Simultaneous Formation of Two Alternative Enzymically Active Structures by Complementation of Two Overlapping Fragments of Staphylococcal Nuclease

Abstract

Several inactive and structurally disordered fragments of staphylococcal nuclease (149 residues, free of disulfide bonds and sulfhydryl groups) have previously been shown to yield productive complementation through noncovalent interaction. Nuclease-T-(6-48) (residues 6 to 48) binds to nuclease-T-(49-149) to form nuclease-T'. Nuclease-T' possesses enzymic activity at a level approximately 8% that of nuclease and appears to have a structure similar to the native enzyme. A second type of enzymically active structure, similar to nuclease-T', is formed in approximately 50% yield when nuclease-T-(49-149) binds to the portion of nuclease-(1-126) that contains residues 6 to 48. The redundant portion (residues 49-126) of nuclease-(1-126) seems to protrude flexibly from the otherwise ordered structure of the complex. The present study shows that nuclease-(1-126) can bind to the cyanogen bromide fragment, nuclease-(99-149), to form a complex with ordered structure which also exhibits enzymic activity similar to that of nuclease-T'. Limited digestion with trypsin in the presence of the ligands, deoxythymidine 3',5'-diphosphate and Ca++, removes residues 99 to 110 from the complex without destruction of enzymic activity. The derived complex is composed of nuclease-(1-126) and nuclease-(111-149). These two fragments have been isolated, identified, and shown to regenerate the enzymic activity upon mixing. Removal by carboxy- and aminopeptidases of residues 124 to 126 and residues 111 to 113 from nuclease-(1-126) and nuclease-(111-149), respectively, appears to cause no diminution in the activity of the complex formed from the two shortened fragments. The studies reported here have also shown that two types of active, complementing structures are formed simultaneously and in approximately equal amounts when nuclease-T-(49-149) is mixed with nuclease-(1-126). In one of the alternative structures, nuclease-(1-126) binds to the portion of nuclease-T-(49-149) that contains residues 111 to 149. In the second, the nuclease-T-(49-149) combines with the portion of nuclease-(1-126) that contains residues 1 to 48. The respective redundant portions (residues 49 to 110 of nuclease-T-(49-149) and residues 49 to 126 of nuclease-(1-126)) appear to be flexible and independent of the ordered portions of the complexes, and are susceptible to specific removal by trypsin in the presence of ligands. The fragments, nuclease-(1-48) and nuclease-(111-149), participating in the two active complexes, ((1-48) + (49-149)) and ((1-126) + (111-149)), were isolated from such trypsin digests and were quantitatively identified. The results are consistent with the concept that essentially the entire amino acid sequence is required to furnish the minimum information necessary to determine the stable and functional structure of staphylococcal nuclease. It also appears that stable structures closely resembling the native enzyme can be formed in several ways from flexible and disordered fragments when the minimum informational requirement is fulfilled.