Ancestral sequence reconstruction dissects structural and functional differences among eosinophil ribonucleases

Abstract

Evolutionarily conserved structural folds can give rise to diverse biological functions, yet predicting atomic-scale interactions that contribute to the emergence of novel activities within such folds remains challenging. Pancreatic-type ribonucleases illustrate this complexity, sharing a core structure that has evolved to accommodate varied functions. In this study, we used ancestral sequence reconstruction to probe evolutionary and molecular determinants that distinguish biological activities within eosinophil members of the RNase 2/3 subfamily. Our investigation unveils functional, structural, and dynamical behaviors that differentiate the evolved ancestral ribonuclease (AncRNase) from its contemporary eosinophil RNase orthologs. Leveraging the potential of ancestral reconstruction for protein engineering, we used AncRNase predictions to design a minimal 4-residue variant that transforms human RNase 2 into a chimeric enzyme endowed with the antimicrobial and cytotoxic activities of RNase 3 members. This work provides unique insights into mutational and evolutionary pathways governing structure, function, and conformational states within the eosinophil RNase subfamily, offering potential for targeted modulation of RNase-associated functions.

Keywords: RNase 2; RNase 3; X-ray crystallography; ancestral RNase; ancestral sequence reconstruction; chimeragenesis; eosinophil; eosinophil cationic protein; eosinophil-derived neurotoxin; molecular dynamics; protein evolution; structural biology.

Figure 1

Ancestral sequence reconstruction within the eosinophil RNase branch.A, phylogenetic tree showing the position of AncRNase relative to contemporary sequences of human RNase 2 (RN2_Human, HsR2), human RNase 3 (RN3_Human, HsR3), and RNase 3 from the crab-eating macaque (Macaca fascicularis, RN3_Macaq2, MfR3). The scale bar at the bottom represents substitutions per site. B, clustal Omega sequence alignment (63) between HsR2, HsR3, MfR3, and AncRNase. Sequence numbering and secondary structure elements of HsR3 are traced on top of the alignment using springs and arrows to represent α-helices and β-strands, respectively. Figure was prepared using EsPript 3.0 (62). AncRNase, ancestral ribonuclease; HsR, human ribonuclease.

Figure 2

Ribonucleolytic, antibacterial, and cytotoxic activity of AncRNase and subfamily orthologs.A, Michaelis–Menten kinetics of RNases against yeast tRNA. HsR2, HsR3, MfR3, AncRNase, and the RNase 2/3 chimera (ChimR23) are colored blue, green, purple, black, and red, respectively. All assays were performed with 16.67 nM enzyme concentration, except for HsR2 (0.83 nM). Error bars represent variability in reaction rates corresponding to each tRNA concentration. All enzyme kinetic assays were performed in triplicate and each individual Michaelis–Menten curves are presented in Fig. S2. B, antibacterial assays performed against Escherichia coli DH5α. C, cytotoxicity assays performed against HeLa cells. D, comparative analysis between antibacterial and cytotoxic activity illustrates the unique functional behavior of AncRNase and ChimR23 relative to the contemporary HsR2/3 enzymes. All antibacterial and cytotoxic assays are expressed as percentage of the negative control (PBS 1X-treated cells, blue dashed lines) and represent the mean ± SD. Scatter plots are shown to report the reproducibility of independent biological replicates within our data sets (red dots). Significance was assessed using one-way ANOVA with ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. AncRNase, ancestral ribonuclease; HsR, human ribonuclease; BtRA, bovine ribonuclease A.

Figure 3

Residue identity between RNases 2/3 subfamily members provide functional information for the design of antibacterial chimeras.Top structure: Nine strictly conserved residues among the antibacterial AncRNase and RNase 3 members are distinct in the nontoxic HsR2, delineating positions essential for antibacterial activity. N-terminal (white spheres) and C-terminal (black spheres) positions are mapped on the structure of HsR2 (PDB 1GQV). Residue positions are labeled as single letter code according to their sequence identity in HsR2/HsR3, respectively. HsR2 structure is colored using the following N- to C-terminal color spectrum: red, orange, yellow, green, cyan, blue. Bottom structure: Alternate view showing N-terminal HsR2 residues T13, Q21, Q22, and Q28 (red), which confer antibacterial and cytotoxic activity to ChimR23. Active-site residues H15, K38, and H129 are shown in green. PDB 1GQV exhibits electronic density for more than one side-chain conformation in residues Q22, Q28, and H129. AncRNase, ancestral ribonuclease; ChimR23, ribonuclease 2/3 chimera; HsR, human ribonuclease; PDB, Protein Data Bank.

Figure 4

Ligand 5′-AMP adopts an alternate conformation in the active site of HsR2 (PDB8F5X).A, molecular interactions between 5′-AMP and HsR2 are shown as sticks in the zoomed panel, with 5′-AMP carbon atoms in yellow and HsR2 carbon atoms in cyan. Oxygen, nitrogen, and phosphate atoms are shown in red, blue, and orange, respectively. Distances in Å between heavy atoms are numbered and shown as yellow dashed lines. B, the alternate binding orientation of 5′-AMP in the HsR2 active site (red, PDB 8F5X) contrasts with the typical B₂ and P₁ positions normally occupied by the adenine and phosphate moieties (blue for ligand 5′-ATP, PDB 2C01). C, closed and open conformations adopted by loop 1 in HsR2 (yellow, PDB 8F5X) and HsR3 (dark blue, PDB 1QMT), respectively. Like HsR3, the MfR3 ortholog adopts the open conformation (green, PDB 7TY1), while AncRNase adopts the closed conformation (red, PDB 8G9A). 5′-AMP bound to the active site is shown as ball-and-stick representation (PDB 8F5X). AncRNase, ancestral ribonuclease; HsR, human ribonuclease; PDB, Protein Data Bank.

Figure 5

Microsecond MD simulations of HsR2, HsR3, and AncRNase. Loops (L) and α-helices (α) are numbered and labeled. Color spectrum and putty width illustrate areas of lower to higher flexibility ranging from blue, cyan, green, yellow, and orange to red. Models were simulated from the X-ray structures of HsR2 (PDB 1GQV), HsR3 (PDB 1QMT), and AncRNase (PDB 8G9A). AncRNase, ancestral ribonuclease; HsR, human ribonuclease; MD, molecular dynamics; PDB, Protein Data Bank.