A range of catalytic efficiencies with avian retroviral protease subunits genetically linked to form single polypeptide chains

Molecular modeling based on the crystal structure of the Rous sarcoma virus (RSV) protease dimer has been used to link the two identical subunits of this enzyme into a functional, single polypeptide chain resembling the nonviral aspartic proteases. Six different linkages were selected to test the importance of different interactions between the amino acids at the amino and carboxyl termini of the two subunits. These linkages were introduced into molecular clones of fused protease genes and the linked protease dimers were expressed in Escherichia coli and purified. Catalytically active proteins were obtained from the inclusion body fraction after renaturation. The linked protease dimers exhibited a 10-20-fold range in catalytic efficiencies (V(max)/K(m)) on peptide substrates. Both flexibility and ionic interactions in the linkage region affect catalytic efficiency. Some of the linked protease dimers were 2-3-fold more active than the nonlinked enzyme purified from bacteria, although substrate specificities were unchanged. Similar relative efficiencies were observed using a polyprotein precursor as substrate. Mutation of one catalytic Asp in the most active linked protease dimer inactivated the enzyme, demonstrating that these proteins function as single polypeptide chains rather than as multimers

Substrate-controlled allotropic phases and growth orientation of TiO2 epitaxial thin films

International audienceTiO2 thin films were grown by pulsed laser deposition on a wide variety of oxide single-crystal substrates and characterized in detail by four-circle X-ray diffraction. Films grown at 873 K on (100)-oriented SrTiO3 and LaAlO3 were (001)-oriented anatase, while on (100) MgO they were (100)-oriented. On (110) SrTiO3 and MgO, (102) anatase was observed. On M-plane and R-plane sapphire, (001)- and (101)-oriented rutile films were obtained, respectively. On C-plane sapphire, the coexistence of (001) anatase, (112) anatase and (100) rutile was found; increasing the deposition temperature tended to increase the rutile proportion. Similarly, films grown at 973 K on (100) and (110) MgO showed the emergence, besides anatase, of (110) rutile. All these films were epitaxically grown, as shown by ' scans and/or pole figures, and the various observed orientations were explained on the basis of misfit considerations and interface arrangement

Comparison of the substrate-binding pockets of the Rous sarcoma virus and human immunodeficiency virus type 1 proteases

A steady state kinetic analysis of the avian myeloblastosis virus/Rous sarcoma virus (AMV/RSV) and human immunodeficiency virus Type 1 (HIV-1) retroviral proteases (PRs) was carried out using a series of 40 peptide substrates that are derivatives of the AMV/RSV nucleocapsid-PR cleavage site. These peptides contain single amino acid substitutions in each of the seven positions of the minimum length substrate required by the PR for specific and efficient cleavage. These peptide substrates are distinguished by the individual enzyme subsites of the AMV/RSV and HIV-1 PRs. The molecular basis for similarities and differences of the individual subsites for both proteases is discussed using steady state kinetic data and modeling based on crystal structures

Mechanism of inhibition of the retroviral protease by a Rous sarcoma virus peptide substrate representing the cleavage site between the gag p2 and p10 proteins

The activity of the avian myeloblastosis virus (AMV) or the human immunodeficiency virus type 1 (HIV-1) protease on peptide substrates which represent cleavage sites found in the gag and gag-pol polyproteins of Rous sarcoma virus (RSV) and HIV-1 has been analyzed. Each protease efficiently processed cleavage site substrates found in their cognate polyprotein precursors. Additionally, in some instances heterologous activity was detected. The catalytic efficiency of the RSV protease on cognate substrates varied by as much as 30-fold. The least efficiently processed substrate, p2- p10, represents the cleavage site between the RSV p2 and p10 proteins. This peptide was inhibitory to the AMV as well as the HIV-1 and HIV-2 protease cleavage of other substrate peptides with K(i) values in the 5-20 μM range. Molecular modeling of the RSV protease with the p2-p10 peptide docked in the substrate binding pocket and analysis of a series of single-amino acid- substituted p2-p10 peptide analogues suggested that this peptide is inhibitory because of the potential of a serine residue in the P1' position to interact with one of the catalytic aspartic acid residues. To open the binding pocket and allow rotational freedom for the serine in P1', there is a further requirement for either a glycine or a polar residue in P2' and/or a large amino acid residue in P3'. The amino acid residues in P1-P4 provide interactions for tight binding of the peptide in the substrate binding pocket

Human immunodeficiency virus, type 1 protease substrate specificity is limited by interactions between substrate amino acids bound in adjacent enzyme subsites

The specificity of the retroviral protease is determined by the ability of substrate amino acid side chains to bind into eight individual subsites within the enzyme. Although the subsites are able to act somewhat independently in selection of amino acid side chains that fit into each pocket, significant interactions exist between individual subsites that substantially limit the number of clearable amino acid sequences. The substrate peptide binds within the enzyme in an extended anti-parallel β sheet conformation with substrate amino acid side chains adjacent in the linear sequence extending in opposite directions in the enzyme-substrate complex. From this geometry, we have defined both cis and trans steric interactions, which have been characterized by a steady state kinetic analysis of human immunodeficiency virus, type-1 protease using a series of peptide substrates that are derivatives of the avian leukosis/sarcoma virus nucleocapsid-protease cleavage site. These peptides contain both single and double amino acid substitutions in seven positions of the minimum length substrate required by the retroviral protease for specific and efficient cleavage. Steady state kinetic data from the single amino acid substituted peptides were used to predict effects on protease-catalyzed cleavage of corresponding double substituted peptide substrates. The calculated Gibbs' free energy changes were compared with actual experimental values in order to determine how the fit of a substrate amino acid in one subsite influences the fit of amino acids in adjacent subsites. Analysis of these data shows that substrate specificity is limited by steric interactions between pairs of enzyme subsites. Moreover, certain enzyme subsites are relatively tolerant of substitutions in the substrate and exert little effect on adjacent subsites, whereas others are more restrictive and have marked influence on adjacent cis and trans subsites

Mutations that alter the activity of the Rous sarcoma virus protease

Mutations designed by analysis of the Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV)-1 protease (PR) crystal structures were introduced into 1) the substrate binding pocket, 2) the substrate enclosing 'flaps,' and 3) surface loops of RSV PR. Each mutant PR was expressed in Escherichia coli. Changes in activity were detected by following cleavage of a truncated (NC-PR) precursor polypeptide in E. coli and cleavage of synthetic peptide substrates representing RSV and HIV-1 PR cleavage sites in vitro. Mutations in the substrate binding pocket exchanged amino acid residues located close to the substrate in the HIV-1 PR for structurally equivalent residues in the RSV PR. Changing histidine 65 to glycine (H65G) gave an inactive enzyme, while a double mutant R105P, G106V, as well as the triple mutant, H65G, R105P, G106V, produced enzymes which showed significant activity toward a substrate that represented a HIV-1 cleavage site. Mutating the catalytic aspartate (D37S) or an adjacent conserved alanine to threonine (A40T), produced inactive enzymes. In contrast, the substitution A40S was active, but showed a reduced rate of catalysis. Mutations in the flaps of conserved glycines (G69L, G70L) produced inactive PRs. Two extended RSV PR surface loops were shortened to the size found in HIV-1 PR and resulted in drastically reduced activity. These results have confirmed some of the basic predictions made from structural models but have also revealed unexpected roles and interactions in the protein

Analysis of substrate interactions of the Rous sarcoma virus wild type and mutant proteases and human immunodeficiency virus-1 protease using a set of systematically altered peptide substrates

In the preceding study, mutant Rous sarcoma virus (RSV) proteases are described in which three amino acids found in the human immunodeficiency virus-1 (HIV-1) protease (PR) were substituted into structurally comparable positions (Grinde, B., Cameron, C. E., Leis, J., Weber, I., Wlodawer, A., Burstein, H., Bizub, D., and Skalka, A. M. (1992) J. Biol. Chem. 267, 9481- 9490). In this report, the activity of the wild type and these mutant PRs are compared using a set of RSV NC-PR peptide substrates with single amino acid substitutions in each of the P4 to P3' positions. With most substrates, the relative activities of the two active mutants followed that of the RSV PR. Substitutions in the P1 and P1' positions were an exception; in this case, the mutants behaved more like the HIV-1 PR. These results confirm predictions from structural analyses which indicate that residues 105 and 106 of the RSV PR are important in forming the S1 and S1' binding subsites. These results, further analyzed with the aid of computer modeling of the RSV PR with different substrates, provide an explanation for why only partial HIV-1 PR- like behavior was introduced into the above RSV PR mutants

Programming the Rous sarcoma virus protease to cleave new substrate sequences

The Rous sarcoma virus protease displays a high degree of specificity and catalyzes the cleavage of only a limited number of amino acid sequences. This specificity is governed by interactions between side chains of eight substrate amino acids and eight corresponding subsite pockets within the homodimeric enzyme. We have examined these complex interactions in order to learn how to introduce changes into the retroviral protease (PR) that direct it to cleave new substrates. Mutant enzymes with altered substrate specificity and wild-type or greater catalytic rates have been constructed previously by substituting single key amino acids in each of the eight enzyme subsites with those residues found in structurally related positions of human immunodeficiency virus (HIV)-1 PR. These individual amino acid substitutions have now been combined into one enzyme, resulting in a highly active mutant Rous sarcoma virus (RSV) protease that displays many characteristics associated with the HIV-1 enzyme. The hybrid protease is capable of catalyzing the cleavage of a set of HIV-1 viral polyprotein substrates that are not recognized by the wild-type RSV enzyme. Additionally, the modified PR is inhibited completely by the HIV-1 PR-specific inhibitor KNI-272 at concentrations where wild-type RSV PR is unaffected. These results indicate that the major determinants that dictate RSV and HIV-1 PR substrate specificity have been identified. Since the viral protease is a homodimer, the rational design of enzymes with altered specificity also requires a thorough understanding of the importance of enzyme symmetry in substrate selection. We demonstrate here that the enzyme homodimer acts symmetrically in substrate selection with each enzyme subunit being capable of recognizing both halves of a peptide substrate equally

Mutational analysis of the substrate binding pockets of the Rous sarcoma virus and human immunodeficiency virus-1 proteases

Mutations, designed by analysis of the crystal structures of Rous sarcoma virus (RSV) and human immunodeficiency virus type 1 (HIV-1) protease (PR), were introduced into the substrate binding pocket of RSV PR. The mutations substituted nonconserved residues of RSV PR, located within 10 Å of the substrate, for those in structurally equivalent positions of HIV-1 PR. Changes in the activity of purified mutants were detected in vitro by following cleavage of synthetic peptides representing wild-type and modified RSV and HIV-1 gag and pol polyprotein cleavage sites. Substituting threonine for valine 104 (V104T), S107N, I44V, Q63M or deletion of residues 61-63 produced enzymes that were 2.5-7-fold more active than the wild type RSV PR. Substituting I42D, M73V, and A100L produced enzymes with lower activity, whereas a mutant that included both M73V and A100L was as active as wild type. Several substitutions altered the specificity for substrate. These include I42D and I44V, which contribute to the S2 and S2' subsites. These proteins exhibited HIV-1 PR specificity for P2- or P2'-modified peptide substrates but unchanged specificity with P4-, P3-, P1-, P1'-, and P3'- modified substrates. Changes in specificity in the S4 subsite were detected by deletion of residues 61-63. These results confirm the hypothesis that the subsites of the substrate binding pocket of the retroviral protease are capable of acting independently in the selection of substrate amino acids

Photoproduction of D∗±D^{*\pm} mesons associated with a leading neutron

The photoproduction of $D^{*\pm} (2010)$ mesons associated with a leading neutron has been observed with the ZEUS detector in $ep$ collisions at HERA using an integrated luminosity of 80 pb$^{-1}$. The neutron carries a large fraction, {$x_L>0.2$}, of the incoming proton beam energy and is detected at very small production angles, {$\theta_n<0.8$ mrad}, an indication of peripheral scattering. The $D^*$ meson is centrally produced with pseudorapidity {$|\eta| 1.9$ GeV}, which is large compared to the average transverse momentum of the neutron of 0.22 GeV. The ratio of neutron-tagged to inclusive $D^*$ production is $8.85\pm 0.93({\rm stat.})^{+0.48}_{-0.61}({\rm syst.})\%$ in the photon-proton center-of-mass energy range {$130 <W<280$ GeV}. The data suggest that the presence of a hard scale enhances the fraction of events with a leading neutron in the final state.Comment: 28 pages, 4 figures, 2 table