![]() ![]() (1) In the mechanism for classic serine proteases, a tetrahedral oxyanion intermediate is formed during catalysis, which is favorably stabilized by a structural feature known as “oxyanion hole.” In the trypsin family, the NH groups of Gly193 and Ser195, one of the residues of the catalytic triad, build this pocket. Binding of the activator induces rearrangement of this loop, which in turn adopts a well-defined conformation. In the peptide-free form, the major part of this loop was highly flexible and not defined by electron density. The most pronounced conformational changes are observed for loop L2. Furthermore, superposition of peptide-bound and peptide-free DegS indicated conformational changes of the distant active site loops L1, L2, and LD ( Figure 2D). Its terminal amide nitrogen hydrogen bonds to the main chain carbonyl of Thr184, thereby pulling the flexible loop 元 15 Å away from its previous location. Most interestingly, the glutamine in −1 position interacts with the protease domain ( Figure 2C). Presumably, this residue interacts with a main chain carbonyl of the PDZ domain. By contrast, the side chain of the tyrosine residue at position −2 was not well defined by electron density. In the DegS-activator complex, the phenylalanine in 0 position is bound in a hydrophobic pocket constructed by residues Ile259, Thr318, Met319, and Val322. ), where pocket 0 anchors the side chain of the carboxy-terminal residue. At low concentration, e.g., 1 μM, the monomeric form is undetectable as judged by gel filtration (data not shown). Together, these interactions extensively stabilize the DegS trimer, an observation that is consistent with the stability of the trimer in solution. On the concave side of the funnel, the side chain of Leu164* is bound in a hydrophobic pocket of the molecular neighbor lined by residues Ile232, Phe234, and Pro229. The hydrophobic core is enclosed by an intersubunit hydrogen bonding network constructed by residues Gln48*, Ser174, Asp193, Asn197, and Glu230*. In the center of the trimer, a large hydrophobic cluster is observed that is formed by residues Gly152, Ile151, Ile172, Tyr47*, Val51*, Leu156*, and Ile168*. On the convex side of the funnel, a two-stranded β sheet (βg, βN*, the asterisk denotes the participation of the neighboring subunit) is formed between different subunits this sheet is further stabilized by specific side chain interactions involving Ser46*, Asp153, and Ser208. Our results suggest a novel regulatory role for PDZ domains and unveil a novel mechanism of reversible protease activation.Īs in other HtrAs, formation of the trimer is exclusively mediated by the serine protease domains, in particular by residues of the N-terminal segment and of strands βg and βh. Taken together, the structural data illustrate in molecular detail how DegS acts as a periplasmic stress sensor. Backsoaking of crystals containing the DegS-activator complex revealed the presence of an active/inactive hybrid structure and demonstrated the reversibility of activation. Binding of stress-signaling peptides to its PDZ domain induces a series of conformational changes that activates protease function. DegS alone exists in a catalytically inactive form. Here, we present the crystal structures of three distinct states of DegS from E. DegS senses misfolded protein in the periplasm, undergoes autoactivation, and cleaves the antisigma factor RseA. ![]() Activation of σE is controlled by a proteolytic cascade that is initiated by the DegS protease. Gram-negative bacteria respond to misfolded proteins in the cell envelope with the σE-driven expression of periplasmic proteases/chaperones.
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