair, generated after spontaneous deamination of methylated cytosines, and cleaves the phosphate backbone on the 5' side of the thymine. Extensive interactions between the DNA and the protein characterize a novel recognition mechanism, where three aromatic residues intercalate from the major groove into the DNA to strikingly deform the base pair stacking. With the presence of a cleaved DNA intermediate in the active center, the structure of the Vsr/DNA complex provides detailed insights into the catalytic mechanism for endonuclease activity.     

1H NMR (500 MHz) identification of aromatic residues of gene 32 protein involved in DNA binding by use of protein containing perdeuterated aromatic residues and by site-directed mutagenesis

Preparation of gene 32 protein containing perdeuterated tyrosyl and phenylalanyl residues has allowed the resolution of separate 1H NMR signals for the Tyr and Phe residues of the protein by NMR difference spectra. Upfield shifts in the chemical shifts of a number of aromatic protons previously observed to accompany deoxyoligonucleotide complex formation with gene 32 protein [Prigodich, R. V., Casas-Finet, J., Williams, K. R., Konigsberg, W., & Coleman, J. E. (1984) Biochemistry 23, 522-529] can be assigned to five Tyr and two Phe residues that must form part of the DNA binding domain. Site-directed mutation of Tyr-115 to Ser-115 results in the disappearance of a set of 2,6 and 3,5 tyrosyl protons that are among those moved upfield by oligonucleotide complex formation. These findings suggest that the amino acid sequence from Tyr-73 to Tyr-115 which contains six of the eight Tyr residues of the protein forms part of the DNA binding surface.     

Crystal structure of Bacillus sp. GL1 xanthan lyase,which acts on the side chains of xanthan

Xanthan lyase, a member of polysaccharide lyase family 8, is a key enzyme for complete depolymerization of a bacterial heteropolysaccharide, xanthan, in Bacillus sp. GL1. The enzyme acts exolytically on the side chains of the polysaccharide. The x-ray crystallographic structure of xanthan lyase was determined by the multiple isomorphous replacement method. The crystal structures of xanthan lyase and its complex with the product (pyruvylated mannose) were refined at 2.3 and 2.4 A resolution with final R-factors of 17.5 and 16.9%, respectively. The refined structure of the product-free enzyme comprises 752 amino acid residues, 248 water molecules, and one calcium ion. The enzyme consists of N-terminal alpha-helical and C-terminal beta-sheet domains, which constitute incomplete alpha(5)/alpha(5)-barrel and anti-parallel beta-sheet structures, respectively. A deep cleft is located in the N-terminal alpha-helical domain facing the interface between the two domains. Although the overall structure of the enzyme is basically the same as that of the family 8 lyases for hyaluronate and chondroitin AC, significant differences were observed in the loop structure over the cleft. The crystal structure of the xanthan lyase complexed with pyruvylated mannose indicates that the sugar-binding site is located in the deep cleft, where aromatic and positively charged amino acid residues are involved in the binding. The Arg(313) and Tyr(315) residues in the loop from the N-terminal domain and the Arg(612) residue in the loop from the C-terminal domain directly bind to the pyruvate moiety of the product through the formation of hydrogen bonds, thus determining the substrate specificity of the enzyme.     

Caught after the Act: a human A-type metallocarboxypeptidase in a product complex with a cleaved hexapeptide

A/B-type metallocarboxypeptidases (MCPs) are among the most thoroughly studied proteolytic enzymes, and their catalytic mechanisms have been considered as prototypes even for several unrelated metalloprote(in)ase families. It has long been postulated that the nature of the side chains of at least five substrate residues, i.e., P4-P1', influence Km and kcat and that once the peptide or protein substrate is cleaved, both products remain in the first instance bound to the active-site cleft of the enzyme in a double-product complex. Structural details of binding of substrate to the nonprimed side of the cleft have largely relied on complexes with protein inhibitors and peptidomimetic small-molecule inhibitors that do not span the entire groove. In the former, the presence of N-terminal globular protein domains participating in large-scale interactions with the surface of the cognate catalytic domain outside the active-site cleft mostly conditions the way their C-terminal tails bind to the cleft. Accordingly, they may not be accurate models for a product complex. We hereby provide the structural details of a true cleaved double-product complex with a hexapeptide of an MCP engaged in prostate cancer, human carboxypeptidase A4, employing diffraction data to 1.6 A resolution (Rcryst and Rfree = 0.159 and 0.176, respectively). These studies provide detailed information about subsites S5-S1' and contribute to our knowledge of the cleavage mechanism, which is revisited in light of these new structural insights.     

C-C-A-G-G-C-m5C-T-G-G. Helical fine structure, hydration, and comparison with C-C-A-G-G-C-C-T-G-G.

The self-complementary DNA duplex C-C-A-G-G-C-m5C-T-G-G has been refined against 1.75-A x-ray diffraction data to an R value of 17.4%. In the crystal of space group P6, 10-base pair DNA fragments with characteristic sequence-related fine structure stack end to end to form long antiparallel B-type double helices. As shown by a structure analysis at lower resolution (Heinemann, U., and Alings, C. (1991) EMBO J. 10, 35-43), the overall geometry of C-C-A-G-G-C-m5C-T-G-G is similar to that of the unmethylated analog C-C-A-G-G-C-C-T-G-G despite a different crystal environment. The present high resolution structure analysis permits a detailed comparison of the two duplexes and their hydration spheres. Helical parameters are significantly correlated between both molecules, with the exception of the base pair propeller. Sugar pucker and backbone torsion angles alpha, gamma, delta, and chi show similar mean values, but their individual values deviate significantly between duplexes. In contrast, torsion angles beta, epsilon, and zeta change along the strands of both duplexes in much the same way. The effect of single-site methylation on DNA conformation appears to be small and limited to the base pairs directly involved. Methylation tends to push base pairs toward the minor groove of the helix. A regular minor groove hydration pattern involves dual hydrogen bonding of water molecules to O-4' and base atoms of C-C-A-G-G-C-m5C-T-G-G.     

1H NMR (500 MHz) of gene 32 protein--oligonucleotide complexes

In concentrated solutions, gene 32 single-stranded DNA binding protein from bacteriophage T4 (gene 32P) forms oligomers with long rotational correlation times, rendering 1H NMR signals from most of the protons too broad to be detected. Small flexible N- and C-terminal domains are present, however, the protons of which give rise to sharp resonances. If the C-terminal A domain (48 residues) and the N-terminal B domain (21 residues) are removed, the resultant core protein of 232 residues (gene 32P) retains high affinity for ssDNA and remains a monomer in concentrated solution, and most of the proton resonances of the core protein can now be observed. Proton NMR spectra (500 MHz) of gene 32P and its complexes with ApA, d(pA)n (n = 2, 4, 6, 8, and 10), and d(pT)8 show that the resonances of a group of aromatic protons shift upfield upon oligonucleotide binding. Proton difference spectra show that the 1H resonances of at least one Phe, one Trp, and five Tyr residues are involved in the chemical shift changes observed with nucleotide binding. The number of aromatic protons involved and the magnitude of the shifts change with the length of the oligonucleotide until the shifts are only slightly different between the complexes with d(pA)8 and d(pA)10, suggesting that the binding groove accommodates approximately eight nucleotide bases. Many of the aromatic proton NMR shifts observed on oligonucleotide complex formation are similar to those observed for oligonucleotide complex formation with gene 5P of bacteriophage fd, although more aromatic residues are involved in the case of gene 32P.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complex of fd gene 5 protein and double-stranded RNA

We report the formation of complexes of the single-stranded DNA binding protein encoded by gene 5 of fd virus, with natural double-stranded RNAs. In the first direct visualization of a complex of the fd gene 5 protein with a double-stranded nucleic acid, we show by electron microscopy that the double-stranded RNA complex has a structure which is distinct from that of complexes with single-stranded DNA and is consistent with uniform coating of the exterior of the double-stranded RNA helix by the protein. Circular dichroism spectral data demonstrate that the RNA double helix in the complex is undisrupted, and that perturbation of the 228-nm circular dichroism assigned to protein tyrosines can occur in the absence of intercalation of nucleotide bases with protein aromatic residues. Our findings emphasize the potential importance of interaction with the sugar-phosphate polynucleotide backbone in binding of the fd gene 5 protein to nucleic acids.     

Interactions of quinoxaline antibiotic and DNA: the molecular structure of a triostin A-d(GCGTACGC) complex

The crystal structure of a DNA octamer d(GCGTACGC) complexed to an antitumor antibiotic, triostin A, has been solved and refined to 2.2 A resolution by x-ray diffraction analysis. The antibiotic molecule acts as a true bis intercalator surrounding the d(CpG) sequence at either end of the unwound right-handed DNA double helix. As previously observed in the structure of triostin A-d(CGTACG) complex (A.H.-J. Wang, et. al., Science, 225, 1115-1121 (1984)), the alanine amino acid residues of the drug molecule form sequence-specific hydrogen bonds to guanines in the minor groove. The two central A.T base pairs are in Hoogsteen configuration with adenine in the syn conformation. In addition, the two terminal G.C base pairs flanking the quinoxaline rings are also held together by Hoogsteen base pairing. This is the first observation in an oligonucleotide of. Hoogsteen G.C base pairs where the cytosine is protonated. The principal functional components of a bis-intercalative compound are discussed.     

Photosensitized splitting of thymine dimers in DNA by gene 32 protein from phage T 4

The binding of denatured DNA to the protein coded by gene 32 of phage T 4 is accompanied by a quenching of the fluorescence of the protein tryptophyl residues. Gene 32 protein also binds to UV-irradiated DNA and photosensitizes the splitting of thymine dimers. Thymine bases are regenerated by this photosensitized reaction both in double stranded and in heat denatured DNA. No photosensitized splitting of thymine dimers is observed when the complex formed by gene 32 protein with UV-irradiated DNA is dissociated at high ionic strength. These results are discussed with respect to the possible stacking interaction of tryptophyl residues of gene 32 protein with bases in single stranded DNA.     

Functionally important substructures of circadian clock protein KaiB in a unique tetramer complex

KaiB is a component of the circadian clock molecular machinery in cyanobacteria, which are the simplest organisms that exhibit circadian rhythms. Here we report the x-ray crystal structure of KaiB from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. The KaiB crystal diffracts at a resolution of 2.6 A and includes four subunits organized as a dimer of dimers, each composed of two non-equivalent subunits. The overall shape of the tetramer is an elongated hexagonal plate, with a single positively charged cleft flanked by two negatively charged ridges whose surfaces includes several terminal chains. Site-directed mutagenesis of Synechococcus KaiB confirmed that alanine substitution of residues Lys-11 or Lys-43 in the cleft, or deletion of C-terminal residues 95-108, which forms part of the ridges, strongly weakens in vivo circadian rhythms. Characteristics of KaiB deduced from the x-ray crystal structure were also confirmed by physicochemical measurements of KaiB in solution. These data suggest that the positively charged cleft and flanking negatively charged ridges in KaiB are essential for the biological function of KaiB in the circadian molecular machinery in cyanobacteria.     

Structural effects of DNA sequence on T.A recognition by hydroxypyrrole/pyrrole pairs in the minor groove

Synthetic polyamides composed of three types of aromatic amino acids, N-methylimidazole (Im), N-methylpyrrole (Py) and N-methyl-3-hydroxypyrrole (Hp) bind specific DNA sequences as antiparallel dimers in the minor groove. The side-by-side pairings of aromatic rings in the dimer afford a general recognition code that allows all four base-pairs to be distinguished. To examine the structural consequences of changing the DNA sequence context on T.A recognition by Hp/Py pairs in the minor groove, crystal structures of polyamide dimers (ImPyHpPy)(2) and the pyrrole counterpart (ImPyPyPy)(2) bound to the six base-pair target site 5'-AGATCT-3' in a ten base-pair oligonucleotide have been determined to a resolution of 2.27 and 2.15 A, respectively. The structures demonstrate that the principles of Hp/Py recognition of T.A are consistent between different sequence contexts. However, a general structural explanation for the non-additive reduction in binding affinity due to introduction of the hydroxyl group is less clear. Comparison with other polyamide-DNA cocrystal structures reveals structural themes and differences that may relate to sequence preference.     

Tyrosyl-base-phenylalanyl intercalation in gene 5 protein-DNA complexes: proton nuclear magnetic resonance of selectively deuterated gene 5 protein.

The interactions of oligodeoxynucleotides with the aromatic residues of gene 5 protein in complexes with d(pA)8 and d(pT)8 have been determined by 1H NMR of the protein in which the five tyrosyl residues have been selectively deuterated either in the 2,6 or the 3,5 positions. Only the 3,5 protons of the three surface tyrosyls (26, 41, and 56) interact with the bases. The remainder of the aromatic protons undergoing base-dependent upfield ring-current shifts on complex formation are phenylalanyl protons, assigned to Phe(13) on the basis of model building. 19F NMR of the complexes of the m-fluorotyrosyl-labeled protein with d(pT)4 and d(pA)8 confirms the presence of ring-current perturbations of nuclei at the 3,5-tyrosyl positions of the three surface tyrosyls. Differential expression of the 19F(1H) nuclear Overhauser effect confirms the presence of two buried and three surface tyrosyl residues. A new model of the DNA binding groove is presented involving Tyr(26)-base-Phe(13) intercalation.