title: NDK Interacts with FtsZ and Converts GDP to GTP to Trigger FtsZ Polymerisation--A Novel Role for NDK.
abstract: BACKGROUND
  Nucleoside diphosphate kinase (NDK), conserved across bacteria to humans, synthesises NTP from NDP and ATP. The eukaryotic homologue, the NDPK, uses ATP to phosphorylate the tubulin-bound GDP to GTP for tubulin polymerisation. The bacterial cytokinetic protein FtsZ, which is the tubulin homologue, also uses GTP for polymerisation. Therefore, we examined whether NDK can interact with FtsZ to convert FtsZ-bound GDP and/or free GDP to GTP to trigger FtsZ polymerisation.
  
  
  METHODS
  Recombinant and native NDK and FtsZ proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis were used as the experimental samples. FtsZ polymersation was monitored using 90° light scattering and FtsZ polymer pelleting assays. The γ32P-GTP synthesised by NDK from GDP and γ32P-ATP was detected using thin layer chromatography and quantitated using phosphorimager. The FtsZ bound 32P-GTP was quantitated using phosphorimager, after UV-crosslinking, followed by SDS-PAGE. The NDK-FtsZ interaction was determined using Ni2+-NTA-pulldown assay and co-immunoprecipitation of the recombinant and native proteins in vitro and ex vivo, respectively.
  
  
  RESULTS
  NDK triggered instantaneous polymerisation of GDP-precharged recombinant FtsZ in the presence of ATP, similar to the polymerisation of recombinant FtsZ (not GDP-precharged) upon the direct addition of GTP. Similarly, NDK triggered polymerisation of recombinant FtsZ (not GDP-precharged) in the presence of free GDP and ATP as well. Mutant NDK, partially deficient in GTP synthesis from ATP and GDP, triggered low level of polymerisation of MsFtsZ, but not of MtFtsZ. As characteristic of NDK's NTP substrate non-specificity, it used CTP, TTP, and UTP also to convert GDP to GTP, to trigger FtsZ polymerisation. The NDK of one mycobacterial species could trigger the polymerisation of the FtsZ of another mycobacterial species. Both the recombinant and the native NDK and FtsZ showed interaction with each other in vitro and ex vivo, alluding to the possibility of direct phosphorylation of FtsZ-bound GDP by NDK.
  
  
  CONCLUSIONS
  Irrespective of the bacterial species, NDK interacts with FtsZ in vitro and ex vivo and, through the synthesis of GTP from FtsZ-bound GDP and/or free GDP, and ATP (CTP/TTP/UTP), triggers FtsZ polymerisation. The possible biological context of this novel activity of NDK is presented.
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
authors.forename: Saurabh
authors.initials: S
authors.lastname: Mishra
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
authors.forename: Kishor
authors.initials: K
authors.lastname: Jakkala
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
authors.forename: Ramanujam
authors.initials: R
authors.lastname: Srinivasan
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
authors.forename: Muthu
authors.initials: M
authors.lastname: Arumugam
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
authors.forename: Raghavendra
authors.initials: R
authors.lastname: Ranjeri
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
authors.forename: Prabuddha
authors.initials: P
authors.lastname: Gupta
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
authors.forename: Haryadi
authors.initials: H
authors.lastname: Rajeswari
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
authors.forename: Parthasarathi
authors.initials: P
authors.lastname: Ajitkumar
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000096:biosynthesis*; D046529:Microscopy, Electron, Transmission; D020102:Mycobacterium smegmatis / Q000235:genetics / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism; D009701:Nucleoside-Diphosphate Kinase / Q000235:genetics / Q000378:metabolism*; D058105:Polymerization; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; C071525:FtsZ protein, Bacteria; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D009701:Nucleoside-Diphosphate Kinase