Article Title: Mechanistic insights into intramembrane proteolysis by E. coli site-2 protease homolog RseP
Figure Lengend Snippet: Involvement of TM4 in the substrate cleavage and dependence on residue D446 (a) Amino acid sequence alignment of the C-terminal regions of nine RseP orthologues classified as γ -proteobacteria. The UniProtKB accession number of each orthologue is shown (Ec, Escherichia coli ; Kk, Kangiella koreensis ; Se, Salmonella enterica serovar Typhimurium; Yp, Yersinia pestis ; Vc, Vibrio cholerae ; Hi, Haemophilus influenzae ; Pm, Pasteurella multocida ; Pa, Pseudomonas aeruginosa ; Xf, Xylella fastidiosa ). Conserved and similar residues are boxed in black and gray, respectively. The positions of TM segments, structural elements and two featured residues are shown based on those of Ec RseP. (b) Complementation assay. Cultures of E. coli KK31 [Δ rseP /pKK6 (PBAD- rseP )] cells carrying plasmids for Ec RseP (pYH825) or its variants were serially diluted and spotted on L agar plates containing IPTG (left) to test complementation or containing L-arabinose (right) as a control for total-cell count. A representative result from three biological replicates is shown. ΔTM4 and ΔCTail indicate the F426amber and D446amber mutations, respectively. (c) In vivo cleavage assay with long induction. E. coli KA306 (Δ rseA Δ rseP Δ clpP ) cells harboring one plasmid for HA-MBP-RseA148 (pKA65) and one for Ec RseP (pYH825) or its variants were grown at 30°C for 3 h with 1 mM IPTG and 1 mM cAMP in M9-based medium. The cleavage efficiency was determined from immunoblots (images) as in Fig 2e . Bar plots and error bars represent the means ± S.D. from three biological replicates. (d) Conserved residues on TM4. The residues shown as sphere models were mutated to alanine (A442 was mutated to serine) to examine their roles in the proteolytic activity, as shown in Fig. S14. Another mutated residue, R449, was disordered in the electron density map as indicated by dotted lines. The ΔTM4 mutant in (b) lacks the C-terminal residues downstream of F426 (white sphere) while ΔCTail lacks the periplasmic tail region (D446 to the C-terminus). Batimastat and active site residues are shown as stick models. The zinc ion is shown as a sphere. (e) Specific interaction between D446 and PCT-H2. TM4 and the PCT-loop, including PCT-SH and PCT-H2, are shown as stick models in magenta, pink, and salmon, respectively. D446 interacts with the electropositive N-terminal end of the PCT-H2 helix dipole where the side chain of D446 and the main chain N-H group of S363 (each in white) form a hydrogen bond.
Article Snippet: 50 or 100 nL aliquots of the protein-monoolein mixture were dispensed onto 96-well glass plates and overlaid with 800 nL of crystallization solution using a mosquito LCP (TTP LabTech) or Crystal Gryphon LCP (Art Robbins Instruments).
Techniques: Sequencing, Cell Counting, In Vivo, Cleavage Assay, Plasmid Preparation, Western Blot, Activity Assay, Mutagenesis