Prem N. S. Yadav

Insertion of a peptide from MuLV RT into the connection subdomain of HIV-1 RT results in a functionally active chimeric enzyme in monomeric conformation.

The natural form of the human immunodeficiency virus type one reverse transcriptase (HIV‐1 RT) found in virion particles is a heterodimer composed of the p66 and p51 subunits. The catalytic activity resides in the larger subunit in the heterodimeric (p66/p51) enzyme while in the monomeric form it is inactive. In contrast, Murine leukemia virus RT (MuLV RT) is functionally active in the monomeric form. In the primary amino acid sequence alignment of MuLV RT and HIV‐1 RT, we have identified three specific regions in MuLV RT, that were missing in HIV‐1 RT. In a separate study, we have shown that a chimeric RT construct comprising of the polymerase domain of HIV‐1 RT and RNase-H domain of MuLV RT is functionally active as monomer [20]. In this communication, we demonstrate that insertion of a peptide (corresponding to amino acid residues 480–506) from the connection subdomain of MuLV RT into the connection subdomain of HIV‐1 RT (between residues 429 and 430) results in a functionally active monomeric chimeric RT. Furthermore, this chimeric enzyme does not dimerize with exogenously added p51 subunit of HIV‐1 RT. Functional analysis of the chimeric RT revealed template specific variations in its catalytic activity. The chimeric enzyme catalyzes DNA synthesis on both heteropolymeric DNA and homopolymeric RNA (poly rA) template but curiously lacks reverse transcriptase ability on heteropolymeric RNA template. Similar to MuLV RT, the polymerase activity of the chimeric enzyme is not affected by acetonitrile, a reagent which dissociates dimeric HIV‐1 RT into inactive monomers. These results together with a proposed 3‐D molecular model of the chimeric enzyme suggests that the insertion of the missing region may induce a change in the spatial position of RNase H domain such that it is functionally active in monomeric conformation.

Structure-based design of inhibitors of HIV-1 reverse transcriptase: a potential class of bidentate nonnucleoside inhibitors

Abstract A design for nonnucleoside compounds with high specificity towards HIV-1 reverse transcriptase (RT) has been developed by means of a structure-based drug design approach. Crystal structures of HIV-1 RT complexed with different nonnucleoside inhibitors served as the basis for this design. An important feature of the model inhibitors is that they not only retain the structural and electronic specificity of the nonnucleosides to bind to the structurally conserved region of RT but also have the ability to interact, by virtue of their additional structural motif, with one of the catalytically important and conserved aspartate residues at the polymerase active site of the enzyme. Computations of the energy of interactions and the free energy of solvation have suggested that this bidentate class of nonnucleoside inhibitors will have significantly higher binding affinity than their parent nonnucleoside inhibitor compounds.

Binding of DNA to large fragment of DNA polymerase I: identification of strong and weak electrostatic forces and their biological implications.

Examination of the electrostatic potential of a modeled complex, consisting of the Klenow fragment of E. coli DNA polymerase I and DNA template-primer, suggested the presence of two distinct interacting regions. The one displaying a strong electropositive potential field is generated by side chains of basic amino acid pairs and is directed towards the major groove site in DNA. The second electrostatic potential field around DNA is somewhat weaker and appears to be exerted by a pair of vicinal side chains of acidic and basic amino acids. The distribution of charges in this manner appears well suited for the binding of enzyme to the template-primer required in the enzymatic synthesis of DNA.