A single amino acid substitution alters ClpS2 binding specificity

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• ClpS2 protein is a developing probe for recognizing N-terminal amino acids in N-degron peptide fragments.
• Understanding the structural basis of ClpS2's amino acid recognition is crucial for its application.

Purpose of the Study:

• To elucidate the structural mechanisms underlying ClpS2's specificity for N-terminal amino acids.
• To predict and experimentally validate the effect of a specific mutation on ClpS2's substrate preference.

Main Methods:

• All-atom molecular dynamics (MD) simulations were performed on a stable ClpS2 mutant (PROSS).
• Computational predictions were made regarding the impact of an L-to-N amino acid substitution on substrate specificity.
• Experimental validation utilized a fluorescent yeast-display assay to assess binding affinities.

Main Results:

• MD simulations provided insights into the structural basis of ClpS2-amino acid interactions.
• A leucine to asparagine substitution in PROSS ClpS2 was predicted to shift specificity from phenylalanine to tyrosine.
• Experimental results confirmed an increased binding of the mutant to tyrosine compared to phenylalanine.

Conclusions:

• The study successfully demonstrated the feasibility of altering ClpS2 specificity through targeted amino acid substitution.
• This research contributes to the development of ClpS2 as a versatile probe for N-terminal amino acid recognition.