The cocrystal structures of two zinc-stabilized DNA-binding domains illustrate different ways of achieving sequence-specific DNA recognition.
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Amino Acid Sequence↗
Biomedical subjects
J T Finch
Publications and source records attributed to J T Finch.
Binding of the dye congo red to the amyloid protein pig insulin reveals a novel homology amongst amyloid-forming peptide sequences.
The three-dimensional structure has been determined of a complex of the dye Congo Red, a specific stain for amyloid deposits, bound to the amyloid protein insulin. One dye molecule intercalates between two globular insulin molecules at an interface formed by a pair of anti-parallel beta-strands. This result, together with analysis of the primary sequences of other amyloidogenic proteins and peptides suggests that this mode of dye-binding to amyloid could be general. Moreover, the structure of this dye-binding interface between protein molecules provides an insight into the polymerization of amyloidogenic proteins into amyloid fibres. Thus the detailed characterization, at a resolution of 2.5 A, of the dye binding site in insulin could form a basis for the design of agents targeted against a variety of amyloid deposits.
Amino Acid Sequence↗
A novel keratan sulphate proteoglycan from a human embryonal carcinoma cell line.
We describe here the purification and partial characterization of a 200 kDa keratan sulphate proteoglycan found in the pericellular matrix of human embryonal carcinoma cells. Previously we have shown that this molecule is recognized by a monoclonal antibody (GCTM-2). The antigen was isolated using ion-exchange chromatography and gel filtration, purification being monitored by e.l.i.s.a. using GCTM-2. Metabolic labelling of GCT 27 C-4 embryonal carcinoma cells with sodium [35S]sulphate resulted in the incorporation of [35S]sulphate into the purified molecule. Throughout the purification procedure, the peaks of 35S radioactivity were coincident with the peaks of immunoreactivity, and this label was released both by digestion with keratanase and chondroitinase, confirming the proteoglycan nature of the antigen. The intact molecule ran as a single broad band of 200 kDa, which has been identified by silver staining and immunoblotting following gel electrophoresis. Amino acid analysis of the purified antigen indicated a high content of serine, glycine and aspartic acid/asparagine residues. Visualization by rotary-shadowing electron microscopy suggests that the purified material forms large aggregates, even under denaturing conditions. Deglycosylation of this preparation with trifluoromethanesulphonic acid yielded a major band of 55 kDa and a minor band of 48 kDa. The biochemical nature of the molecule described here, along with tissue distribution studies using GCTM-2, indicates that the antigen is not related to previously described keratan sulphate proteoglycans.
Amino Acids↗
The mechanism of Z alpha 1-antitrypsin accumulation in the liver.
Most northern Europeans have only the normal M form of the plasma protease inhibitor alpha 1-antitrypsin, but some 4% are heterozygotes for the Z deficiency variant. For reasons that have not been well-understood, the Z mutation results in a blockage in the final stage of processing of antitrypsin in the liver such that in the Z homozygote only 15% of the protein is secreted into the plasma. The 85% of the alpha 1-antitrypsin that is not secreted accumulates in the endoplasmic reticulum of the hepatocyte; much of it is degraded but the remainder aggregates to form insoluble intracellular inclusions. These inclusions are associated with hepatocellular damage, and 10% of newborn Z homozygotes develop liver disease which often leads to a fatal childhood cirrhosis. Here we demonstrate the molecular pathology underlying this accumulation and describe how the Z mutation in antitrypsin results in a unique molecular interaction between the reactive centre loop of one molecule and the gap in the A-sheet of another. This loop-sheet polymerization of Z antitrypsin occurs spontaneously at 37 degrees C and is completely blocked by the insertion of a specific peptide into the A-sheet of the antitrypsin molecule. Z antitrypsin polymerized in vitro has identical properties and ultrastructure to the inclusions isolated from hepatocytes of a Z homozygote. The concentration and temperature dependence of this loop-sheet polymerization has implications for the management of the liver disease of the newborn Z homozygote.
Circular Dichroism↗
Direct visualization of the structure of the "20 S" aggregate of coat protein of tobacco mosaic virus. The "disk" is the major structure at pH 7.0 and the Proto-helix at lower pH.
We have employed the rapid-freeze technique to prepare specimens for electron microscopy of a coat protein solution of tobacco mosaic virus at equilibrium at pH 7.0 and 6.8, ionic strength 0.1 M and 20 degrees C. The former are the conditions for the most rapid assembly of the virus from its isolated protein and RNA. At both pH values, the equilibrium mixture contains approximately 80% of a "20 S" aggregate and 20% of a "4 S" aggregate (the so-called A-protein). The specimens were prepared either totally unstained or positively stained with methyl mercury nitrate, which binds to an amino acid residue (Cys27) internally located within the subunit, which we show not to affect the virus assembly. The images in the electron microscope are compatible only with the major structure for the "20 S" aggregate at pH 7.0 containing two rings of subunits and these aggregates display the same binding contacts as those seen between the aggregate that forms the asymmetric unit in the crystal, which has been shown by X-ray crystallography to be a disk containing two rings, each of 17 subunits, oriented in the same direction. In contrast, the images from specimens prepared at pH 6.8 show the major structure to be a proto-helix at this slightly lower pH, demonstrating that the technique of cryo-electron microscopy is capable of distinguishing between these aggregates of tobacco mosaic virus coat protein. The main structure in solution at pH 7.0 must therefore be very similar to that in the crystal, although slight differences could occur and there are probably other, minor, components in a mixture of species sedimenting around 20 S under these conditions. The equilibrium between aggregates is extremely sensitive to conditions, with a drop of 0.2 pH unit tipping the disk to proto-helix ratio from approximately 10:1 at pH 7.0 to 1:10 at pH 6.8. This direct determination of the structure of the "20 S" aggregate in solution, under conditions for virus assembly, contradicts some recent speculation that it must be helical, and establishes that, at pH 7.0, it is in fact predominantly a two-layer disk as it had been modelled before.
Capsid↗
Cooperative binding of the globular domains of histones H1 and H5 to DNA.
In view of the likely role of H1-H1 interactions in the stabilization of chromatin higher order structure, we have asked whether interactions can occur between the globular domains of the histone molecules. We have studied the properties of the isolated globular domains of H1 and the variant H5 (GH1 and GH5) and we have shown (by sedimentation analysis, electron microscopy, chemical cross-linking and nucleoprotein gel electrophoresis) that although GH1 shows no, and GH5 little if any, tendency to self-associate in dilute solution, they bind highly cooperatively to DNA. The resulting complexes appear to contain essentially continuous arrays of globular domains bridging 'tramlines' of DNA, similar to those formed with intact H1, presumably reflecting the ability of the globular domain to bind more than one DNA segment, as it is likely to do in the nucleosome. Additional (thicker) complexes are also formed with GH5, probably resulting from association of the primary complexes, possibly with binding of additional GH5. The highly cooperative nature of the binding, in close apposition, of GH1 and GH5 to DNA is fully compatible with the involvement of interactions between the globular domains of H1 and its variants in chromatin folding.
Animals↗
Single crystals of long DNA molecules.
We have grown single crystals of long DNA molecules, 54 and 65 base pairs in length which encompass the binding site of the Xenopus protein transcription factor IIIA (TFIIIA). These molecules are considerably longer than those previously crystallised. X-ray diffraction shows that the crystals are ordered to a resolution of 9 A. The DNA molecules are arranged side to side in layers which are orientated at 120 degrees with respect to adjacent layers. The phosphate backbones of molecules in adjacent layers are interdigitated in both the major and minor grooves. The intensity distribution indicates that the average structure is B-like, although CD spectra in solution are more consistent with the structure, intermediate between A- and B-, found in crystals of a nonamer fragment of the binding site. The B-character in our crystals may therefore be impressed by the tight packing between layers.
Base Composition↗
Crystal structure of uncleaved ovalbumin at 1.95 A resolution.
Ovalbumin, the major protein in avian egg-white, is a non-inhibitory member of the serine protease inhibitor (serpin) superfamily. The crystal structure of uncleaved, hen ovalbumin was solved by the molecular replacement method using the structure of plakalbumin, a proteolytically cleaved form of ovalbumin, as a starting model. The final refined model, including four ovalbumin molecules, 678 water molecules and a single metal ion, has a crystallographic R-factor of 17.4% for all reflections between 6.0 and 1.95 A resolution. The root-mean-square deviation from ideal values in bond lengths is 0.02 A and in bond angles is 2.9 degrees. This is the first crystal structure of a member of the serpin family in an uncleaved form. Surprisingly, the peptide that is homologous to the reactive centre of inhibitory serpins adopts an alpha-helical conformation. The implications for the mechanism of inhibition of the inhibitory members of the family is discussed.
Animals↗
Human immunodeficiency virus type 1 regulator of virion expression, rev, forms nucleoprotein filaments after binding to a purine-rich "bubble" located within the rev-responsive region of viral mRNAs.
The human immunodeficiency virus type 1 rev protein binds with high affinity (Kd less than 1-3 nM) to a purine-rich "bubble" containing bulged GG and GUA residues on either side of a double-helical RNA stem-loop located toward the 5' end of rev-response element RNA. High-affinity rev binding is maintained when the bubble is placed in heterologous stem-loop structures, but rev binding is reduced when either the bulged residues or flanking base pairs in the stem are altered. Rev binding to the purine-rich bubble nucleates assembly of long filamentous ribonucleoprotein structures containing polymers of rev bound to flanking RNA sequences. It is proposed that rev regulates human immunodeficiency virus RNA expression by selectively packaging viral transcripts carrying the rev-response element sequence into rod-like nucleoprotein complexes that block splicing of the packaged mRNAs.
Base Sequence↗
Histone-DNA interactions and their modulation by phosphorylation of -Ser-Pro-X-Lys/Arg- motifs.
The sea urchin sperm-specific histones H1 and H2B are multiply phosphorylated in spermatids, dephosphorylated in the final stages of spermatogenesis to give mature sperm, and rephosphorylated upon fertilization. Phosphorylation in spermatids, and probably at fertilization, occurs at repeated -Ser-Pro-X-Basic-motifs in the distinctive N-terminal basic domains of both histones and at the end of the much longer C-terminal domain of H1. Here we identify the consequences of multiple phosphorylation through comparison of some physical and biochemical properties of spermatid (phosphorylated) and sperm (dephosphorylated) chromatin and histones. Study of the DNA binding properties of the intact histones and isolated basic domains suggests that phosphorylation at three dispersed sites in the C-terminal tail of H1 has little effect on its overall DNA binding affinity, whereas, strikingly, binding of the N-terminal domains of H2B and H1 is abolished by phosphorylation at four or six tandemly repeated sites respectively. Together with the relative timing of events in vivo, this suggests that phosphorylation/dephosphorylation of the N-terminal (and distal end of the C-terminal) tail of H1, and/or the N-terminal tail of H2B, effectively controls intermolecular interactions between adjacent chromatin filaments, and hence chromatin packing in the sperm nucleus.
Amino Acid Sequence↗
RNA binding by the tat and rev proteins of HIV-1.
HIV-1 tat protein binds specifically to HIV-1 TAR RNA. A Scatchard analysis of tat binding has shown that the purified protein forms a one-to-one complex with HIV-1 TAR RNA with a dissociation constant of Kd = 12 nM. Tat binding in vitro is dependent upon the presence of 3 non-base paired U residues which produce a 'bulge' in the TAR RNA stem-loop structure. Deletion of the uridine residues in the bulge or substitution with guanine residues produced RNAs with a 6 to 8-fold lower affinity than wild-type TAR. By contrast, mutations that alter the sequence of the 6 nucleotide-long loop at the tip of TAR RNA structure, and mutations which alter the sequence of the stem whilst preserving Watson-Crick base pairing, do not affect tat binding significantly. There is a direct correlation between the ability of tat to bind to TAR RNA and to activate HIV transcription. Viral LTRs encoding TAR sequences known to bind tat weakly, are not stimulated efficiently by tat in vivo. HIV-1 regulator of virion expression (rev) protein binds specifically to RNA transcripts containing the 223 nucleotide-long RRE sequence with an apparent dissociation constant of 1-3 nM. The minimum binding site for rev is a 'bubble' containing 2 G residues on one side and the sequence AGU on the other. Rev is able to bind efficiently to this restricted site in the context of the RRE sequence as well as in the context of a stable RNA duplex with a sequence unrelated to that found in the RRE.(ABSTRACT TRUNCATED AT 250 WORDS)
Base Sequence↗
Crystal structure of ovalbumin as a model for the reactive centre of serpins.
The serpins are a widely distributed family of proteins with diverse functions; they include the key serine protease inhibitors of human plasma as well as noninhibitory homologues such as hormone-binding globulins, angiotensinogen and egg-white ovalbumin. Sequence alignment based on the crystal structure. On the cleaved form of the archetypal serpin, alpha 1-antitrypsin, indicates that the serpins share a common highly ordered structure. On cleavage of the reactive centre peptide bond, they characteristically undergo a remarkable conformational change, the newly generated C terminus moving some 70 A to the opposite pole of the molecule. The structure of this post-cleavage form is known, but the conformation of the intact serpins and in particular that of their reactive centre is not. Wright et al.'s structure of plakalbumin (ovalbumin cleaved by subtilisin) has provided evidence for the conformational change that results from cleavage. We have now determined the structure of native ovalbumin to 1.95 A resolution and have found that the intact peptide loop forming the analogue to the reactive centre of the inhibitory serpins takes the unexpected form of a protruding, isolated helix. This model of the intact structures of the serpins suggests how they may interact with their target proteases.
Amino Acid Sequence↗
Resolution and analysis of 'native' and 'activated' properdin.
A rapid and reproducible procedure for the resolution of 'native' and 'activated' forms of properdin (a component of the alternative activation pathway of complement), by gel filtration on the polyvinyl matrix Fractogel TSK HW-55(S), is reported. This fractionation permitted effective screening of samples for conditions that cause activation. Only 'native' properdin was detected in serum, even after activation of the alternative pathway by yeast cell walls. Transformation of 'native' into 'activated' properdin in vitro was produced by freeze-thawing of the protein, but not upon binding to and dissociation from the C3 convertase, C3bBb. Electron microscopy showed that only the 'native' population contained the discrete cyclic structures described previously by Smith, Pangburn, Vogel & Müller-Eberhard [(1984) J. Biol. Chem. 259, 4582-4588]. 'Activated' properdin, which was eluted from the gel-filtration column close to the breakthrough peak, was mainly composed of large amorphous aggregates. We therefore conclude that properdin 'activation' is not a physiological event that occurs in serum on complement activation, but is an artifact of isolation. Fractionation of properdin on Fractogel TSK HW-55(S) has, however, enabled detailed analysis of functional heterogeneity within the 'native' population.
Cell Wall↗
Oligonucleotide binding to the coat protein disk of tobacco mosaic virus. Possible steps in the mechanism of assembly.
Binding of the oligoribonucleotides AAG, AAGAAG and AAGAAGUUG to the disk aggregate of tobacco mosaic virus coat protein has been studied in solution under conditions favourable for virus assembly. The two longer oligomers bind strongly with Kd around 1 microM, approach complete saturation of binding sites and cause the formation of long, nicked helical rods resembling the virus. It is suggested that the binding of these oligomers, with sequences chosen from the assembly origin of the viral RNA, simulates the tobacco mosaic virus assembly process. No binding could be detected for AAG, indicating that chain length is a crucial determinant in the interaction. The binding of AAGAAG to coat protein crystals is very much weaker than that observed in solution, and the crystals crack at high oligomer concentrations. The corresponding oligodeoxyribonucleotide, d(AAGAAG), shows no binding to the protein in solution; the interaction is extremely specific for RNA.
Base Sequence↗
Studies of tobacco mosaic virus reassembly with an RNA tail blocked by a hybridised and cross-linked probe.
Segments of cloned cDNA to tobacco mosaic virus RNA, 150--300-bases long, have been hybridised and cross-linked to the RNA, which has then been used for reassembly experiments. This enables the elongation reaction, which does not encapsidate the double-stranded region generated, to be stopped at specific regions along the RNA and the resulting particles to be characterised, by measuring the lengths of the rods in the electron microscope. With hybridisation to the 3'-tail the entire RNA contiguous to the nucleation region is encapsidated, from the 5'-terminus up to the modified region. When the double-stranded region is on the 5'-side of the nucleation region, the mean length of the particles corresponds to a situation in which the double-stranded region is unable to enter the central hole of the growing rod, but the 3'-tail of the RNA is completely encapsidated. The longest particles hybridised on the 5'-tail (i.e. in a class longer than the mean length) show an effect complementary to those with a 3'-block, and have lengths which correspond to encapsidation from the modified region to the 3'-terminus, despite the continued presence of the 5'-tail up the rod. In all cases where there is a remaining 5'-tail the lengths observed can only be explained if elongation has occurred substantially, or probably completely, along the 3'-tail. Hence elongation must have occurred simultaneously along both the 5' and 3'-tails of the tobacco mosaic virus RNA after initiation on the internal nucleation region.
Binding Sites↗
Clathrin cubes: an extreme variant of the normal cage.
Clathrin triskelions form polyhedral cages with hexagonal and pentagonal faces when dialyzed against suitable assembly buffers. However, when the buffer is made 12% saturated in ammonium sulfate and the dialysis is performed at 4 degrees C, clathrin polymerizes into cubes. The cube is constructed from eight triskelions with one at each corner. The edge length of the cube is approximately 45 nm, equivalent to the length of the leg of a triskelion. Thus, each edge of the cube is composed of two antiparallel legs overlapping over their whole length. The interactions between the legs in the cube are a subset of those postulated to occur in cages. Indeed, the cube can be derived from a pentagonal dodecahedron by removing 12 of the 20 triskelions with only slight adjustment of the legs of the remaining triskelions. The cube forms regular arrays and appears to be a favorable species for crystallization of clathrin.
Clathrin↗
Higher-order structure of long repeat chromatin.
The higher-order structure of chromatin isolated from sea urchin sperm, which has a long nucleosomal DNA repeat length (approximately 240 bp), has been studied by electron microscopy and X-ray diffraction. Electron micrographs show that this chromatin forms 300 A filaments which are indistinguishable from those of chicken erythrocytes (approximately 212 bp repeat); X-ray diffraction patterns from partially oriented samples show that the edge-to-edge packing of nucleosomes in the direction of the 300 A filament axis, and the radial disposition of nucleosomes around it, are both similar to those of the chicken erythrocyte 300 A filament, which is described by the solenoid model. The invariance of the structure with increased linker DNA length is inconsistent with many other models proposed for the 300 A filament and, furthermore, means that the linker DNA must be bent. The low-angle X-ray scattering in the 300-400 A region both in vitro and in vivo differs from that of chicken erythrocyte chromatin. The nature of the difference suggests that 300 A filaments in sea urchin sperm in vivo are packed so tightly together that electron-density contrast between individual filaments is lost; this is consistent with electron micrographs of the chromatin in vitro.
Animals↗
Crystal structure of the nucleosome core particle at 16 A resolution.
The crystal structure of the nucleosome core particle has been studied by neutron diffraction to a resolution of 16 A. By using H2O/D2O solvent contrast variation, the structures of the DNA and histone core were analysed separately. The DNA, as seen at this resolution, forms a super-helix of pitch 25.8 A, radius 42.1 A and 1.8 turns in length. The histone core itself is approximately helical and follows the DNA along the inside of the super-helix, giving the nucleosome core particle an overall 2-fold axis of symmetry. Four regions can be distinguished in the protein density, which we interpret as dimers of histones within the octameric core. The dimers have been assigned on the basis of other evidence as being of two kinds, (H2A-H2B) and (H3-H4). Because solvent contrast variation can distinguish between hydrophobic and hydrophilic regions in the protein density, our results suggest that the interface between the monomers of each dimer is probably quite hydrophobic in character, while the interaction between dimers is weaker and/or more hydrophilic. The protein is in contact with most of the DNA and there are some regions where it may penetrate between the turns of the super-helix. In particular, the tetramer (H4-H3)-(H3-H4) is in close contact with the central part of the DNA, but significant contacts are seen also between the histones H3 and the extremities of the super-helix, thus explaining the stability of a nucleosome-like particle depleted of H2A and H2B. Significant departures from the molecular 2-fold axis of symmetry occur in the relative arrangements of the two (H2A-H2B) dimers.
Crystallography↗