title: In silico analysis and experimental verification of OSR1 kinase - Peptide interaction.
abstract: The oxidative-stress-responsive kinase 1 (OSR1) and the STE20/SPS1-related proline/alanine-rich kinase (SPAK) are key enzymes in a signaling cascade regulating the activity of Na(+)-K(+)-2Cl(-) cotransporters (NKCC1-2) and Na(+)-Cl(-) cotransporter (NCC). Both kinases have a conserved carboxyl-terminal (CCT) domain, which recognizes a unique peptide motif present in OSR1- and SPAK-activating kinases (with-no-lysine kinase 1 (WNK1) and WNK4) as well as their substrates (NKCC1, NKCC2, and NCC). Utilizing various modalities of the Rosetta Molecular Modeling Software Suite including flexible peptide docking and protein design, we comprehensively explored the sequence space recognized by the CCT domain. Specifically, we studied single residue mutations as well as complete unbiased designs of a hexapeptide substrate. The computational study started from a crystal structure of the CCT domain of OSR1 in complex with a hexapeptide derived from WNK4. Point mutations predicted to be favorable include Arg to His or Trp substitutions at position 2 and a Phe to Tyr substitution at position 3 of the hexapeptide. In addition, de novo design yielded two peptides predicted to bind to the CCT domain: FRFQVT and TRFDVT. These results, which indicate a little bit more freedom in the composition of the peptide, were confirmed through the use of yeast two-hybrid screening.
authors.affiliation: Department of Anesthesiology, Vanderbilt University School of Medicine, Nashville, TN 37232, United States.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Austin
authors.affiliation: Department of Chemistry, Vanderbilt University, Nashville, TN, United States.
authors.forename: David P
authors.initials: DP
authors.lastname: Nannemann
authors.affiliation: Department of Chemistry, Vanderbilt University, Nashville, TN, United States.
authors.forename: Samuel L
authors.initials: SL
authors.lastname: Deluca
authors.affiliation: Department of Chemistry, Vanderbilt University, Nashville, TN, United States; Departments of Pharmacology and Bioinformatics, Vanderbilt University, Nashville, TN, United States.
authors.forename: Jens
authors.initials: J
authors.lastname: Meiler
authors.affiliation: Department of Anesthesiology, Vanderbilt University School of Medicine, Nashville, TN 37232, United States. Electronic address: eric.delpire@vanderbilt.edu.
authors.forename: Eric
authors.initials: E
authors.lastname: Delpire
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D015870:Gene Expression; D006801:Humans; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D011993:Recombinant Fusion Proteins; C486407:Stk39 protein, mouse; C534421:OXSR1 protein, mouse; D017346:Protein Serine-Threonine Kinases
keywords: Chloride transport; Computer modeling; Peptide conformation; Protein design; Rosetta Modeling Suite; Ste20 kinases