Conformation of a plasmid replication initiator protein affects its proteolysis by ClpXP system

Abstract

Proteins from the Rep family of DNA replication initiators exist mainly as dimers, but only monomers can initiate DNA replication by interaction with the replication origin (ori). In this study, we investigated both the activation (monomerization) and the degradation of the broad-host-range plasmid RK2 replication initiation protein TrfA, which we found to be a member of a class of DNA replication initiators containing winged helix (WH) domains. Our in vivo and in vitro experiments demonstrated that the ClpX-dependent activation of TrfA leading to replicationally active protein monomers and mutations affecting TrfA dimer formation, result in the inhibition of TrfA protein degradation by the ClpXP proteolytic system. These data revealed that the TrfA monomers and dimers are degraded at substantially different rates. Our data also show that the plasmid replication initiator activity and stability in E. coli cells are affected by ClpXP system only when the protein sustains dimeric form.

Figure 1

TrfA stability in E. coli cells (panel A). The stability of TrfA protein was analyzed after inhibition of translation as described under “Materials and Methods.” The intracellular stability of His6-TrfA was tested in E. coli wild-type and clpX, clpP, clpXP mutant strains. Densitometry analysis of Western blots enabled an estimation of TrfA half-life time in the analyzed strains, displayed in table. The stability of DnaB protein was analyzed in E. coli wild-type cells similarly as for TrfA protein (panel B). The dnaB gene was expressed constitutively from it's natural promoter. Anti-DnaB and anti-TrfA sera giving similar detection level, SuperSignal West Pico and chemiluminescence substrate kit was used for TrfA and DnaB analysis.

Figure 2

TrfA is proteolyzed by ClpXP. A proteolytic assay containing native 33-kDa TrfA as the subtrate was performed as described in the “Materials and Methods.” Time course (panel A) and control experiment where TrfA was incubated with ClpX or ClpP alone (panel B) are presented. Reactions were analyzed by SDS-PAGE and Coomassie staining. White arrow indicates the position of TrfA protein, black arrow indicates the position of the TrfA proteolysis product, TrfA*.

Figure 3

Activation by ClpX inhibits TrfA proteolysis. The experimental design is presented in panel (A). For activation (first step), His6-TrfA protein was incubated with or without (control reaction) ClpX. After activation, a portion of the reaction mixture was analyzed for TrfA activity in the FI* assay (see Materials and Methods) (panel B). The appearance of the FI* band indicates that the TrfA protein has been activated. The TrfA activation reaction was followed by the proteolytic assay (second step), which was initiated by the addition of ClpP or ClpXP. After incubation for the indicated time, samples were analyzed by SDS-PAGE and Coomassie staining (panel C) as described under “Materials and Methods.” The quantity of TrfA protein was estimated by densitometry and plotted (panel C).

Figure 4

Mutations altering the TrfA dimer formation affect replication activity and requirement for the ClpX-dependent activation. The His6-TrfA(S257F) and His6-TrfA(G254D/S267L) were analyzed along with His6-TrfA in glycerol gradients for dimer and monomer formation (A). Densitometry analysis of Western blots enabling an estimation of protein position in gradient is plotted in panel A. The chemical denaturation with guanidine HCl profiles of the His6-TrfA(S257F), the His6-TrfA(G254D/S267L), and the His6-TrfA were determined by the changes in ellipticity at 222 nm (B). Panel C shows comparison of the ClpX-dependent activation of the TrfA(S257F), the His6-TrfA(G254D/S267L), and the His6-TrfA. Panel D shows isothermal circular dichroism spectra of the analyzed TrfA variants in a range from 195 to 260 nm.

Figure 5

TrfA shares homology with members of the Rep initiators family. A model of the TrfA dimer (aa 190-382; coordinates are available from ftp://genesilico.pl/iamb/models/), with individual protomers in blue and green, showing residues that interfere with dimerization (A). A combination of two substitutions G254D/S267L (orange) results in protein that is monomeric. The exchange S257F (shown in gray) produces a protein with a more stable dimer interface. Panel B shows TrfA monomer bound to DNA. Substitutions P314S and D198N result in TrfA mutants defective in DNA binding. The substitution E361K results in a mutant protein that binds DNA more efficiently). Table in C shows properties of TrfA mutants: G254D/S267L, S257F, P314S, D198N, E361K, and CΔ305. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 6

Mutations within the TrfA dimer interface inhibit the protein proteolysis by ClpXP. Proteolytic reactions containing His6-TrfA (A), His6-TrfA(S257F) (B), and His6-TrfA(G254D/S267L) (C) were assembled as described under “Materials and Methods.” Incubation was performed for indicated time and products analyzed by SDS-PAGE and Coomassie staining.

Figure 7

TrfA mutant proteins with S257F and G254D/S267L substitutions exhibit increased intracellular stability in E. coli. The stability of His6-TrfA, His6-TrfA(S257F), and His6-TrfA(G254D/S267L) was tested in E. coli wild-type strain as described under “Materials and Methods.” The results of densitometry analysis of Western blots enabling an estimation of TrfA mutant proteins half-life times are displayed in the table.

Figure 8

Binding of ClpX to TrfA conformational variants. Protein interactions were tested using ELISA reaction according to procedure described under “Materials and Methods.” Immobilized proteins: His6-TrfA (filled circles), His6-TrfA(G254D/S267L) (open rectangles), His6-TrfA(S257F) (filled rectangles), λO (opened circle), and BSA (filled triangles) were incubated with 62.5, 125, 250, 500, or 750 ng of ClpX in the presence of ATP. Plate wells contained 0.5 μg of the relevant protein.