Figure 1. Schematic diagram showing proposed isomerization and initial p66 homodimer formation.
The subunit conformations are color coded (extended, green; compact, blue). Primes are introduced after homodimer formation to allow subunit identification and indicate the subunit destined to be proteolyzed. The palm loop E conformation becomes the primer grip.
Figure 1—figure supplement 1. Ribbon diagram representations of reverse transcriptase (RT) monomer and dimer structures.
(A) The monomer structure of p66 is based on the crystal structure of p51∆PL (pdb: 4KSE) and NMR data showing that it also contains a folded ribonuclease H (RH) domain linked by residues derived from an unfolded α-helix M. Domains are identified as fingers (green), palm (blue), thumb (red), connection (magenta), and RH (gray). (B) Ribbon diagram of the RT heterodimer structure (pdb: 1S9E, Das et al., 2004). For this panel, we used an NNRTI complex containing the palm loop, so the position of the p66 thumb domain differs from that in panels C and D. Color coding: p66 subunit: fingers/palm (red), thumb (orange), connection (pink), RH (magenta); p51 subunit: fingers/palm (blue), thumb (green), connection (cyan), palm loop (yellow). (C) Ribbon representation of the p51/p51' homodimer derived from the p66/p51 heterodimer structure (pdb: 1DLO) by deletion of the p66 RH domain and replacement of the p51 subunit with the p51∆PL monomer. Color coding: p51 subunit: fingers/palm (red), thumb (orange), connection (pink); p51' subunit: fingers/palm (blue), thumb (green), connection (cyan). Note that α-helix M' is unfolded in the p51' subunit of the homodimer, as it is in the monomer. (D) Initial p66/p66' homodimer structure based on NMR results and modeled from the p66/p51 heterodimer by replacing the p51 subunit with the p51∆PL monomer, and adding an additional, folded RH' domain. The disordered helix M' residues (418'–430') observed in the crystal structure of p51∆PL have been moved to avoid structural conflict with the p66 subunit and are linked to the supernumerary RH' domain (purple).
Figure 1—figure supplement 2. Structural comparison of connection domains.
(A) Overlay of the ribbon diagrams for the connection domains observed in the p66 subunit of RT (green), the p51 subunit of RT (blue), and the monomer (orange). Based on pdb files 1DLO and 4KSE. (B) Ribbon diagrams for the RT heterodimer (p66, green; RH domain, red; p51, blue), and the connection domain of the p51 monomer (orange) in which the p66 connection domain is overlayed with the connection domain of the monomer. As illustrated in B, although an initial domain repositioning of the p66 monomer domains could place the connection domain in position to interact with a second monomer, it would not be in the E conformation characteristic of the mature heterodimer. Formation of additional interfaces within the p66 subunit and with p51 may facilitate the straightening of helix αL. For example, interactions between Glu404 and Lys431 and/or Gln509 on the RH domain may facilitate this conformational change. Stacking of Trp406 with Pro420' can also facilitate the conformational change required for formation of the mature heterodimer.
Figure 1—figure supplement 3. Alternate conformations of helix αM'.
The figure shows a ribbon representation of helix αM' from the p51 subunits of two RT structures: pdb: 3QIP (chain B, green) and 1SV5 (chain B, blue). Both structures correspond to NNRTI-RT complexes. In 3QIP, helix αM' adopts a more standard geometry, with five strong hydrogen bonds (≤3.2 Å), while the helix in 1SV5 has only one strong hydrogen bond and adopts a more extended conformation in which Tyr427 is at the position of Gln426 in the 3QIP structure. These alternate conformations correspond to an alternate set of interactions within the p51 subunit. A similar conclusion supporting a conformational mixture also results from re-analysis of the electron density for some individual structures (not shown). The ability of the helix to adopt alternate registrations results from the fact that nearly all of the residues are hydrophobic. The ability of αM' to adopt these conformations facilitates transfer of residues from the RH' domain, allowing recruitment of Tyr427' by the connection' domain when thermal fluctuations release it from RH'.