His92Ala mutation in ribonuclease T1 induces segmental flexibility. An X-ray study

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Ribonuclease T1 (RNase T1) is a crucial enzyme in RNA processing.
• The active site histidine (His92) is essential for RNase T1 activity.
• Understanding enzyme structure-function relationships aids in drug design and biotechnology.

Purpose of the Study:

• To investigate the structural consequences of the His92Ala mutation in RNase T1.
• To characterize the crystal structures of RNase T1 His92Ala obtained using different crystallization conditions.
• To elucidate the impact of the mutation on enzyme flexibility and substrate binding site conformation.

Main Methods:

• Site-directed mutagenesis to create RNase T1 His92Ala.
• X-ray crystallography to determine the three-dimensional structures of the mutant enzyme.
• Molecular replacement and stereochemically restrained least-squares refinement for structure solution.
• Comparison of crystal structures with wild-type RNase T1 and its complexes.

Main Results:

• Two crystal forms of RNase T1 His92Ala were obtained using polyethylene glycol (PEG) and phosphate precipitants, with altered unit cell dimensions compared to wild-type.
• The His92Ala mutation disrupts a key hydrogen bond, leading to increased flexibility in loop 91-101.
• Conformational changes were observed in the guanine binding site, resembling those in wild-type RNase T1 complexed with guanosine derivatives.

Conclusions:

• The His92Ala mutation significantly impacts RNase T1 structure, leading to enzyme inactivation and altered conformational states.
• Crystallization conditions influence the stabilization of different loop conformations in the mutant enzyme.
• The study provides insights into the structural basis of RNase T1 activity and substrate recognition.