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Thomas Austin , David P YJSBI 6577 No. of Pages 8, Model 5G 12 May 2014 Journal of Structural Biology xxx (2014) xxx–xxx 1 Contents lists available at ScienceDirect Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi 5 6 3 In silico analysis and experimental verification of OSR1 kinase – Peptide 4 interaction a b b b,c a,⇑ 7 Q1 Thomas Austin , David P. Nannemann , Samuel L. Deluca , Jens Meiler , Eric Delpire 8 Q2 a Department of Anesthesiology, Vanderbilt University School of Medicine, Nashville, TN 37232, United States 9 b Department of Chemistry, Vanderbilt University, Nashville, TN, United States 10 c Departments of Pharmacology and Bioinformatics, Vanderbilt University, Nashville, TN, United States 11 article info abstract 2713 14 Article history: The oxidative-stress-responsive kinase 1 (OSR1) and the STE20/SPS1-related proline/alanine-rich kinase 28 15 Received 10 December 2013 (SPAK) are key enzymes in a signaling cascade regulating the activity of Na +–K+–2ClÀ cotransporters 29 16 Received in revised form 25 April 2014 (NKCC1–2) and Na+–ClÀ cotransporter (NCC). Both kinases have a conserved carboxyl-terminal (CCT) 30 17 Accepted 4 May 2014 domain, which recognizes a unique peptide motif present in OSR1- and SPAK-activating kinases (with- 31 18 Available online xxxx no-lysine kinase 1 (WNK1) and WNK4) as well as their substrates (NKCC1, NKCC2, and NCC). Utilizing 32 various modalities of the Rosetta Molecular Modeling Software Suite including flexible peptide docking 33 19 Keywords: and protein design, we comprehensively explored the sequence space recognized by the CCT domain. 34 20 Computer modeling Specifically, we studied single residue mutations as well as complete unbiased designs of a hexapeptide 35 21 Protein design 22 Peptide conformation substrate. The computational study started from a crystal structure of the CCT domain of OSR1 in com- 36 23 Ste20 kinases plex with a hexapeptide derived from WNK4. Point mutations predicted to be favorable include Arg to His 37 24 Rosetta Modeling Suite or Trp substitutions at position 2 and a Phe to Tyr substitution at position 3 of the hexapeptide. In addi- 38 25 Chloride transport tion, de novo design yielded two peptides predicted to bind to the CCT domain: FRFQVT and TRFDVT. 39 26 These results, which indicate a little bit more freedom in the composition of the peptide, were confirmed 40 through the use of yeast two-hybrid screening. 41 Ó 2014 Published by Elsevier Inc. 42 43 44 45 46 1. Introduction and substrates involves a 90 residue domain located at the C-ter- 62 minal tail of the kinase and a short conserved peptide located 63 47 SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxi- within the substrate (Piechotta et al., 2002). Yeast 2-hybrid analy- 64 48 dative stress response 1 kinase) are members of the Ste20-related ses indicated that the peptide needed to be nine residues long, at 65 49 family of protein kinases (Dan et al., 2001; Delpire, 2009). They least when located at the extreme C-terminus of the bait protein. 66 50 modulate the activity of cation-chloride cotransporters (Gagnon Amino acid alignment between the cytosolic N-terminal tails of 67 51 et al., 2006; Grimm et al., 2012; Lin et al., 2011; McCormick membrane transporters (related targets) revealed the preliminary 68 52 et al., 2011; Rafiqi et al., 2010), which are involved ion secretion conserved sequence: Arg-Phe-Xaa-Val (Piechotta et al., 2002). Fol- 69 53 (Kurihara et al., 2002; Matthews et al., 1993) and reabsorption lowing a large yeast 2-hybrid screen that used the conserved car- 70 54 (Gimenez and Forbush, 2005; Pacheco-Alvarez et al., 2006) across boxyl-terminal domain of SPAK as bait (Piechotta et al., 2003), 71 55 a variety of epithelia, ClÀ homeostasis in neurons (Austin and the motif was expanded to [Val/Ser/Gly]-Arg-Phe-Xaa-[Val/Iso]- 72 56 Delpire, 2011; Blaesse et al., 2009; Delpire, 2000; Delpire and Xaa-Xaa-[Thre/Ser/Val/Iso]. A whole mouse proteome search iden- 73 57 Austin, 2010), and cell volume maintenance and regulation in tified some 170 proteins containing this expanded motif (Delpire 74 58 many cells (Gagnon and Delpire, 2012). Molecular studies have and Gagnon, 2007). 75 59 demonstrated that kinase binding to the substrate is a pre- The crystal structure of the 90 amino acid human OSR1 76 60 requisite for the function of SPAK and OSR1 (Gagnon et al., 2006; domain, which includes an embedded GRFQVT hexapeptide from 77 61 Piechotta et al., 2003, 2002). The interaction between SPAK/OSR1 human WNK4, was resolved at 1.95 Å (PDB ID: 2v3s, (Villa et al., 78 2007)). This domain, which was termed conserved carboxyl-termi- 79 nal (CCT) (Villa et al., 2007) or protein fold 2 (PF2) (Lee et al., 2009), 80 ⇑ Corresponding author. Address: Department of Anesthesiology, Vanderbilt is represented in Fig. 1. The most salient feature is a hydrophobic 81 University School of Medicine, T-4202 Medical Center North, 1161 21st Avenue South, Nashville, TN 37232, United States. Fax: +1 (615) 343 3916. groove that accommodates the Phe and Val residues of the peptide. 82 E-mail address: [email protected] (E. Delpire). While the protein fold was originally thought to be unique to SPAK 83 http://dx.doi.org/10.1016/j.jsb.2014.05.001 1047-8477/Ó 2014 Published by Elsevier Inc. Please cite this article in press as: Austin, T., et al. In silico analysis and experimental verification of OSR1 kinase – Peptide interaction. J. Struct. Biol. (2014), http://dx.doi.org/10.1016/j.jsb.2014.05.001 YJSBI 6577 No. of Pages 8, Model 5G 12 May 2014 2 T. Austin et al. / Journal of Structural Biology xxx (2014) xxx–xxx each residue of the hexapeptides was mutated into the other 19 120 canonical amino acids at all six positions, resulting in a total of 121 114 mutated peptides for each relaxed structure. The backbone 122 atoms of all of these mutants had similar three-dimensional coor- 123 dinates as their starting hexapeptides with the alterations only 124 occurring at the side chains. These engineered peptides were then 125 recombined with their corresponding unbound CCT domains (CCT- 126 peptide complexes with peptides removed). 127 The mutated hexapeptides, along with the native forms, were 128 then docked into the CCT domains by utilizing the FlexPepDock 129 application of Rosetta 3.4 (London et al., 2011; Raveh et al., 130 2010) with the following flags: -use_input_sc, -ex1, -ex2, -pep_re- 131 fine, and -unboundrot. Two hundred models were produced for 132 each relaxed structure with a total of one thousand models created 133 for each mutated hexapeptide. A low-resolution pre-optimization 134 flag (-lowres_preoptimize) was employed in half of the docking 135 runs in order to sample a larger peptide conformational space. A 136 Rosetta binding energy (ddG) was calculated to assess the stability 137 of the docked protein–peptide complex. The top ten models for 138 each mutation, regardless of initial relaxed structure, were deter- 139 Fig.1. PyMol rendering of the hydrophobic pocket of OSR1 with the GRFQVT mined based on ddG and reweighted total energy score. The 140 peptide of WNK4. The surface representation of the OSR1 domain highlights reweighted score is a combination of interface score, peptide score, 141 negative (red), positive (blue), and polar (green) moieties. (For interpretation of the 142 references to colour in this figure legend, the reader is referred to the web version of and total score and is a better scoring function than score12 in the this article.) case of flexible peptides docking onto their receptors (Raveh et al., 143 2011). After averaging the scores of the ten models, each mutant 144 was compared to the native CCT-peptide and other mutated pep- 145 tide complexes. 146 84 and OSR1 (Villa et al., 2007), we later showed that it is also present, Separately, the design module (Jha et al., 2010; Kuhlman and 147 85 at least partially, downstream of the catalytic domain of WNK4 Baker, 2000) of Rosetta 3.4.1 was applied to the native hexapeptide 148 86 (Delpire and Gagnon, 2008; Gagnon and Delpire, 2012). Availabil- in order to sample the sequence space consistent with CCT binding 149 87 ity of this domain – peptide structure allowed us to use the dock- in a more unbiased fashion. In our procedure, only the hexapeptide 150 88 ing and design applications of Rosetta, a protein structure was targeted for redesign while the CCT domain was left 151 89 prediction and functional design software package, to assess the untouched. High resolution docking of these designed peptides 152 90 binding of various hexapeptides in the pocket and estimate binding into the binding pocket of the CCT domain was achieved through 153 91 energies. Thus, this analysis allows us to better understand the the use of FlexPepDock with similar options as previously 154 92 amino acid requirements for the interaction by ranking peptides described. After docking, another iterative round of design was 155 93 with favorable to unfavorable energies. It also comprehensively performed on the hexapeptide to further refine the list of mutable 156 94 determines the peptide sequences consistent with OSR1 and SPAK residues. Again, two hundred decoys were produced for each 157 95 interaction thereby identifying potential sequences that have not relaxed structure with a total of one thousand models being cre- 158 96 yet been implied but can now be tested experimentally. ated. The top one hundred models based on ddG were analyzed 159 and compared to the native structure using PyMOL (PyMOL Molec- 160 97 2. Material and methods ular Graphics System, Version 1.5.0.4, Schrödinger, LLC).
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