HIV-1 Reverse Transcriptase

Matthew Goldman (1) and David Marcey (2)
© David Marcey 2001

I. Structural Features of Reverse Transcriptase Å and its Subunits
II. Nucleic Acid-Reverse Transcriptase (RT) Interactions
III. HIV-RT Inhibitors
IV. Similarity of HIV-RT with the Klenow Fragment of Polymerase I
V. References

Note:  This exhibit is best viewed if the cue buttons ( ) are pressed in sequence and if the viewer does not independently manipulate the molecule on the left.

The enzyme reverse transcriptase (RT) is used by retroviruses to transcribe their single-stranded RNA genome into single-stranded DNA and to subsequently construct a complementary strand of DNA, providing a DNA double helix capable of integration into host cell chromosomes. Functional HIV1-RT is a heterodimer containing subunits of 66 kDa (p66) and 51 kDa (p51) . p66 contains two domains, the N-terminal polymerase domain (440 residues) and the C-terminal RNase H domain (120 residues). p51 is processed by proteolytic cleavage of p66 and corresponds to the polymerase domain of the p66 subunit. Portions of both p51 and the polymerase domain of p66 can be described as a "right hand" that contains three subdomains : fingers, palm, and thumb. The connection subdomain connects the hand of the polymerase domain and the RNase H domain in p66, which provides the ribonuclease activity of HIV-RT. Although p51 contains a connection subdomain, it lacks an RNase domain. The connection subdomains and the palm subdomains contain three-stranded b-sheets with a-helices on one side. The thumb subdomains comprise three a-helices . Interestingly, although the two subunits are identical in their primary amino acid sequence (except for length), they are structurally very different. This can be clearly seen by observing the subunits separately . Three catalytic residues are exposed in the nucleic acid binding cleft of p66, but are buried in p51, which lacks a discernable cleft. Another striking difference between the two subunits is the orientation of the connection subdomain; in p51 it is tucked into a central position and contacts all of the other subdomains, but in p66 it contacts only RNase H and the thumb. A question arises as to why HIV has evolved a heterodimer in which the smaller subunit (p51) is a cleavage product of the larger. One speculation (Kohlstaedt, et al., 1992) is that the selection for streamlined genomes in retroviruses has forced the evolution of different protein subunits encoded by the same gene. In the case of HIV-RT, subunits with different structural and functional properties can be produced by proteolytic cleavage of one of two initially identical subunits.

II. Nucleic Acid-Reverse Transcriptase (RT) Interactions

The majority of the template/primer-RT interactions are thought to occur between the sugar-phosphate backbone of the DNA/RNA and p66. The two a-helices of the thumb, in combination with the fingers, serve as a clamp, holding the nucleic acid in place over the palm, which contains the polymerase active site (asterix). In the functional heterodimer, the p51 subunit has been modelled to bind the anticodon stem and loop of tRNA at the start of reverse transcription: the 3' end of tRNA^Lys primes DNA synthesis from the viral RNA template by base pairing to 18 nucleotides of the viral primer binding site. The interactions between an A-form nucleic acid template/primer and the functional heterodimer can be modelled, as shown here. Helix H of the thumb of p66 partially embeds itself in the minor groove of the DNA and the thumb of p51 plus the connection domains of p66 and p51 form the floor of the binding cleft. The active site of p66 contains three catalytic residues in the palm subdomain that may bind metal ions (Asp¹⁸⁵, Asp¹⁸⁶ and Asp¹¹⁰) . This active triad is positioned close to the 3' - OH terminus of the DNA primer.

Because of the importance of RT to HIV replication, inhibitors of this enzyme are potential theraputic agents in the battle against HIV. One class of RT inhibitors are the nucleoside analogs like AZT (=zidovudine, Retrovir), ddI, ddC, and d4T. These dideoxy compounds lack a 3' oxygen, causing DNA chain termination when they are incorporated into a growing DNA strand. Another class of compounds that inhibit HIV-RT are the non-nucleoside inhibitors (NNIs). These inhibitors (e.g., APA) have been shown to bind in a pocket formed between two beta-sheets of the p66 palm, ~10 Šaway from the polymerase active site! (Ren et al. 1995 Nature Struct. Biol. 2, 293-302; Esnouf et al. 1995 Nature Struct. Biol. 2, 303-308; Ren et al. 1995 Structure 3, 915-926). The internal surface of this pocket is predominantly hydrophobic, being constructed primarily from leucine, valine, tryptophan and tyrosine residues . Although the NNIs are chemically diverse compounds, the crystal structures (e.g., Ren et al. 1995 Nature Struct. Biol. 2, 293-302) reveal a common mode of binding. Each compound has a unique structure accomodated by plasticity in regions of the surrounding protein to allow some unfavourable contacts to be relieved without changing the overall binding mode. Depending on the NNI bound, the volume of the pocket varies between ~600 and ~700 ų, with the inhibitors occupying ~250-350 ų. There is a clear matching of NNI shape to fit in this volume and in some cases this is achieved by conformational rearrangement of the compound from its lowest energy structure in solution. These results provide some understanding of the structural basis of the potency of the inhibitors and may suggest possible modifications that could improve interactions with the enzyme.

IV. Similarity of HIV-RT with the Klenow Fragment of Polymerase I

Although sequence similarities among various DNA and RNA polymerases are weak, structural similarities are obvious. A prime example of this is the comparison of HIV-1 reverse transcriptase to the Klenow fragment of DNA polymerase I of E. coli . These two DNA polymerases have insignificant primary sequence homologies (limited to to two short stretches of 14 and 24 amino acids with 4/14 identities and 3/24 identities). Despite this, the two molecules share structural characteristics, especially in their polymerase domains. The pol domains of both proteins form a nucleic acid binding cleft with three subdomains: fingers, palm, and thumb. The structural motifs of these particular subdomains are virtually identical. The palm subdomain of both molecules contain catalytically important residues, Asp ⁸⁸² and Glu ⁸⁸³ in the Klenow fragment , and Asp ¹⁸⁵ and Asp ¹⁸⁶ in HIV-RT . Mutations of these residues significantly reduce the polymerase activity of the particular molecule, indicating an important role in catalysis. In addition to palm subdomain, the thumb subdomain of reverse transcriptase is also quite similar to the thumb of the Klenow fragment: in both molecules the thumb consists of a bundle of a-helices that may engage a groove of double stranded nucleic acid.

V. References

Jacobo-Molina, A., Ding, J., Nanni, R.G., Clark, A.D., Jr., Lu, X., Tantillo, C., Williams, R.L., Kamer, G., Ferris, A.L., Clark, P., Hizi, A., Hughes, S.H., and Arnold, E. (1993). Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 Å resolution shows bent DNA. Proc. Natl. Acad. Sci. USA Vol. 90: 6320-6324

Kohlstaedt, L.A., Wang, J., Friedman, J.M., Rice, P.A., and Steitz, T.A. (1992). Crystal Structure at 3.5 Å Resolution of HIV-1 Reverse Transcriptase Complexed with an inhibitor. Science Vol. 256: 1783-1790

1, Kenyon College, Gambier, Ohio. A first draft of this exhibit was created for D. Marcey's Molecular Biology class, Biology 63.

2, Kenyon College, Gambier, Ohio. Present address: California Lutheran University. Address correspondence to this author (see below).

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