Engineered human angiogenin mutations in the placental ribonuclease inhibitor complex for anticancer therapy: Insights from enhanced sampling simulations

Abstract

Targeted human cytolytic fusion proteins (hCFPs) represent a new generation of immunotoxins (ITs) for the specific targeting and elimination of malignant cell populations. Unlike conventional ITs, hCFPs comprise a human/humanized target cell-specific binding moiety (e.g., an antibody or a fragment thereof) fused to a human proapoptotic protein as the cytotoxic domain (effector domain). Therefore, hCFPs are humanized ITs expected to have low immunogenicity. This reduces side effects and allows long-term application. The human ribonuclease angiogenin (Ang) has been shown to be a promising effector domain candidate. However, the application of Ang-based hCFPs is largely hampered by the intracellular placental ribonuclease inhibitor (RNH1). It rapidly binds and inactivates Ang. Mutations altering Ang's affinity for RNH1 modulate the cytotoxicity of Ang-based hCFPs. Here we perform in total 2.7 µs replica-exchange molecular dynamics simulations to investigate some of these mutations-G85R/G86R (GGRRmut ), Q117G (QGmut ), and G85R/G86R/Q117G (GGRR/QGmut ). GGRRmut turns out to perturb greatly the overall Ang-RNH1 interactions, whereas QGmut optimizes them. Combining QGmut with GGRRmut compensates the effects of the latter. Our results explain the in vitro finding that, while Ang GGRRmut -based hCFPs resist RNH1 inhibition remarkably, Ang WT- and Ang QGmut -based ones are similarly sensitive to RNH1 inhibition, whereas Ang GGRR/QGmut -based ones are only slightly resistant. This work may help design novel Ang mutants with reduced affinity for RNH1 and improved cytotoxicity.

Keywords: angiogenin; apoptosis; cytolytic fusion protein; molecular dynamics; mutagenesis; replica exchange; ribonuclease inhibitor; targeted therapy.

Figure 1

Representative structure of the RNH1‐Ang WT trajectory (green) superimposed on the X‐ray structure. The red to blue color indicates low to high Debye–Waller factors in the X‐ray structure.35 The representative structure is obtained by cluster analysis of the trajectory using 2.5‐Å cutoff on Ang Cα's RMSD. It corresponds to the middle structure of the most populated cluster (including 54% of the frames).

Figure 2

Cα's RMSF values of (A) RNH1 and (B) the Ang variants, in RNH1‐Ang WT (black), RNH1‐Ang GGRR_mut (blue), RNH‐Ang QG_mut (green), and RNH1‐Ang GGRR/QG_mut (red).

Figure 3

Representative structures (from a cluster analysis) of (A) RNH1‐Ang WT, (B) RNH1‐Ang GGRR_mut, (C) RNH1‐Ang QG_mut, and (D) RNH1‐Ang GGRR/QG_mut around Ang Residues 85–89. RNH1 and Ang backbones are shown in gray and yellow ribbons, respectively. Residues discussed in the main text are shown in sticks. Magenta dashed lines indicate H‐bonds. Water molecules are not shown for clarity.

Figure 4

Salt bridge and H‐bond contacts (magenta dashed lines) around the C‐terminus of Ang. The RNH1 backbone is shown in gray ribbon. The Ang backbone is shown in cartoon and colored by secondary structure. Positively and negatively charged residues that form salt bridges are labeled in blue and red, respectively. Main‐chain and side‐chain atoms are shown in sticks and balls, respectively. (A) RNH1‐Ang WT preserves a salt‐bridge/H‐bond network throughout the simulation. (B) In RNH1‐Ang GGRR_mut, ^AngQ117 side chain swings between the original position (such as in RNH1‐Ang WT) and a solvent‐exposed one (shown here) where it forms an H‐bond with ^RNH1N406. (C) RNH1‐Ang QG_mut maintains the same C‐terminal conformation and intra‐/intermolecular interactions as those in RNH1‐Ang WT, except for the H‐bonds involving the mutated Ang residue 117. (D) Ang GGRR/QG_mut C‐terminus adjusts its position with respect to RNH1 and forms stable salt bridges with the latter via ^AngR121 and ^AngR122.

Figure 5

Superimposition of representative structures of RNH1‐Ang GGRR_mut (blue) and RNH1‐Ang WT (yellow) obtained by cluster analysis on the trajectories using 2.5 Å cutoff on Ang Cα's RMSD. The most populated cluster of RNH1‐Ang GGRR_mut contains 39% of the frames. The mutation sites are indicated by red dots.

Figure 6

Superimposition of representative structures of RNH1‐Ang QG_mut (green) and RNH1‐Ang WT (yellow). Cluster analysis on the RNH1‐Ang QG_mut trajectories used 1.1 Å cutoff on Ang Cα's RMSD. The most populated cluster contains 68% of the frames. The mutation site is indicated by a red dot.

Figure 7

Representative structure of RNH1‐Ang GGRR/QG_mut (red) from the most populated cluster (including 43% of the frames) superimposed on that of RNH1‐Ang WT (yellow). The mutation sites are indicated by blue dots.