PMC

Residue 148 of RseA plays an important role for Site-2 cleavage. (A) Mutation of Val-148 to Thr or Ile, but not His or Lys, allowed Site-2 cleavage. DegS and OMP peptide were added together to the reactions where DegS is indicated. (B) Mutation of Val-148 to Thr or Ile in RseA allowed a more robust envelope stress response than mutation of Val-148 to His or Lys.

Conserved mutation of Val-148 in RseA allowed retention of Site-2 cleavage. (A) Mutation of Val-148 to conserved, but not dissimilar or charged, amino acids in RseA allowed retention of Site-2 cleavage. DegS and OMP peptide were added together to the reactions where DegS is indicated. (B) Classification of three categories of amino acids at position 148 of RseA based on their impact on Site-1 and Site-2 cleavages. Mutation of Val-148 to any of the five amino acids—Glu, Asp, Gly, Pro, and Phe—crippled Site-1 cleavage of RseA by DegS. Among the mutations that allow Site-1 cleavage, six (mutation of Val-148 to Lys, His, Arg, Ser, Gln, and Tyr) do not allow Site-2 cleavage.

A proposed model on the mechanism of Site-2 cleavage. (A) Sequence alignment of PDZ domains from representative S2Ps and two non-S2P proteins (PSD-95 and P55). Conserved amino acids are colored yellow. Shared secondary structural elements of RseP PDZ1 and PDZ2 are shown above the sequences. Note the presence of extra amino acids preceding strand β5 of S2P proteins. (B) A proposed model on the mechanism of Site-2 cleavage. After DegS cleavage, the newly exposed carboxyl terminus of RseA directly binds to the PDZ domains of RseP, and this binding is essential for the Site-2 cleavage. This binding may activate RseP or help recruit RseA 1–148 to the protease activity of RseP. (C) Sequence alignment of representative SREBPs reveals a hydrophobic amino acid at the newly exposed carboxyl terminus after the Site-1 cleavage.

In vitro reconstitution of sequential cleavages of RseA by DegS and RseP. (A) A schematic diagram of the proteolytic cascade across the inner membrane of E. coli. After OMP peptide binding to the PDZ domain, DegS is activated and cleaves RseA between Val-148 and Ser-149. Then, the membrane-embedded protease RseP makes the second cleavage after residue 108, at a site that is close to the cytoplasm. (B) A representative SDS/PAGE gel showing the full-length, recombinant proteins DegS, RseA, and RseP. All gels shown in this study were stained by Coomassie blue. (C) Reconstitution of sequential cleavages of RseA by DegS and RseP. Note that RseP only cleaved RseA after it was first cleaved by DegS in the presence of OMP peptides. (D) RseA 1–148 contains the essential factor for Site-2 cleavage. RseP efficiently cleaved the isolated RseA 1–148 (lane 2) but exhibited no activity for the full-length RseA (lane 3). RseP failed to cleave the full-length RseA in the presence of RseA 1–148 but completely cleaved RseA 1–148 (lane 4).

Integrity of the peptide-binding grooves in RseP PDZ domains is essential for Site-2 cleavage. (A) Mutation of residues in the putative peptide-binding groove of RseP PDZ domains led to loss of Site-2 cleavage. Gly-214/Ile-215 and Gly-303/Ile-304 are predicted to be located in the putative peptide-binding grooves of PDZ domains 1 and 2, respectively. (B) A single missense mutation in the putative peptide-binding groove of RseP PDZ domains led to loss of Site-2 cleavage. (C) Structure of the first PDZ domain (PDZ1; cyan) is shown in ribbon diagram (Left) and surface representation (Right). Note the absence of the putative peptide-binding groove. (D) Structure of the second PDZ domain (PDZ2). (Left) The homodimer of PDZ2 in an asymmetric unit. (Right) The surface representation of one PDZ2 domain, with its peptide-binding pocket accommodating Ile-309 from the adjacent PDZ domain. (E) Structure of PDZ2-GKASPV, which has the RseA peptide G₁₄₃KASPV₁₄₈ fused to its carboxyl terminus. Val-148 binds to the surface pocket of PDZ2 through four specific hydrogen bonds (red dashed lines) and a number of van der Waals contacts (black dashed lines). (F) Structure of PDZ2-I304A. (Left) The two molecules of PDZ2-I304A in an asymmetric unit. (Right) The surface representation of PDZ2-I304. The mutation I304A deforms the putative peptide-binding pocket.