Active site mutant Glu-43----Asp in staphylococcal nuclease displays nonlocal structural changes

Area of Science:

• Biochemistry
• Structural Biology
• Protein Engineering

Background:

• Staphylococcal nuclease is a model enzyme for studying protein folding and function.
• Site-directed mutagenesis is a common tool to investigate protein structure-function relationships.

Purpose of the Study:

• To determine the crystal structure of the Glu-43----Asp mutant of staphylococcal nuclease.
• To understand the structural impact of a seemingly conservative amino acid substitution on enzyme activity and active site environment.

Main Methods:

• X-ray crystallography was used to determine the structure at 1.74 A resolution.
• Restrained least-squares refinement was employed to refine the crystal structure.
• Analysis of the mutant structure was compared to the wild-type protein.

Main Results:

• The Glu-43----Asp mutation caused significant perturbations in a loop adjacent to the active site.
• Binding of the essential calcium ion (Ca2+) was loosened in the mutant.
• The network of bound water molecules in the active site was less extensive compared to the wild-type.

Conclusions:

• Even conservative mutations can have drastic and unpredictable effects on protein structure and function.
• Structural data is crucial for accurate interpretation of site-directed mutagenesis experiments.
• The study serves as a cautionary example for structure-function relationship studies.