Molecular mechanics calculations on HIV-1 protease with peptide substrates correlate with experimental data

Weber, Irene T.; Harrison, Robert W.

doi:10.1093/protein/9.8.679

Cited by 35 publications

(34 citation statements)

References 4 publications

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“…All the other enzyme-substrate structures were built from this model by altering the side chain(s) of the appropriate residue(s). Each of the side chain torsion angles for substituted residues in the peptide substrate was rotated through 360°in steps of 15°to find the conformation with the smallest nonbonded energy as described (20).…”

Section: Methodsmentioning

confidence: 99%

Studies on the Symmetry and Sequence Context Dependence of the HIV-1 Proteinase Specificity

Tőzsér¹,

Bagossi²,

Weber³

et al. 1997

Journal of Biological Chemistry

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Two major types of cleavage sites with different sequence preferences have been proposed for the human immunodeficiency virus type 1 (HIV-1) proteinase. To understand the nature of these sequence preferences better, single and multiple amino acid substitutions were introduced into a type 1 cleavage site peptide, thus changing it to a naturally occurring type 2 cleavage site sequence. Our results indicated that the previous classification of the retroviral cleavage sites may not be generally valid and that the preference for a residue at a particular position in the substrate depends strongly on the neighboring residues, including both those at the same side and at the opposite side of the peptide backbone of the substrate. Based on these results, pseudosymmetric (palindromic) substrates were designed. The retroviral proteinases are symmetrical dimers of two identical subunits; however, the residues of naturally occurring cleavage sites do not show symmetrical arrangements, and no obvious symmetrical substrate preference has been observed for the specificity of HIV proteinase. To examine the role of the asymmetry created by the peptide bonds on the specificity of the respective primed and nonprimed halves of the binding site, amino acid substitutions were introduced into a palindromic sequence. In general, the results suggested that the asymmetry does not result in substantial differences in specificity of the S 3 and S 3 subsites, whereas its effect is more pronounced for the S 2 and S 2 subsites. Although it was possible to design several good palindromic substrates, asymmetrical arrangements may be preferred by the HIV proteinase.

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Section: Methodsmentioning

confidence: 99%

Studies on the Symmetry and Sequence Context Dependence of the HIV-1 Proteinase Specificity

Tőzsér¹,

Bagossi²,

Weber³

et al. 1997

Journal of Biological Chemistry

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“…The missing loops were produced by obtaining initial C␣-C␣ distances from a dynamic programming search of overlapping 30-mers generated from the whole PDB protein structure data base (51). The program AMMP (52) was used with the current all-atom sp4 potential set (53,54). The charge generation parameters were taken from Bagossi et al (55).…”

Section: Reagents-[␥-mentioning

confidence: 99%

Identification of Amino Acids in HIV-1 and Avian Sarcoma Virus Integrase Subsites Required for Specific Recognition of the Long Terminal Repeat Ends

Chen

Weber

Harrison

et al. 2006

Journal of Biological Chemistry

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A tetramer model for HIV-1 integrase (IN) with DNA representing 20 bp of the U3 and U5 long terminal repeats (LTR) termini was assembled using structural and biochemical data and molecular dynamics simulations. It predicted amino acid residues on the enzyme surface that can interact with the LTR termini. A separate structural alignment of HIV-1, simian sarcoma virus (SIV), and avian sarcoma virus (ASV) INs predicted which of these residues were unique. To determine whether these residues were responsible for specific recognition of the LTR termini, the amino acids from ASV IN were substituted into the structurally equivalent HIV-12 DNA integration is a concerted process that occurs in defined stages. After assembly of a stable complex of the viral integrase (IN) and host cell proteins with specific DNA sequences at the end of the HIV-1 LTR, the terminal dinucleotides are removed from each 3Ј end by endonucleolytic processing. The viral DNA 3Ј ends are then covalently linked to the host cell target DNA in a concerted reaction. Biochemical details concerning the processing and joining steps in the integration process have been reported for several retroviruses including avian sarcomaleukosis virus (ASV), murine leukemia virus (MuLV), and HIV-1. Specific DNA sequences at the 3Ј end of the viral LTR are required for recognition by the assembled viral integrase complex. Typically, the terminal 12-20 nucleotides are sufficient. After 3Ј processing of the CAXX sequence from the ends, viral DNA ends are joined by the viral integrase and the gapped intermediates are repaired, and unpaired viral 5Ј ends are excised by host nucleases. This results in a 4 -6-base pair duplication of host cell DNA, depending upon the virus, flanking the integrated viral DNA. Both the 3Ј processing and viral DNA-host DNA joining steps require integrase to be assembled on the specific viral DNA substrate. Integration of viral into host DNA occurs with limited sequence specificity (1). Several cell proteins have been reported to affect the integration process (2-8).Much of the information available concerning the molecular mechanism of integration comes from the use of reconstituted systems employing duplex oligodeoxyribonucleotides (oligos). The ASV IN, for example, catalyzes specific cleavage at the 3Ј end of the strands adjacent to the conserved CA dinucleotide using 15-bp substrates corresponding to the ASV U3 or U5 termini (9, 10). Similar substrates were used to demonstrate the joining reaction in which one oligo integrated into another (11-12). For HIV-1 duplex oligo substrates of comparable size, selected nucleotide substitutions in the U5 and U3 LTR regions were shown to affect one or both of the catalytic functions of HIV-1 integrase. Nucleic acid substitutions in the HIV-1 U5 LTR region, for example, can inhibit 3Ј processing (e.g. positions 1-5 and 9 -11) or not (e.g. positions 6 -8 and 12-14) (13). Other changes at specific nucleic acid positions in the HIV-1 U3 and U5 LTR regions can affect each of the catalytic reactions, althou...

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“…The molecular mechanics calculations used the UFF potential set (Rappe et al, 1992) with parameters modified as described in Weber and Harrison (1996). In addition, the force constants for the planarity terms of the carbonyl and aromatic carbon atoms were increased from 6 kcal/mol-A to 150 and 100 kcal/mol-8,, respectively.…”

Section: Energy Minimization Calculationmentioning

confidence: 99%

“…This change significantly improved the agreement between the calculated normal mode frequencies and observed infrared absorptions for formaldehyde and benzene. Charges for the nonstandard groups in the tetrahedral intermediate were generated as described for HTV-1 protease (Weber & Harrison, 1996). The charges were generated for each tetrahedral intermediate in the complex with RSV protease before the minimization.…”

Section: Energy Minimization Calculationmentioning

confidence: 99%

“…The RSV protease structure with non-hydrogen atoms from the crystal structure, modeled flaps, and minimized hydrogen atoms was combined with the RSV NC-PR peptide substrate that had been minimized in a complex with HIV-1 protease (Weber & Harrison, 1996). Each of the side-chain torsion angles for residues in the peptide substrate was rotated through 360" in steps of 15" to search for alternate conformations of these residues.…”

Section: Energy Minimization Calculationmentioning

confidence: 99%

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Molecular mechanics calculations on rous sarcoma virus protease with peptide substrates

Weber

Harrison

1997

Protein Science

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Molecular models of Rous sarcoma virus (RSV) protease and 20 peptide substrates with single amino acid substitutions at positions from P4 to P3', where the scissile bond is between P1 and Pl', were built and compared with kinetic measurements. The unsubstituted peptide substrate, Pro-Ala-Val-Ser-Leu-Ala-Met-Thr, represents the NC-PR cleavage site of RSV protease. Models were built of two intermediates in the catalytic reaction, RSV protease with peptide substrate and with the tetrahedral intermediate. The energy minimization used an algorithm that increased the speed and eliminated a cutoff for nonbonded interactions. The calculated protease-substrate interaction energies showed correlation with the relative catalytic efficiency of peptide hydrolysis. The calculated interaction energies for the 8 RSV protease-substrate models with changes in P1 to P1' next to the scissile bond gave the highest correlation coefficient of 0.79 with the kinetic measurements, whereas all 20 substrates showed the lower, but still significant correlation of 0.46. Models of the tetrahedral reaction intermediates gave a correlation of 0.72 for the 8 substrates with changes next to the scissile bond, whereas a correlation coefficient of only 0.34 was observed for all 20 substrates. The differences between the energies calculated for the tetrahedral intermediate and the bound peptide gave the most significant correlation coefficients of 0.90 for models with changes in PI and Pl', and 0.56 for all substrates. These results are compared to those from similar calculations on HIV-1 protease and discussed in relation to the rate-limiting steps in the catalytic mechanism and the entropic contributions.Keywords: aspartic proteases; calculated interaction energies; RSV protease; substrate binding; transition state The Rous sarcoma virus (RSV) protease is a member of the aspartic protease family and is enzymatically active as a dimer. The crystal structure of RSV protease Jaskolski et al., 1991) has a three-dimensional fold similar to that of HIV-1 protease Weber, 1990). Although there are many crystal structures of HIV protease with different inhibitors (for review, see Wlodawer & Erickson, 1993), there is no crystal structure of RSV protease in the complex with an inhibitor. Instead, the structure of the RSV protease-substrate complex has been modeled by analogy to the structures of HIV protease with peptide-like inhibitors, as described in Grinde et al. (1992). RSV and HIV-1 proteases hydrolyze different cleavage sites in their viral polyprotein substrates (Oroszlan & Luftig, 1990), and RSV protease does not cleave peptides representing the natural substrates of HIV-I protease . The molecular basis of substrate specificity of RSV and HIV-1 proteases is not obvious, due to the lack of a clear consensus sequence in the different cleavage sites. The substrate specificity of RSV protease has been analyzed by kinetic measurements of hydrolysis of peptides that represent the natural cleavage sites, and variations of these peptides with dif...

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Molecular mechanics calculations on HIV-1 protease with peptide substrates correlate with experimental data

Cited by 35 publications

References 4 publications

Studies on the Symmetry and Sequence Context Dependence of the HIV-1 Proteinase Specificity

Studies on the Symmetry and Sequence Context Dependence of the HIV-1 Proteinase Specificity

Identification of Amino Acids in HIV-1 and Avian Sarcoma Virus Integrase Subsites Required for Specific Recognition of the Long Terminal Repeat Ends

Molecular mechanics calculations on rous sarcoma virus protease with peptide substrates

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