Conformational features of a truncated staphylococcal nuclease R (SNR135) and their implications for catalysis

Conformational features of a truncated (14 amino acid residues deleted from the C-terminus) staphylococcal nuclease R (SNR135) and the ternary complex of SNR135-Ca2+-pdTp were studied using circular dichroism (CD) spectra and 1-anilinonaphthalene-8-sulfonate (ANS)-binding fluorescence spectra under different conditions. Kinetic parameters such as KDNAM, KDNAS, KCaM, KCaA, and KpdTpd of SNR135 were also determined. The results show that SNR135 contains some residual secondary structure and some tertiary structure elements as indicated by far-UV and near-UV CD spectra and that it has the ability to fold into a native-like state in the presence of pdTp and Ca2+, but there are obvious differences both in secondary structure and in tertiary structure between the SNR135-Ca2+-pdTp complex and SNase R. The unfolding curves in Gdn-HCl show that the stability of the native-like conformation of the SNR135-Ca2+-pdTp complex is much less than that of SNase R though the ligand (Ca2+, pdTp) binding increases the stability of the SNR135-Ca2+-pdTp complex to some extent. Comparison of the kinetic parameters of SNR135 with those of the full-length nuclease shows that both SNR135 and SNase R have the same value of KpdTpd and very similar values of KCaM and KCaA, but SNR135 has larger values of KDNAM and KDNAS than SNase R. Such results indicate that the C-terminal deletion for SNR135 does not greatly affect the ligand (Ca2+, pdTp) binding and decreases the binding affinity of the DNA substrate to the nuclease, implying that the amino acid residues at the ligand binding sites in SNR135 are probably arranged in a similar topology to those in SNase R and that effective binding of the DNA substrate to the enzyme needs the conformational integrity of the entire enzyme molecule. Furthermore, it is suggested that the binding sites of pdTp and DNA substrate may overlap but are not exactly the same. This paper also provides evidence obtained by monitoring ANS-binding fluorescence that the partially unfolded conformation of SNR135 is not in the molten globule state.