ACCOMMODATION OF INSERTION MUTATIONS ON THE SURFACE AND IN THE INTERIOR OF STAPHYLOCOCCAL NUCLEASE

Alignment of homologous amino acid sequences reveals that insertion mu tations are fairly common in evolution. Hitherto, the structural conse quences of insertion mutations on the surface and in the interior of p roteins of known structure have received little attention. We report h ere the high-resolution X-ray crystal structures of 2 site-directed in sertion mutants of staphylococcal nuclease. The structure of the first insertion mutant, in which 2 glycine residues were inserted on the pr otein surface in the amino-terminal beta-strand, has been solved to 1. 70 angstrom resolution and refined to a crystallographic R value of 0. 182. The inserted residues are accommodated in a special 3-residue be ta-bulge. A bridging water molecule in the newly created cavity satisf ies the hydrogen bonding requirements of the beta-sheet by forming a b ifurcated hydrogen bond to 1 beta-strand, and a single hydrogen bond t o the other beta-strand. The second insertion mutant contains a single leucine residue inserted at the end of the third beta-strand. The str ucture was solved to 2.0 angstrom resolution and refined to a final R value of 0.196. The insertion is accommodated in a register shift that changes the conformation of the flexible loop portion of the molecule , relaxing and widening the omega turn. This structural alteration res ults in changes in position and coordination of a bound calcium ion im portant for catalysis. These structures illustrate important differenc es in how amino acid insertions are accommodated: as localized bulges, and as extensive register shifts.