Vito Graziano

Identification of two DNA-binding sites on the globular domain of histone H5.

The nature of the complexes of histones H1 and H5 and their globular domains (GH1 and GH5) with DNA suggested two DNA‐binding sites which are likely to be the basis of the preference of H1 and H5 for the nucleosome, compared with free DNA. More recently the X‐ray and NMR structures of GH5 and GH1, respectively, have identified two basic clusters on opposite sides of the domains as candidates for these sites. Removal of the positive charge at either location by mutagenesis impairs or abolishes the ability of GH5 to assemble cooperatively in ‘tramline’ complexes containing two DNA duplexes, suggesting impairment or loss of its ability to bind two DNA duplexes. The mutant forms of GH5 also fail to protect the additional 20 bp of nucleosomal DNA that are characteristically protected by H1, H5 and wild‐type recombinant GH5. They still bind to H1/H5‐depleted chromatin, but evidently inappropriately. These results confirm the existence of, and identify the major components of, two DNA‐binding sites on the globular domain of histone H5, and they strongly suggest that both binding sites are required to position the globular domain correctly on the nucleosome.

Regulation of a viral proteinase by a peptide and DNA in one-dimensional space. IV. viral proteinase slides along DNA to locate and process its substrates

Background: The adenovirus proteinase and its precursor protein substrates are all sequence independent DNA binding proteins. Results: The proteinase slides along DNA to locate and process its substrates. Conclusion: Processing of precursor proteins by the adenovirus proteinase occurs on DNA. Significance: This is a new way an enzyme not involved in nucleic acid metabolism interacts with its substrates: sliding on DNA via one-dimensional diffusion. Precursor proteins used in the assembly of adenovirus virions must be processed by the virally encoded adenovirus proteinase (AVP) before the virus particle becomes infectious. An activated adenovirus proteinase, the AVP-pVIc complex, was shown to slide along viral DNA with an extremely fast one-dimensional diffusion constant, 21.0 ± 1.9 × 106 bp2/s. In principle, one-dimensional diffusion can provide a means for DNA-bound proteinases to locate and process DNA-bound substrates. Here, we show that this is correct. In vitro, AVP-pVIc complexes processed a purified virion precursor protein in a DNA-dependent reaction; in a quasi in vivo environment, heat-disrupted ts-1 virions, AVP-pVIc complexes processed five different precursor proteins in DNA-dependent reactions. Sliding of AVP-pVIc complexes along DNA illustrates a new biochemical mechanism by which a proteinase can locate its substrates, represents a new paradigm for virion maturation, and reveals a new way of exploiting the surface of DNA.

Turned on for degradation: ATPase-independent degradation by ClpP

Clp is a barrel-shaped hetero-oligomeric ATP-dependent protease comprising a hexameric ATPase (ClpX or ClpA) that unfolds protein substrates and translocates them into the central chamber of the tetradecameric proteolytic component (ClpP) where they are degraded processively to short peptides. Chamber access is controlled by the N-terminal 20 residues (for Escherichia coli) in ClpP that prevent entry of large polypeptides in the absence of the ATPase subunits and ATP hydrolysis. Remarkably, removal of 10-17 residues from the mature N-terminus allows processive degradation of a large model unfolded substrate to short peptides without the ATPase subunit or ATP hydrolysis; removal of 14 residues is maximal for activation. Furthermore, since the product size distribution of Delta14-ClpP is identical to ClpAP and ClpXP, the ATPases do not play an essential role in determining this distribution. Comparison of the structures of Delta14-ClpP and Delta17-ClpP with other published structures shows R15 and S16 are labile and that residue 17 can adopt a range of rotomers to ensure protection of a hydrophobic pocket formed by I19, R24 and F49 and maintain a hydrophilic character of the pore.

Reconstitution of chromatin higher-order structure from histone H5 and depleted chromatin

Reconstitution of the 30 nm filament of chromatin from pure histone H5 and chromatin depleted of H1 and H5 has been studied using small-angle neutron-scattering. We find that depleted, or stripped, chromatin is saturated by H5 at the same stoichiometry as that of linker histone in native chromatin. The structure and condensation behavior of fully reconstituted chromatin is indistinguishable from that of native chromatin. Both native and reconstituted chromatin condense continuously as a function of salt concentration, to reach a limiting structure that has a mass per unit length of 6.4 nucleosomes per 11 nm. Stripped chromatin at all ionic strengths appears to be a 10 nm filament, or a random coil of nucleosomes. In contrast, both native and reconstituted chromatin have a quite different structure, showing that H5 imposes a spatial correlation between neighboring nucleosomes even at low ionic strength. Our data also suggest that five to seven contiguous nucleosomes must have H5 bound in order to be able to form a higher-order structure.

Enzymatic activity of the SARS coronavirus main proteinase dimer.

The enzymatic activity of the SARS coronavirus main proteinase dimer was characterized by a sensitive, quantitative assay. The new, fluorogenic substrate, (Ala‐Arg‐Leu‐Gln‐NH)2‐Rhodamine, contained a severe acute respiratory syndrome coronavirus (SARS CoV) main proteinase consensus cleavage sequence and Rhodamine 110, one of the most detectable compounds known, as the reporter group. The gene for the enzyme was cloned in the absence of purification tags, expressed in Escherichia coli and the enzyme purified. Enzyme activity from the SARS CoV main proteinase dimer could readily be detected at low pM concentrations. The enzyme exhibited a high K m, and is unusually sensitive to ionic strength and reducing agents.

Processing of the L1 52/55k Protein by the Adenovirus Protease: a New Substrate and New Insights into Virion Maturation

ABSTRACT Late in adenovirus assembly, the viral protease (AVP) becomes activated and cleaves multiple copies of three capsid and three core proteins. Proteolytic maturation is an absolute requirement to render the viral particle infectious. We show here that the L1 52/55k protein, which is present in empty capsids but not in mature virions and is required for genome packaging, is the seventh substrate for AVP. A new estimate on its copy number indicates that there are about 50 molecules of the L1 52/55k protein in the immature virus particle. Using a quasi-in vivo situation, i.e., the addition of recombinant AVP to mildly disrupted immature virus particles, we show that cleavage of L1 52/55k is DNA dependent, as is the cleavage of the other viral precursor proteins, and occurs at multiple sites, many not conforming to AVP consensus cleavage sites. Proteolytic processing of L1 52/55k disrupts its interactions with other capsid and core proteins, providing a mechanism for its removal during viral maturation. Our results support a model in which the role of L1 52/55k protein during assembly consists in tethering the viral core to the icosahedral shell and in which maturation proceeds simultaneously with packaging, before the viral particle is sealed.

Crystallization of the globular domain of histone H5.

The globular domain of histone H1/H5 binds to the nucleosome and is crucial for the formation of chromatin higher order structure. We have expressed in Escherichia coli a gene that codes for the globular domain of H5. The protein produced in E. coli is functional in nucleosome binding assays. We have obtained crystals of the protein that diffract to beyond 2.5 A (1 A = 0.1 nm) resolution. The crystals are orthorhombic with unit cell dimensions of a = 80.1 A, b = 67.5 A and c = 38.0 A.

Regulation of a Viral Proteinase by a Peptide and DNA in One-dimensional Space II. ADENOVIRUS PROTEINASE IS ACTIVATED IN AN UNUSUAL ONE-DIMENSIONAL BIOCHEMICAL REACTION

Background: pVIc, an 11-amino acid peptide from the C terminus of adenovirus precursor protein pVI, activates the adenovirus proteinase (AVP). Results: pVI slides on DNA into AVP, which excises and then covalently binds pVIc thereby rendering AVP fully active. Conclusion: Activation of AVP requires pVI in cis on DNA. Significance: These results indicate a new way a protein substrate interacts with a proteinase, via one-dimensional diffusion on DNA. Late in an adenovirus infection, the viral proteinase (AVP) becomes activated to process virion precursor proteins used in virus assembly. AVP is activated by two cofactors, the viral DNA and pVIc, an 11-amino acid peptide originating from the C terminus of the precursor protein pVI. There is a conundrum in the activation of AVP in that AVP and pVI are sequence-independent DNA-binding proteins with nm equilibrium dissociation constants such that in the virus particle, they are predicted to be essentially irreversibly bound to the viral DNA. Here, we resolve that conundrum by showing that activation of AVP takes place on the one-dimensional contour of DNA. In vitro, pVI, a substrate, slides on DNA via one-dimensional diffusion, D1 = 1.45 × 106 bp2/s, until it binds to AVP also on the same DNA molecule. AVP, partially activated by being bound to DNA, excises pVIc, which binds to the AVP molecule that cut it out. pVIc then forms a disulfide bond with AVP forming the fully active AVP-pVIc complex bound to DNA. In vivo, in heat-disrupted immature virus, AVP was also activated by pVI in DNA-dependent reactions. This activation mechanism illustrates a new paradigm for virion maturation and a new way, by sliding on DNA, for bimolecular complexes to form among proteins not involved in DNA metabolism.

Regulation of a Viral Proteinase by a Peptide and DNA in One-dimensional Space I. BINDING TO DNA AND TO HEXON OF THE PRECURSOR TO PROTEIN VI, pVI, OF HUMAN ADENOVIRUS

Background: The C terminus of pVI activates the adenovirus proteinase. pVI escorts hexon into the nucleus. Results: pVI binds tightly to DNA independent of sequence, Kd = 46 nm. pVI binds tightly to hexon, Kd = 1.1 nm. Conclusion: DNA binding of pVI is the first step in the activation of adenovirus proteinase. Significance: This step links pVI, hexon, viral DNA, and the adenovirus proteinase in virion maturation. The precursor to adenovirus protein VI, pVI, is a multifunctional protein with different roles early and late in virus infection. Here, we focus on two roles late in infection, binding of pVI to DNA and to the major capsid protein hexon. pVI bound to DNA as a monomer independent of DNA sequence with an apparent equilibrium dissociation constant, Kd(app), of 46 nm. Bound to double-stranded DNA, one molecule of pVI occluded 8 bp. Upon the binding of pVI to DNA, three sodium ions were displaced from the DNA. A ΔG00 of −4.54 kcal/mol for the nonelectrostatic free energy of binding indicated that a substantial component of the binding free energy resulted from nonspecific interactions between pVI and DNA. The proteolytically processed, mature form of pVI, protein VI, also bound to DNA; its Kd(app) was much higher, 307 nm. The binding assays were performed in 1 mm MgCl2 because in the absence of magnesium, the binding to pVI or protein VI to DNA was too tight to determine a Kd(app). Three molecules of pVI bound to one molecule of the hexon trimer with an equilibrium dissociation constant Kd(app) of 1.1 nm.

Interaction of HMG14 with chromatin

Neutron scattering has been used to study the interaction of HMG14 with chromatin. Chromatin depleted of H1/H5 was reconstituted separately with histones H1 and H5, and complexed with HMG14. We have also studied the conformation of complexes formed by the binding of HMG14 to nucleosome dimers without linker DNA. Our data on the binding of HMG14 to linkerless nucleosome dimers argue against a significant change in the exit and entry angles of nucleosomal core DNA. Data on the condensation of chromatin into a higher-order structure suggest that there is no dramatic difference between the roles of H1 and H5 in their influence on HMG14 complex formation. However, there is a decrease of about 25% in the mass per unit length of chromatin fibers on HMG14 binding, which is not accompanied by a change in the fiber repeat distance. This is evidence that there are fewer nucleosomes per repeat in HMG14 containing chromatin fibers than in normal chromatin. Alteration of chromatin structure in this manner may be part of the role of HMG14 in actively transcribed chromatin.