Substrate Recognition through a PDZ Domain in Tail-Specific Protease

Abstract

Tail-specific protease (Tsp) is a periplasmic enzyme that selectively degrades proteins bearing a nonpolar C-terminus. Its substrate specificity suggests that Tsp may contain a substrate recognition domain, which selectively binds to the nonpolar C-termini of substrate proteins, separate from its catalytic site. In this work, we show that substrate recognition of Tsp is mediated by a PDZ domain, a small protein module that promotes protein−protein interactions by binding to internal or C-terminal sequences of their partner proteins. Partial proteolysis by V8 protease at a single peptide bond immediately N-terminal to the PDZ domain resulted in two distinct and relatively stable fragments and complete loss of catalytic activity. Photoaffinity labeling with a fluorescent nonpolar peptide caused the covalent attachment of the peptide to a single site on the Tsp protein. Systematic deletion mutagenesis of Tsp localized the binding site to amino acids 206−307, a region that completely encompasses the putative PDZ domain (217−301). The isolated PDZ domain (amino acids 206−334) is capable of folding into a well-behaved structure and binds to a nonpolar peptide with a dissociation constant (K_D) of 1.9 μM, similar to that of the intact Tsp protein. Site-directed mutagenesis of a surface residue at the peptide binding site of the PDZ domain, valine 229, to Glu or Gln resulted in an increase in the K_M value but had no effect on the k_cat value. The use of a separate substrate recognition domain such as a PDZ domain may be a general mechanism for achieving selective protein degradation.