Structures of RT
To retrieve HIV RT structures from the PDB, we performed an advanced query for structures having similar sequences (search parameters: BLAST search, e-value cutoff = 10-20, query sequence), and exported the data into a spreadsheet using the menu options on the left side of the page.
Overall, 124 structures were identified including 122 HIV-1 and 2 HIV-2. Some structures included the complete p51/p66 heterodimer, whereas others consisted of important RT subdomains. The structures could be categorized as follows: (i) unliganded RT, (ii) RT bound to primer/template, (iii) RT bound to primer/template and incoming dNTP, (iv) RT bound to a non-nucleoside RT inhibitor, (v) RT bound to AZT, pre- and post- translocation, (vi) RT bound to TDF, pre- and post- binding, (vii) RT structures with NRTI resistance mutations and (viii) RT structures with NNRTI resistance mutations.
|RT bound to primer/template||
|RT bound to primer/template and incoming dNTP||
|RT bound to NNRTI||
|AZT: pre- and post- translocation||
|TDF: pre- and post- binding||
Structure of a covalently trapped catalytic complex of HIV-1 RT published by Huang H et al, Science 1998.
The HIV reverse transcriptase (RT) enzyme is responsible for RNA-dependent DNA polymerization and DNA-dependent DNA polymerization. RT is a heterodimer consisting of of p66 and p51 subunits. The p66 subunit contains 560 amino acids, whereas the p51 subunit is contains only the first 440 residues. Although the amino acid sequence of p51 is identical to the first 440 residues of the p66 subunit, it adopts a markedly different structural conformation. The p66 subunit contains the DNA-binding groove and the active site; the p51 subunit displays no enzymatic activity and functions as a scaffold for the enzymatically active p66 subunit (Sarafinos et al, J Mol Biol 2008). The p66 subunit has subdomains including the fingers, palm, and thumb subdomains that participate in polymerization, and the connection and RNasH subdomains.
The above figure contains a representation of the X-ray cyrstallographic structure reported by Huang et al, 1RTD which the enzyme is captured in register with the nucleic acid template (yellow) / primer (orange) and incoming dNTP shown in yellow spacefill mode. The enzyme active site which consists of three catalytic aspartates, D110, D185, and D186 is shown in white spacefill mode.
The p51 monomer is shown in grey. The p66 monomer is colored as follows: fingers (cyan), palm (green), thumb (red), connection (blue), RNAseH (purple). RNAseH has an active site which is responsible for degrading the RNA template from the RNA-DNA hybrid created during reverse transcription. The binding cleft is configured so that he nucleic contacts both the polymerase ad the RNAseH active sites; these are located about 17 or 18 bp apart.
The p66 subunit of HIV-1 RT consists of 27 beta strands, 17 alpha helixes, and many loops or coils. The above structure contains a representation in which beta strands are depicted by flattened arrows and alpha helices are depicted by cylinders. The fingers (blue), palm (red), thumb (green), connection (yellow), and RNAseH (magenta) subdomains are also indicated.
There are two biochemical mechanisms of NRTI drug resistance. The one mechanism is mediated by mutations that increase the hydrolytic removal of a chain-terminating NRTI (primer unbrocking). This category of mutations are usually selected by the thymidine analogs zidovudine and stavudine (thymidine analog mutations; TAMs). Another mechanism is mediated by mutations that allow the RT enzyme to discriminate against NRTIs, thereby preventing their addition to the growing DNA chain.
Figure1 shows the positions of the most common TAMs: M41, D67, K70, L210, T215, K219. Of note K70E is associated with NRTI discrimination whereas K70R is associated with primer unblocking. Figure2 shows the positions of several of the most common discriminatory mutations occur: K65, K70, L74, V75, Y115, and M184.
There are two patterns of mutations that result in high-level resistance to most NRTIs. Figure3 shows the one pattern of mutations including Q151M in combination with mutations at positions A62, V75, F77, F116 +/- M184V. Figure4 illustrates the pattern of mutations which includes the combination of a double amino acid insertion at position 69 (most commonly T69S_SS) in combination with multiple TAMs +/- M184V.
Figure 1. Thymidine analog mutations (TAMs)
Figure 2. Major discriminatory mutations
Figure 3. Multidrug resistance: Q151M Pathway
Figure 4. Multidrug resistance : TAMs + T69insertions
The NNRTIs inhibit HIV-1 RT allosterically by binding to a hydrophobic pocket about 10 angstroms under the active site. The above figure shows the 5’ polymerase-coding region of HIV-1 RT encompassing the fingers, palm, and thumb subdomains of the p66 subunit bound to the NNRTI nevirapine (Kohlstaedt) PDB #. Positions associated with NNRTI resistance that make up the central NNRTI binding pocket are shown: L100, K101, K103, V106, V108, V179, Y181, Y188, G190, F227. Additional positions that make up the pocket include E138 which is contributed by the p51 subunit) not shown and M230, L234, P236, K238, and L318 which form part of an extended pocket. Additional accessory NNRTI-resistance abutting positions that form the NNRTI binding pocket include A98 and P225.