Accommodation of insertion mutations on the surface and in the interior of staphylococcal nuclease

Open Access
Publisher Website
Google Scholar
Abstract
Alignment of homologous amino acid sequences reveals that insertion mutations are fairly common in evolution. Hitherto, the structural consequences of insertion mutations on the surface and in the interior of proteins of known structure have received little attention. We report here the high-resolution X-ray crystal structures of 2 site-directed insertion mutants of staphylococcal nuclease. The structure of the first insertion mutant, in which 2 glycine residues were inserted on the protein surface in the amino-terminal β-strand, has been solved to 1.70 Å resolution and refined to a crystallographic R value of 0.182. The inserted residues are accommodated in a special 3-residue β-bulge. A bridging water molecule in the newly created cavity satisfies the hydrogen bonding requirements of the β-sheet by forming a bifurcated hydrogen bond to 1 β-strand, and a single hydrogen bond to the other β-strand. The second insertion mutant contains a single leucine residue inserted at the end of the third β-strand. The structure was solved to 2.0 Å 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 results in changes in position and coordination of a bound calcium ion important for catalysis. These structures illustrate important differences in how amino acid insertions are accommodated: as localized bulges, and as extensive register shifts.