ABSTRACT Human ribonuclease 7 (HsR7) is a potent antimicrobial member of the RNase A superfamily with little cytotoxic activity, in contrast to the closely related eosinophil cationic protein (HsR3). To determine which structural elements underlie these divergent functions, we engineered a chimeric RNase 7 variant in which loop 4-5 residues 61-77 were replaced by the corresponding segment from HsR3. This chimera acquired cytotoxicity toward HeLa cells while retaining antibacterial activity against Escherichia coli and Mycobacterium smegmatis, identifying this segment as a key transferable determinant of RNase function. Consistent with this result, a peptide spanning RNase 3 residues 61-77 also displayed antibacterial activity, supporting a direct contribution of this region to host-defense activity and functional modulation within the RNase scaffold. To define the structural basis of RNase 7 activity, we determined crystal structures of RNase 7 in apo and 5'-AMP-bound forms and combined these with NMR titrations and molecular docking. These analyses revealed a canonical RNase fold together with an unexpected adenine-binding pose in the B1 subsite. Complementary binding analyses showed that RNase 7 preferentially recognizes pyrimidines, yet can also accommodate purine-containing ligands such as UpA in more than one binding mode. Together, these findings establish loop 4-5 as a portable functional module that can reprogram biological activity within the human RNase scaffold and provide a structural framework for engineering multifunctional RNases with tailored properties.
Tran et al. (Fri,) studied this question.