Abstract
The Escherichia coli envelope stress response is controlled by the alternative sigma factor, sigma(E), and is induced when unfolded outer membrane proteins accumulate in the periplasm. The response is initiated by sequential cleavage of the membrane-spanning antisigma factor, RseA. RseB is an important negative regulator of envelope stress response that exerts its negative effects onsigma(E) activity through its binding to RseA. In this study, we analyze the interaction between RseA and RseB. We found that tight binding of RseB to RseA required intact RseB. Using programs that performed global and local sequence alignment of RseB and RseA, we found regions of high similarity and performed alanine substitution mutagenesis to test the hypothesis that these regions were functionally important. This protocol is based on the hypothesis that functionally dependent regions of two proteins co-evolve and therefore are likely to be sequentially conserved. This procedure allowed us to identify both an N-terminal and C-terminal region in RseB important for binding to RseA. We extensively analyzed the C-terminal region, which aligns with a region of RseA coincident with the major RseB binding determinant in RseA. Both allele-specific suppression analysis and cysteine-mediated disulfide bond formation indicated that this C-terminal region of similarity of RseA and RseB identifies a contact site between the two proteins. We suggest a similar protocol can be successfully applied to pairs of non-homologous but functionally linked proteins to find specific regions of the protein sequences that are important for establishing functional linkage.
Highlights
FEBRUARY 20, 2009 VOLUME 284 NUMBER 8 partment of the cell (1–3). E regulon members encode periplasmic chaperones and proteases, the machinery for inserting -barrel proteins into the outer membrane and components controlling the synthesis and assembly of LPS (4 – 6)
During steady state growth, E is inhibited by its antisigma factor, RseA, a membrane-spanning protein whose cytoplasmic domain binds to E with picomolar affinity (10 –13)
Accumulation of unassembled porin monomers serves as a signal to activate the DegS protease to cleave RseA in its periplasmic domain (14, 15)
Summary
Media and Antibiotics—Luria-Bertani (LB) was prepared as described (26). When required, the medium was supplemented with 30 g/ml kanamycin (Kan), 20 g/ml chloramphenicol (Cm), and/or 100 g/ml ampicillin (Amp). Expression, and Purification of RseB—The coding sequence of mature RseB (residues 24 –318) was amplified by PCR and cloned between HindIII and EcoRI sites of the vector pFLAG-MAC (Sigma-Aldrich) or pFLAG-ATS (Sigma-Aldrich) to make the construct pRseB-MAC or pRseB-ATS, respectively Both these constructs have coding sequence for FLAG tag at the 5Ј-end of the gene, and the expression of the cloned gene is under the control of an IPTG-inducible tac promoter. Expression and Purification of RseA—To express His-tagged RseA-peri, the construct pLC234 was transformed in E. coli BL21(DE3) cells (11) Cells bearing this plasmid were grown at 30 °C to an A600 of 0.3 in LB containing 30 g/ml Kan. IPTG was added to a final concentration of 0.5 mM, and induction was allowed for 3 h at 30 °C. RseB was allowed to interact with RseA for 15 min at room temperature, and unbound protein was removed by washing the column with PBS.
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