title: Differential binding between PatS C-terminal peptide fragments and HetR from Anabaena sp. PCC 7120.
abstract: Heterocyst differentiation in the filamentous cyanobacterium Anabaena sp. strain PCC 7120 occurs at regular intervals under nitrogen starvation and is regulated by a host of signaling molecules responsive to availability of fixed nitrogen. The heterocyst differentiation inhibitor PatS contains the active pentapeptide RGSGR (PatS-5) at its C-terminus considered the minimum PatS fragment required for normal heterocyst pattern formation. PatS-5 is known to bind HetR, the master regulator of heterocyst differentiation, with a moderate affinity and a submicromolar dissociation constant. Here we characterized the affinity of HetR for several PatS C-terminal fragments by measuring the relative ability of each fragment to knockdown HetR binding to DNA in electrophoretic mobility shift assays and using isothermal titration calorimetry (ITC). HetR bound to PatS-6 (ERGSGR) >30 times tighter (K(d) = 7 nM) than to PatS-5 (K(d) = 227 nM) and >1200 times tighter than to PatS-7 (DERGSGR) (K(d) = 9280 nM). No binding was detected between HetR and PatS-8 (CDERGSGR). Quantitative binding constants obtained from ITC measurements were consistent with qualitative results from the gel shift knockdown assays. CW EPR spectroscopy confirmed that PatS-6 bound to a MTSL spin-labeled HetR L252C mutant at a 10-fold lower concentration compared to PatS-5. Substituting the PatS-6 N-terminal glutamate to aspartate, lysine, or glycine did not alter binding affinity, indicating that neither the charge nor size of the N-terminal residue's side chain played a role in enhanced HetR binding to PatS-6, but rather increased binding affinity resulted from new interactions with the PatS-6 N-terminal residue peptide backbone.
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, United States.
authors.forename: Erik A
authors.initials: EA
authors.lastname: Feldmann
authors.forename: Shuisong
authors.initials: S
authors.lastname: Ni
authors.forename: Indra D
authors.initials: ID
authors.lastname: Sahu
authors.forename: Clay H
authors.initials: CH
authors.lastname: Mishler
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Levengood
authors.forename: Yegor
authors.initials: Y
authors.lastname: Kushnir
authors.forename: Robert M
authors.initials: RM
authors.lastname: McCarrick
authors.forename: Gary A
authors.initials: GA
authors.lastname: Lorigan
authors.forename: Blanton S
authors.initials: BS
authors.lastname: Tolbert
authors.forename: Sean M
authors.initials: SM
authors.lastname: Callahan
authors.forename: Michael A
authors.initials: MA
authors.lastname: Kennedy
mesh_terms: D019943:Amino Acid Substitution; D017033:Anabaena; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001483:Base Sequence; D002151:Calorimetry; D004578:Electron Spin Resonance Spectroscopy; D024202:Electrophoretic Mobility Shift Assay; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; C115380:PatS protein, Anabaena; D010446:Peptide Fragments; C067403:HetR protein, Bacteria