Nonnucleoside Inhibitor Binding Affects the Interactions of the Fingers Subdomain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase with DNA

Abstract

Site-directed photoaffinity cross-linking experiments were performed by using human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with unique cysteine residues at several positions (i.e., positions 65, 67, 70, and 74) in the fingers subdomain of the p66 subunit. Since neither the introduction of the unique cysteine residues into the fingers nor the modification of the SH groups of these residues with photoaffinity cross-linking reagents caused a significant decrease in the enzymatic activities of RT, we were able to use this system to measure distances between specific positions in the fingers domain of RT and double- Human immunodeficiency virus type 1 (HIV-1), like other retroviruses, has a single-stranded RNA genome that is converted into double-stranded DNA (dsDNA) in the cytoplasm of a newly infected cell. This conversion is carried out by the viral enzyme reverse transcriptase (RT). HIV-1 RT is a homodimeric enzyme composed of two related subunits, p66 and p51. p66 folds into two domains: polymerase and RNase H. The polymerase domain of p66 is divided into four subdomains: fingers, palm, thumb, and connection. p51 contains the first 440 amino acids of p66. This corresponds closely, but not exactly, to the polymerase domain. p51 contains the same four subdomains as the polymerase subdomain, but the relative arrangement of the subdomains is different in p66 and p51. The polymerase and RNase H active site are in the p66 subunit; the role of p51 is primarily structural.There are a number of different types of structures of HIV-1 RT available. These include the structures of unliganded HIV-1 RT (17, 36): RT bound to a DNA-DNA substrate (7,20,21), to a pseudoknot RNA inhibitor (22), and to an RNA-DNA substrate (38). The structure of a ternary complex with a DNA-DNA substrate and an incoming deoxynucleoside triphosphate (dNTP) has also been determined (18,19). Several structures of RT-nonnucleoside RT inhibitor (NNRTI) complexes have been published (6, 8, 9, 11, 12, 14-16, 24, 30, 31, 33, 34, 39). Comparison of these structures has provided insights into the mechanism of polymerization and evidence for the flexibility of the enzyme. For example, the position of the fingers subdomain of p66 changes when unliganded HIV-1 RT binds a nucleic acid substrate and the fingers move when the incoming dNTP binds at the polymerase active site.RT is a major target for anti-HIV-1 drugs. There are two classes of anti-RT drugs: nucleoside RT inhibitors (NRTIs) and NNRTIs. Despite the large number of RT-NNRTI structures, the mechanism of action of the NNRTIs is not well understood (for reviews, see references 10, 23, and 28). The NNRTI binding pocket does not exist in the absence of the inhibitor. The binding of an NNRTI is associated with the formation of a hydrophobic pocket, which distorts the region near the polymerase active site. The binding of different NNRTIs has similar, although not identical, effects on the structure of HIV-1 RT (see supplemental material, which includes a comparison of five ...

Domain structure and three-dimensional model of a group II intron-encoded reverse transcriptase

“…The template (top) strand (green in Fig. 8) continues around the back of the fingers (Peletskaya et al 2004) and is extended as B-form duplex DNA from position −14 to −30, a region known to be double-stranded in complex with Ll.LtrB RNPs (Singh and Lambowitz 2001). Although the modeled DNA is clearly a simplification of the actual structure in which the intron RNA is base-paired to the top strand between positions −12 and +2 (Singh and Lambowitz 2001), it provides insight into distance considerations.…”

“…The target-site DNA was docked to the LtrA model based on the HIV-1 RT model structure described in Peletskaya et al (2004;1R0A.pdb), which adds a 5Ј-extension of the template strand to the template-primer DNA in the crystal structure of Huang et al (1998). The DNA was extended farther along the same trajectory by adding straight B-form DNA created in InsightII (Accelrys Inc.), and its sequence was changed to match that of the LtrA target site.…”

“…The trajectory of the DNA backbone was modeled based on the HIV-1 RT model structure described in Peletskaya et al (2004;1R0A.pdb), which adds a 5Ј-extension to the DNA template strand in the crystal structure of Huang et al (1998;1RTD.pdb). This structure includes a 40°bend with a change from B-form to A-form DNA 5 bp to the primer side of the RT active site.…”

“…In both the open and closed structures, the template-primer undergoes a transition from A-form to B-form near the RT active site accompanied by a bend of ∼40°, which brings the 5Ј-overhang of the template across the face of the fingers (Ding et al 1998;Huang et al 1998). Farther upstream regions of the template strand are thought to interact with distal regions of the fingers (Huang et al 1998;Peletskaya et al 2004).…”

“…HIV-1 RT is conformationally flexible, with the fingers and thumb in an "open" conformation upon binding template-primer, and the fingers moving down to a "closed" conformation upon binding dNTP (Ding et al 1998;Huang et al 1998;Peletskaya et al 2004). X-ray crystal structures with bound DNA template-primer show that the nucleic acid interacts primarily with the p66 subunit, binding along a groove with positively charged walls that extends from the polymerase active site to the RNase H domain (Bebenek et al 1997;Ding et al 1998;Huang et al 1998).…”