Large Multidomain Protein NMR: HIV-1 Reverse Transcriptase Precursor in Solution

Abstract

NMR studies of large proteins, over 100 kDa, in solution are technically challenging and, therefore, of considerable interest in the biophysics field. The challenge arises because the molecular tumbling of a protein in solution considerably slows as molecular mass increases, reducing the ability to detect resonances. In fact, the typical ¹H-¹³C or ¹H-¹⁵N correlation spectrum of a large protein, using a ¹³C- or ¹⁵N-uniformly labeled protein, shows severe line-broadening and signal overlap. Selective isotope labeling of methyl groups is a useful strategy to reduce these issues, however, the reduction in the number of signals that goes hand-in-hand with such a strategy is, in turn, disadvantageous for characterizing the overall features of the protein. When domain motion exists in large proteins, the domain motion differently affects backbone amide signals and methyl groups. Thus, the use of multiple NMR probes, such as ¹H, ¹⁹F, ¹³C, and ¹⁵N, is ideal to gain overall structural or dynamical information for large proteins. We discuss the utility of observing different NMR nuclei when characterizing a large protein, namely, the 66 kDa multi-domain HIV-1 reverse transcriptase that forms a homodimer in solution. Importantly, we present a biophysical approach, complemented by biochemical assays, to understand not only the homodimer, p66/p66, but also the conformational changes that contribute to its maturation to a heterodimer, p66/p51, upon HIV-1 protease cleavage.

Keywords: HIV; NMR; isotope labeling; protein; reverse transcriptase; ribonuclease H.

Figure 1

p66/p51 reverse transcriptase (RT) structure, highlighting (a) the domain orientation in the p66 subunit, (b) the domain orientation in the p51 subunit, and relative orientation of (c) two finger–palm domains in the p66 and p51 subunits and that of (d) the two connection domains in the p66 and p51 subunits. In panels (a,b), the bar presentations below the structures indicate which domains are highlighted: finger–palm (blue), thumb (green), connection (yellow) and ribonuclease H (RNH) (orange). In panel (c), residues, 10–16 and 86–95, that are at the subunit interface in the p66 subunit, are highlighted with a red color in both subunits. Similarly, in panel (d), residues, 405–412, that are at the subunit interface in the p66 subunit, are highlighted in both subunits. The graphic presentation was made using VMD software [65] and the RT structure (PDB 1DLO [66]).

Figure 2

RNH domain structure, highlighting (a) the p51-RNH processing site (i.e., F440–Y441, red ribbon) and the active site (purple sticks), (b) the Y427 side chain (thick stick), and (c) F440 cleavage site. In panels (b,c) the side chains of the surrounding residues are shown. The graphic presentation was made using VMD software [65] and the RT structures (PDB 3KK2 [72] or 1DLO [66]).

Figure 3

(a) RT maturation process, i.e., the formation of p66/p66 and processing to p66/p51, and (b) three homodimer structural models: asymmetric homodimer with one unfolded RNH domain, proposed based on solution NMR experiments of p66/p66, p66/p51, and p51 [70,101,102]; symmetric homodimer with both RNH domains folded, proposed based on solution NMR experiments of p66/p66 and p51 [100]; or asymmetric homodimer with both RNH domains folded, proposed based on electron spin resonance (ESR) experiments [103].

Figure 4

NMR strategies used to investigate RT: (a) an example of a ¹H-¹⁵N TROSY HSQC spectrum, used to compare p66 with those of isolated domains; (b) an example of an Ile-δ1 methyl ¹H-¹³C HMQC spectrum, used for comparison of the different states of p66/p66; and (c) a one-dimensional ¹⁹F NMR spectrum of a single-site labeled p66/p66. In panel (a), HSQC signals from an isolated RNH (orange) are overlaid as an example.

Figure 5

Calculated longitudinal relaxation time (T₁) for protons in methyl geometry, (a) with and (b) without methyl fast rotation, assuming a rigid molecule (solid line) and a molecule with a domain motion at 4 ns (dashed line), as a function of the molecular rotational correlation time at 900 MHz. In the calculations, a model-free spectral density function with an extended fast motion was assumed [140,141] with a generalized order parameter (S_s²) of 0.8, and with a correlation time for internal motion of 50 ps for a rigid molecule (solid lines) or 4 ns for one that undergoes domain motion (dashed lines). In the calculation in panel (a), an order parameter for methyl fast motion (S_f²) was assumed at 0.25 for methyl protons [117,139]. In the calculation in panel (b), S_f² at 0.9 was assumed to provide a relaxation sink.

Figure 6

Our current model for p66/p51 production from p66/p66: (a) symmetric p66/p66 conformation in which thumb and RNH domains undergo domain motion; (b) asymmetric p66/p66 bound to NNRTI; and (c) conformational change induced by tRNA^Lys3 interaction, regardless of the presence of NNRTI or not [40,100,124]. Note the degrees of conformational asymmetry in (b,c) are hypothetical.