Progress Report 0728’08
ParA/MinD superfamily of the Walker-type ATPases N-terminal Walker A BoxIV Walker B ParA1Scoe ParA1NCScoe MTS MinD highly conserved in ParA and MinD proteins relatively diverse in the various subgroups of the walker type ATPases similar function involving ATP hydrolysis Walker A (P-loop): (GXXXXGKT/S) BoxIV (Switch I):interacts with Mg2+ through a water mediated hydrogen bond network and activates a water molecule for an in-line attack of the γ–phosphate of nucleotide Walker B (switchII)the Asp of ZZZZDsenses the γ–phosphate phosphate in nucleotide binding Muneyuki and Yoshida 2000
Aims: • To study the mechanism of ParA1 mediated-chromosome partition is • in Streptomyces coelicolor • To confirm roles of ParA1 in partition chromosome - in mini-F plasmids - effects on nucleoids in E. coli • 2. To study ParA1 dynamic in live E. coli cells - helical structure formation - nucleoid association - relations to other cytoskeletal structures in vitro • To dissect functional domains of ParA1 • Nucleoid association - N-terminal domain - C-terminal domain - ATPase domain - ParA-ParB interacting domain - Dimerization and oligomerization domain
To confirm roles of ParA1 in partition chromosome Functional assay of miniF construct
2. To study ParA1 dynamic in live E. coli cells - helical structure formation - nucleoid association - relations to other cytoskeletal structures
Localization of full-length ParA1 and ParA1NC in E. coli Live cell Fixed cell YFP DAPI merge DIC MC1000/pSOT1032 (WT/Plac- yfp::parA1) MC1000/pSOT27 (WT/Plac- yfp::parA1NC) The N-terminal domain of ParA1Scoe (amino acids 1-32) dynamics is required for targeting ParA1 to the nucleoids.
ParA1 Localization in the presence of ParB and parS Plac parS merge yfp parA parB YFP DAPI DIC MC1000/pSOT1035 (WT/Plac- yfp::parA1 parB) In addition to the nucleoid localization, ParA1 localizes into bright foci in the presence of ParB.
ParA1 Localization in the presence of ParB and parS Plac parS* yfp parA parB merge DIC YFP DAPI MC1000/pSOT1044 (WT/Plac- yfp::parA1 parB*) parS/parB silent mutation The ParA1 foci are caused by nucleation of ParA1 on the parS-ParB complex formed on the plasmid.
Yfp-ParA1 localizes to nucleoids in E. coli and appears to undergo stochastic movement. Yfp-ParA1NC undergoes stochastic movement in E. coli and forms long-range filaments that wrap around the cell. 3. that may be caused by nucleation of ParA1 on the parS-ParB complex formed on the plasmid. 4. The ParA1 foci are caused by nucleation of ParA1 on the parS-ParB complex formed on the plasmid.
Evidence of ParA1NC can self-organize into filamentous structures in E. coli The Min system MreB, (A22 specifically, rapidly, and reversibly perturbs MreB function) FtsZ
Helical structure Formation of the ParA1NC is independent of the Min system Merge is not effectively showing the point YFP DIC merge RC1 /pSOT27 (Δ minCDE/Plac-yfp::parA1NC) ParA1NCmaintain the filament structure in the absence of minCDE
Formation of the ParA1NC helical structure is independent of MreB time image
ParA1NC forms helical structures in E. coli that is independent of the Min system and MreB.
To dissect functional domains of ParA1 Nucleoid association - N-terminal domain - C-terminal domain ATPase domain
Mutations in the ATPase domain performed in ParA family protein Walker A (P-loop), nucleotide binding Switch I (BoxIV), nucleotide hydrolysis Switch II (Walker B), senses the γ–phosphate • ParA1Scoe (Jakimowicz and Charter 2007) • Loss of function (plasmid stability assay) • No dynamic movement or diffused pattern • Not responding to ParB expression
Alignment among parA familty proteins Walker A 39 40 44 BoxIV Walker B 68 152
To test mutant phenotypes by examining Nucleoid association Filament formation in E. coli
Nucleoid association of the mutant ParA1 proteins Mutations in the ATPase domain merge YFP DAPI DIC In Walker A box yfp::parA1_K44E parA1 _K44E::yfp Plac-yfp::parA1_K44E-parB The mutation of K44E, prevents ParA1Scoe from associating with nucleoids
Mutant localization correspond to nucleoid merge YFP DAPI DIC K39E In Walker A box K44E In Walker B box G40V In box IV D154A The mutation in the Walker A and Walker B motifs prevented ParA1Scoe from associating with nucleoids D68A
Nucleoid association of the mutant ParA1 proteins Mutations in the N-terminal domain merge YFP DAPI DIC R19E R31 and R8? R26E R19E+R26E Q29E Although amino acids 1-31 are required for nucleoid -targeting of ParA1, the positively charged residues do not affect the nucleoid targeting, indicating the N-terminal domain may not directly associate with DNA.
YFP DAPI merge DIC Nucleoid localization of ParA1 is independent of the helical structure formaiton MC1000/pSOT1054 (WT/Plac-yfp::parA1’_G40V) +Aztreonam/ fixed cells From Ref, “ATP is required for the spiral structures formation” ParA1’_G40V retains the ability of filament formation.
Nucleoid association of the mutant ParA1 proteins Mutations in the C-terminal domain Soj (B. Subtilis ) Hester and Lutkenhaus 2007 R247E R218E R247 and R218 in ParA1Scoe are involved in nucleoid association, similar to Soj (ParA) of B. subtilis.
Predicted spatial correlation of N-terminal domain to the DNA association face 2BEJ: Soj N-terminus DNA association face The N-terminal domain is on the opposite site of the direct DNA association face
To confirm roles of ParA1 in partition chromosome - effects on nucleoids in E. coli Describe experimental procedures --Cells treated with aztreonam (FtsA inhibitor)--