Progress Report 0728’08
MinD Walker A BoxIV Walker B ParA1NCScoe MTS N-terminal ParA1Scoe ParA/MinD superfamily of the Walker-type ATPases 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 Streptomycescoelicolor • 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-ParBinteracting domain - Dimerizationand 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 Add illustration of Plac- parABS merge YFP DAPI DIC MC1000/pSOT (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 Add illustration of Plac- parAB merge YFP DAPI DIC MC1000/pSOT (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 inhibit MreB ATPase activity by binding to the nucleotide pocket) 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
Mutations introduced to ParA1 function Domain K39E, G40V, P-loop , nucleotide binding/hydrolysis? K44E, P-loop, nucleotide binding D68A, BoxIV, catalytic carboxylate D152A, Walker B, nucleotide binding/γ–phosphate sensing R218E,, nucleoid localization R247E, C-terminal, nucleoid localizaton
Alignment of the ParA family proteins Walker A K44 (ParA1Scoe) Box IV Walker B
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 yfp::parA1_K44E In Walker A box 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 R31 and R8? merge YFP DAPI DIC R19E 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.
Nucleoid association of the mutant ParA1 proteins Mutations in the C-terminal domain R247E B. Subtilis Soj ref R218E Change to other positive charged residues? 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 N-terminus 2BEJ: Soj Add in mutated residues that affected in function 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)--
Will expression of ParA1 affect E. coli nucleoid? Check growth conditions Example of the cell and nucleoid figures? Growth curve Should calculate the normal cell!! Expression of ParA1 or ParB do not affect the host cell nucleoid length or inter-nucleoid distance.
How many ParA are there on one plasmid? Stoichiometry of ParA on the parS-ParB complex? Can parA nucleate on parS in the absence of ParB? Probably not Do the spots represent parS-ParB-ParA complex? Probably A competition between the parS-ParB complex and nucleoid DNA? How to reconcile the result with Dagmara’s model? Physiological roles of nucleoid localization? How to regulate in between? Why can’t we observe the nucleoid localization in S. coelicolor? The movement of parS containing plasmids? Can ParB activate ParA nucleation on parS?
Comparison of Soj localization (with or without Spo0J) spo0J+ (parB) Δspo0J Soj-GFP GFP-Soj Soj-GFP GFP-Soj DIC GFP DAPI GFP-soj movement in a spo0J+ background Quisel and Grossman 1999
Localization of mutated Soj (with or without Spo0J) Nucleotide binding ATP hydrolysis • Binding of Soj to either ATP or ADP may be required for polar localization • or DNA binding • Soj G12V (remain predominantly in the ATP-bound form) localization to • cell pole is not dependent on Spo0J Quisel and Grossman 1999
Single primer PCR P template Each arm 14-20mer, adjust to similar Tm (40-50 ℃) Gradient PCR (Tm ±5 ℃) with 200ng plasmid DNA Remove the parental plasmid DNA by DpnI digestion Transform into Top10 BioTechniques 29, 970-972 (2000)
Mutants affect Soj (ParA) DNA binding Soj associate with DNA directly through the two C-terminal Arginines. Hester and Lutkenhaus 2007
Soj-DNA association model The dimer interface is parallel to the axis of polymerization. DNA binding surface will be generated upon dimerization which could be cooperative Hester and Lutkenhaus 2007
This nonspecific DNA binding does not require the N-terminal extension that is likely involved in the specific binding required for regulation of the sop operon. The assembly on the DNA is the essential function in partitioning Hester and Lutkenhaus 2007
There are at least 3 factors involved in nucleoid targeting • N-terminal • nucleotide binding • C-terminal positive charged residues
Align the C-terminal of ParA and MinD The DNA association positive charged residues are not conserved in MinD
Compare the structures of Soj and MinD 2BEJ: Soj 1G3R: MinD G186 S216 R218 R247 Although the C-terminal regions are not conserved between ParA and MinD they share structure similarity
The effect of mutations in MinDΔ5 Plac-yfp::NTDparA1 ::MinDΔ5-MinE YFP DAPI merge DIC merge YFP DAPI DIC G186R MC1000 S216R YLS1 Plac-yfp::NTDparA1’ ::MinDΔ5 MC1000 Introduction of NTDparA1 and C-terminal arginines on MinD does not result in nucleoid localizaiton
and Next • Double labeling • YFP-ParA and CFP-ParB • parSA1B on miniF-sopABC • functional assay • FRAP on ParA1 coated nucleoid • ParA1 dynamic movement • Protein purification • in vitro studies
Localization of ParA1scoe • Effects on nucleoid • In the presence of ParB • ParA mutants • N-terminal, R19/R26/Q29 • ATPase domain • Nucleotide binding, K39/G40/K44 • Hydrolysis, D68 • Nucleoid association residues, R218/R247 Name the N-terminal domain
ParA1(N-terminal truncate) forms helical structures independent of MreB and Min system.