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• W2068361985 abstract "Phosphorylation is one of the most common posttranslational modifications controlling cellular protein activity. Here, we describe a combined computational and experimental strategy to design new phosphorylation sites into globular proteins to regulate their functions. We target a peptide recognition protein, the Erbin PDZ domain, to be phosphorylated by cAMP-dependent protein kinase. Comparing the five successful designs to the unsuccessful cases, we find a trade-off between protein stability and the ability to be modified by phosphorylation. In two designs, Erbin's peptide binding function is modified by phosphorylation, where the presence of the phosphate group destabilizes peptide binding. One of these showed an additional switch in specificity by introducing favorable interactions between a designed arginine in the peptide and phosphoserine on the PDZ domain. Because of the diversity of PDZ domains, this opens avenues for the design of related phosphoswitchable domains to create a repertoire of regulatable interaction parts for synthetic biology. Phosphorylation is one of the most common posttranslational modifications controlling cellular protein activity. Here, we describe a combined computational and experimental strategy to design new phosphorylation sites into globular proteins to regulate their functions. We target a peptide recognition protein, the Erbin PDZ domain, to be phosphorylated by cAMP-dependent protein kinase. Comparing the five successful designs to the unsuccessful cases, we find a trade-off between protein stability and the ability to be modified by phosphorylation. In two designs, Erbin's peptide binding function is modified by phosphorylation, where the presence of the phosphate group destabilizes peptide binding. One of these showed an additional switch in specificity by introducing favorable interactions between a designed arginine in the peptide and phosphoserine on the PDZ domain. Because of the diversity of PDZ domains, this opens avenues for the design of related phosphoswitchable domains to create a repertoire of regulatable interaction parts for synthetic biology. ▸ Successful design of phosphorylation sites into a globular protein ▸ Redesigned PDZ domain phosphorylation is associated with local destabilization ▸ Phosphorylation switches peptide binding function ▸ Phosphorylation switches peptide recognition specificity Rational protein design has been increasingly successful at producing proteins with new functions. Many early results involved adapting an existing function for new uses, such as changing enzyme substrate specificity (Wells et al., 1987Wells J.A. Powers D.B. Bott R.R. Graycar T.P. Estell D.A. Designing substrate specificity by protein engineering of electrostatic interactions.Proc. Natl. Acad. Sci. USA. 1987; 84: 1219-1223Crossref PubMed Scopus (214) Google Scholar; Yoshikuni et al., 2006Yoshikuni Y. Ferrin T.E. Keasling J.D. Designed divergent evolution of enzyme function.Nature. 2006; 440: 1078-1082Crossref PubMed Scopus (347) Google Scholar), modifying protein interaction specificity (Shifman and Mayo, 2003Shifman J.M. Mayo S.L. Exploring the origins of binding specificity through the computational redesign of calmodulin.Proc. Natl. Acad. Sci. USA. 2003; 100: 13274-13279Crossref PubMed Scopus (94) Google Scholar; Kortemme et al., 2004Kortemme T. Joachimiak L.A. Bullock A.N. Schuler A.D. Stoddard B.L. Baker D. Computational redesign of protein-protein interaction specificity.Nat. Struct. Mol. Biol. 2004; 11: 371-379Crossref PubMed Scopus (255) Google Scholar; Grigoryan et al., 2009Grigoryan G. Reinke A.W. Keating A.E. Design of protein-interaction specificity gives selective bZIP-binding peptides.Nature. 2009; 458: 859-864Crossref PubMed Scopus (286) Google Scholar; Kapp et al., 2012Kapp G.T. Liu S. Stein A. Wong D.T. Reményi A. Yeh B.J. Fraser J.S. Taunton J. Lim W.A. Kortemme T. Control of protein signaling using a computationally designed GTPase/GEF orthogonal pair.Proc. Natl. Acad. Sci. USA. 2012; 109: 5277-5282Crossref PubMed Scopus (68) Google Scholar), or altering fluorescent properties (Treynor et al., 2007Treynor T.P. Vizcarra C.L. Nedelcu D. Mayo S.L. Computationally designed libraries of fluorescent proteins evaluated by preservation and diversity of function.Proc. Natl. Acad. Sci. USA. 2007; 104: 48-53Crossref PubMed Scopus (76) Google Scholar). More recent efforts have shown remarkable progress in creating proteins with entirely new functions, including enzymatic activity (Jiang et al., 2008Jiang L. Althoff E.A. Clemente F.R. Doyle L. Röthlisberger D. Zanghellini A. Gallaher J.L. Betker J.L. Tanaka F. Barbas 3rd, C.F. et al.De novo computational design of retro-aldol enzymes.Science. 2008; 319: 1387-1391Crossref PubMed Scopus (904) Google Scholar; Röthlisberger et al., 2008Röthlisberger D. Khersonsky O. Wollacott A.M. Jiang L. DeChancie J. Betker J. Gallaher J.L. Althoff E.A. Zanghellini A. Dym O. et al.Kemp elimination catalysts by computational enzyme design.Nature. 2008; 453: 190-195Crossref PubMed Scopus (989) Google Scholar), de novo protein binding (Karanicolas et al., 2011Karanicolas J. Corn J.E. Chen I. Joachimiak L.A. Dym O. Peck S.H. Albeck S. Unger T. Hu W. Liu G. et al.A de novo protein binding pair by computational design and directed evolution.Mol. Cell. 2011; 42: 250-260Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), biomineralization catalysis (Masica et al., 2010Masica D.L. Schrier S.B. Specht E.A. Gray J.J. De novo design of peptide-calcite biomineralization systems.J. Am. Chem. Soc. 2010; 132: 12252-12262Crossref PubMed Scopus (41) Google Scholar), and oxygen transport (Koder et al., 2009Koder R.L. Anderson J.L. Solomon L.A. Reddy K.S. Moser C.C. Dutton P.L. Design and engineering of an O(2) transport protein.Nature. 2009; 458: 305-309Crossref PubMed Scopus (193) Google Scholar). Proteins have also been engineered to adapt and respond to their environment in different ways, such as pH-sensitive antibodies (Murtaugh et al., 2011Murtaugh M.L. Fanning S.W. Sharma T.M. Terry A.M. Horn J.R. A combinatorial histidine scanning library approach to engineer highly pH-dependent protein switches.Protein Sci. 2011; 20: 1619-1631Crossref PubMed Scopus (57) Google Scholar), proteins that change folds in response to pH or other ions (Cerasoli et al., 2005Cerasoli E. Sharpe B.K. Woolfson D.N. ZiCo: a peptide designed to switch folded state upon binding zinc.J. Am. Chem. Soc. 2005; 127: 15008-15009Crossref PubMed Scopus (92) Google Scholar; Palmer et al., 2006Palmer A.E. Giacomello M. Kortemme T. Hires S.A. Lev-Ram V. Baker D. Tsien R.Y. Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs.Chem. Biol. 2006; 13: 521-530Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar; Ambroggio and Kuhlman, 2006Ambroggio X.I. Kuhlman B. Computational design of a single amino acid sequence that can switch between two distinct protein folds.J. Am. Chem. Soc. 2006; 128: 1154-1161Crossref PubMed Scopus (141) Google Scholar), small domains that fold or unfold upon phosphorylation (Balakrishnan and Zondlo, 2006Balakrishnan S. Zondlo N.J. Design of a protein kinase-inducible domain.J. Am. Chem. Soc. 2006; 128: 5590-5591Crossref PubMed Scopus (56) Google Scholar; Riemen and Waters, 2009Riemen A.J. Waters M.L. Controlling peptide folding with repulsive interactions between phosphorylated amino acids and tryptophan.J. Am. Chem. Soc. 2009; 131: 14081-14087Crossref PubMed Scopus (17) Google Scholar), phosphorylation-induced oligomerization of alpha helices (Szilák et al., 1997Szilák L. Moitra J. Vinson C. Design of a leucine zipper coiled coil stabilized 1.4 kcal mol-1 by phosphorylation of a serine in the e position.Protein Sci. 1997; 6: 1273-1283Crossref PubMed Scopus (47) Google Scholar; Signarvic and DeGrado, 2003Signarvic R.S. DeGrado W.F. De novo design of a molecular switch: phosphorylation-dependent association of designed peptides.J. Mol. Biol. 2003; 334: 1-12Crossref PubMed Scopus (47) Google Scholar), and FRET-based reporters of protein kinase activity (Zhang et al., 2001Zhang J. Ma Y. Taylor S.S. Tsien R.Y. Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 14997-15002Crossref PubMed Scopus (491) Google Scholar). Such designs can form the basis of synthetic, posttranslational regulatory machinery for programming cellular behavior. Nature has evolved many mechanisms for modifying and regulating protein function, with one of the most pervasive being phosphorylation. One of the simplest regulatory mechanisms is disruption of a protein-protein interface via addition of a phosphate group. Other proteins create interactions upon phosphorylation, with domain families, such as SH2 and 14-3-3, having specifically evolved for this purpose. Phosphorylation can also alter intramolecular properties that change protein activity, with phosphorylation of the kinase “activation loop” being a canonical example (Steinberg et al., 1993Steinberg R.A. Cauthron R.D. Symcox M.M. Shuntoh H. Autoactivation of catalytic (C alpha) subunit of cyclic AMP-dependent protein kinase by phosphorylation of threonine 197.Mol. Cell. Biol. 1993; 13: 2332-2341Crossref PubMed Google Scholar; Adams et al., 1995Adams J.A. McGlone M.L. Gibson R. Taylor S.S. Phosphorylation modulates catalytic function and regulation in the cAMP-dependent protein kinase.Biochemistry. 1995; 34: 2447-2454Crossref PubMed Scopus (133) Google Scholar). Phosphorylation sites can be readily introduced into proteins by fusion of an existing phosphorylatable domain or disordered region. One of the first reported instances of a phosphorylation site introduced through site-specific mutation was the incorporation of a cAMP-dependent protein kinase (PKA) recognition sequence in the C-terminal tail of human interferon α (IFN-α) (Li et al., 1989Li B.L. Langer J.A. Schwartz B. Pestka S. Creation of phosphorylation sites in proteins: construction of a phosphorylatable human interferon alpha.Proc. Natl. Acad. Sci. USA. 1989; 86: 558-562Crossref PubMed Scopus (62) Google Scholar), with the goal of creating radioactive 32P-labeled proteins for research and clinical use. That work was followed by other examples, including mutation of the C-terminal region of a monoclonal antibody to be recognized by casein kinase I (CKI) (Lin et al., 1999Lin L. Gillies S.D. Schlom J. Pestka S. Construction of phosphorylatable chimeric monoclonal antibody CC49 with a casein kinase I recognition site.Protein Expr. Purif. 1999; 15: 83-91Crossref PubMed Scopus (7) Google Scholar). Another phosphosite was designed in the semiflexible linker region between the Fab and Fc regions of a monoclonal antibody heavy chain (Wu et al., 2004Wu W. Kerrigan J.E. Yadav P. Schwartz B. Izotova L. Lavoie T.B. Pestka S. Design and construction of a phosphorylatable chimeric monoclonal antibody with a highly stable phosphate.Oncol. Res. 2004; 14: 541-558PubMed Google Scholar). Beyond producing radiolabeled proteins, other phosphosite design efforts have aimed at modulating protein-protein interactions and protein regulation. Yeh and colleagues (Yeh et al., 2007Yeh B.J. Rutigliano R.J. Deb A. Bar-Sagi D. Lim W.A. Rewiring cellular morphology pathways with synthetic guanine nucleotide exchange factors.Nature. 2007; 447: 596-600Crossref PubMed Scopus (101) Google Scholar) mutated a peptide recognized by the syntrophin PDZ domain so that it could also be phosphorylated by PKA. Upon phosphorylation the PDZ-peptide interaction was destabilized, which allowed activation of individual guanine exchange factors (GEF) fused between the peptide and PDZ domain, whose interaction conformationally restricted and inactivated the GEF. As illustrated above, most previous studies have focused on introducing phosphorylation sites into sequences outside folded protein domains or in triggering folding/unfolding transitions upon phosphorylation (Balakrishnan and Zondlo, 2006Balakrishnan S. Zondlo N.J. Design of a protein kinase-inducible domain.J. Am. Chem. Soc. 2006; 128: 5590-5591Crossref PubMed Scopus (56) Google Scholar; Riemen and Waters, 2009Riemen A.J. Waters M.L. Controlling peptide folding with repulsive interactions between phosphorylated amino acids and tryptophan.J. Am. Chem. Soc. 2009; 131: 14081-14087Crossref PubMed Scopus (17) Google Scholar). Here, we describe the work that involves phosphorylation of a globular domain itself to modulate its function. This should enable new avenues for designing controllable domain-domain interactions, enzyme activities, and conformational changes. In addition, characterization of design successes and failures may provide clues about how phosphorylation sites evolve in folded domains. We make use of the Erbin PDZ domain, whose peptide interaction specificities have been extensively characterized with phage display (Laura et al., 2002Laura R.P. Witt A.S. Held H.A. Gerstner R. Deshayes K. Koehler M.F. Kosik K.S. Sidhu S.S. Lasky L.A. The Erbin PDZ domain binds with high affinity and specificity to the carboxyl termini of delta-catenin and ARVCF.J. Biol. Chem. 2002; 277: 12906-12914Crossref PubMed Scopus (130) Google Scholar; Tonikian et al., 2008Tonikian R. Zhang Y. Sazinsky S.L. Currell B. Yeh J.H. Reva B. Held H.A. Appleton B.A. Evangelista M. Wu Y. et al.A specificity map for the PDZ domain family.PLoS Biol. 2008; 6: e239Crossref PubMed Scopus (356) Google Scholar; Ernst et al., 2009Ernst A. Sazinsky S.L. Hui S. Currell B. Dharsee M. Seshagiri S. Bader G.D. Sidhu S.S. Rapid evolution of functional complexity in a domain family.Sci. Signal. 2009; 2: ra50Crossref PubMed Scopus (52) Google Scholar). We first apply a computational design strategy to select mutations that are predicted to not disrupt native binding activity in the unphosphorylated form and to be phosphorylated by PKA. Guided by characterization of thermostability, chemical shifts, and solvent exposure, we then evaluate the successes and limitations of the design strategy and generate several additional successful designs. Finally, we characterize the changes in binding affinity and interaction specificity upon phosphorylation with a set of peptide variants. We first wanted to develop and test a computational strategy to generate sites in the Erbin PDZ domain that could be phosphorylated by cAMP dependent protein kinase (PKA), irrespective of whether phosphorylation would affect peptide binding to the PDZ domain. Exhaustive scanning of a degenerate PKA recognition motif yielded over 1,000 potential sequences (see the Experimental Procedures; Figure 1). We used the change in Rosetta score (Smith and Kortemme, 2008Smith C.A. Kortemme T. Backrub-like backbone simulation recapitulates natural protein conformational variability and improves mutant side-chain prediction.J. Mol. Biol. 2008; 380: 742-756Crossref PubMed Scopus (230) Google Scholar) in the presence of the peptide as a proxy for the extent of disruption in fold stability or binding affinity. Our protocol incorporated limited amounts of backbone flexibility shown to better predict side-chain conformations (Smith and Kortemme, 2008Smith C.A. Kortemme T. Backrub-like backbone simulation recapitulates natural protein conformational variability and improves mutant side-chain prediction.J. Mol. Biol. 2008; 380: 742-756Crossref PubMed Scopus (230) Google Scholar). To computationally approximate the probability of phosphorylation, we used pkaPS (Neuberger et al., 2007Neuberger G. Schneider G. Eisenhaber F. pkaPS: prediction of protein kinase A phosphorylation sites with the simplified kinase-substrate binding model.Biol. Direct. 2007; 2: 2Crossref PubMed Scopus (179) Google Scholar), a sequence-based predictor of phosphorylation sites optimized for PKA. Using those computational tools and subsequent manual inspection of a reduced number of candidates (Figure 1), we selected eight sequences for testing (Figure 2; Table 1). Each design was named for the serine residue targeted for phosphorylation (e.g., S13 standing for serine 13).Figure 2Initial Phosphosite DesignsShow full caption(A) Protein sequences of wild-type Erbin PDZ domain and eight designed phosphorylation candidates. Amino acids are numbered such that residue 1 is the first amino acid present in the PDB structure 1MFG (Birrane et al., 2003Birrane G. Chung J. Ladias J.A. Novel mode of ligand recognition by the Erbin PDZ domain.J. Biol. Chem. 2003; 278: 1399-1402Crossref PubMed Scopus (94) Google Scholar) (i.e., residue 1277 in full-length Erbin). Serines targeted for phosphorylation are shown in red, added arginines are shown in blue, and added hydrophobic amino acids are shown in orange. See also Figure S1 for gel filtration chromatography to characterize the oligomeric state of WT and S47.(B) Structural distribution of target serines shown on PDB 1MFG (Birrane et al., 2003Birrane G. Chung J. Ladias J.A. Novel mode of ligand recognition by the Erbin PDZ domain.J. Biol. Chem. 2003; 278: 1399-1402Crossref PubMed Scopus (94) Google Scholar) with C-alpha atoms shown as spheres and the bound peptide shown in purple. All structure figures were rendered with PyMOL (Schrödinger, LLC, 2012Schrödinger, LLC. (2012). The PyMOL Molecular Graphics System, Version 1.5.Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1Scoring of Initial Phosphosite DesignsProteinMutant SequenceWT SequenceRosetta Δ ScorepkaPS ScoreS13RKDSEEKDPE−5.500.96S14KRPSLKDPEL−1.541.31S33FRPSVFRPDD−0.441.09S43RRVSPTRVQP−0.121.76S45VRPSGVQPEG−3.171.08S47RRGSAPEGPA−0.722.06S53KRLSPKLLQP−0.501.37S82RRFSFKTFQN−0.581.15Initial phosphosite designs based on having a negative change in Rosetta score (relative to the wild-type) and a high pkaPS score. Mutated residues are in bold. Open table in a new tab (A) Protein sequences of wild-type Erbin PDZ domain and eight designed phosphorylation candidates. Amino acids are numbered such that residue 1 is the first amino acid present in the PDB structure 1MFG (Birrane et al., 2003Birrane G. Chung J. Ladias J.A. Novel mode of ligand recognition by the Erbin PDZ domain.J. Biol. Chem. 2003; 278: 1399-1402Crossref PubMed Scopus (94) Google Scholar) (i.e., residue 1277 in full-length Erbin). Serines targeted for phosphorylation are shown in red, added arginines are shown in blue, and added hydrophobic amino acids are shown in orange. See also Figure S1 for gel filtration chromatography to characterize the oligomeric state of WT and S47. (B) Structural distribution of target serines shown on PDB 1MFG (Birrane et al., 2003Birrane G. Chung J. Ladias J.A. Novel mode of ligand recognition by the Erbin PDZ domain.J. Biol. Chem. 2003; 278: 1399-1402Crossref PubMed Scopus (94) Google Scholar) with C-alpha atoms shown as spheres and the bound peptide shown in purple. All structure figures were rendered with PyMOL (Schrödinger, LLC, 2012Schrödinger, LLC. (2012). The PyMOL Molecular Graphics System, Version 1.5.Google Scholar). Initial phosphosite designs based on having a negative change in Rosetta score (relative to the wild-type) and a high pkaPS score. Mutated residues are in bold. Using a radiolabeled phosphotransfer assay, the eight designs were tested for phosphorylation by PKA. S47 and S82 both showed significant levels of phosphorylation, with a stronger signal at S47 (Figure 3A). Using mass spectrometry, we confirmed that each domain was singly phosphorylated (i.e., M+80) and found that S47 was phosphorylated to completion in the time used for the gel-based assays, whereas S82 was not. Residue 47 is in the middle of a 15-residue loop between the third and fourth beta strands of the Erbin PDZ domain (Figure 2B). Residue 82 is located on a short loop between the alpha helix and the last beta strand. The pattern of phosphorylation did not strictly follow the pkaPS scores: although S47 had the highest pkaPS score (2.1; Table 1), the designs with next three highest scores (1.8–1.3) did not phosphorylate. However, S82, the construct with the next lowest score (1.2), could be phosphorylated. Given that known phosphorylation sites are often found in disordered regions of proteins (Iakoucheva et al., 2004Iakoucheva L.M. Radivojac P. Brown C.J. O'Connor T.R. Sikes J.G. Obradovic Z. Dunker A.K. The importance of intrinsic disorder for protein phosphorylation.Nucleic Acids Res. 2004; 32: 1037-1049Crossref PubMed Scopus (1126) Google Scholar), we hypothesized that disorder and/or protein stability may play a role in substrate phosphorylation. To determine whether this was the case for our designs, we used apparent melting temperatures from thermal denaturation as a proxy for protein stability. Figure 3B shows melting curves measured by circular dichroism for wild-type Erbin and the eight designs. S82 and S47, the two variants that could be phosphorylated, also had the two lowest melting temperatures of the initial eight designs (Table 2). These lower melting temperatures relative to the wild-type protein were unexpected (based on our design criterion to not destabilize the protein) and indicate known difficulties in accurately estimating the energetic effects of surface mutations. As discussed later, these two designs were still functional and bound to all tested peptides (Table 3). S47 showed a 4- to 6-fold reduction in binding affinity over wild-type and S82 bound slightly tighter than wild-type (less than a 2-fold increase). Reversible GuHCl denaturation of WT and S47 mirrored the thermal denaturation results and showed that S47 had lower fold stability than WT (Figure S2 available online). The qualitative rates of phosphorylation do not necessarily correlate, as S82 was less thermostable than the more rapidly phosphorylated S47.Table 2Apparent Melting Temperatures of Phosphosite DesignsInitial DesignsSecondary DesignsProteinTm,appProteinTm,appS8244S13-A4743S4746S43-A4744S3349S14-A4744S5350S45-A4748S4357S47A50S4558S13-A47a6xHis tag removed. Phosphorylated designs are indicated by “p”.50S1360S14-A47a6xHis tag removed. Phosphorylated designs are indicated by “p”.51S1461pS13-A47a6xHis tag removed. Phosphorylated designs are indicated by “p”.55WT66pS14-A47a6xHis tag removed. Phosphorylated designs are indicated by “p”.55Temperatures (Tm,app) in degrees Celcius from irreversible thermal denaturation measured by circular dichroism signal at 218 nm.a 6xHis tag removed. Phosphorylated designs are indicated by “p”. Open table in a new tab Table 3Binding Affinities of Unphosphorylated and Phosphorylated DesignsPeptidesProteinTGWETWVTGWETRVTGWETDVS13-A470.022 ± 0.00251.4 ± 0.030.71 ± 0.11pS13-A470.072 ± 0.00164.5 ± 0.613.2 ± 0.65S14-A470.017 ± 0.00113.3 ± 0.070.54 ± 0.10pS14-A470.17 ± 0.03222 ± 2.34.7 ± 1.3S43-A470.050 ± 0.002326 ± 0.316 ± 0.2pS43-A470.45 ± 0.03437 ± 2.8123 ± 7.7S470.094 ± 0.01828 ± 0.621 ± 0.6pS470.14 ± 0.00325 ± 1.125 ± 0.8S820.017 ± 0.00083.2 ± 0.031.9 ± 0.12pS820.024 ± 0.00284.5 ± 0.223.1 ± 0.04WT0.022 ± 0.00165.2 ± 0.303.5 ± 0.18Dissociation constants (Kd) in μM derived from fluorescence polarization experiments for unphosphorylated and phosphorylated PDZ domains with three peptides differing at the −1 position. Standard errors from multiple independent experiments are given. See also Figure S5 for individual binding curves. Phosphorylated designs are indicated by “p”. Open table in a new tab Temperatures (Tm,app) in degrees Celcius from irreversible thermal denaturation measured by circular dichroism signal at 218 nm. Dissociation constants (Kd) in μM derived from fluorescence polarization experiments for unphosphorylated and phosphorylated PDZ domains with three peptides differing at the −1 position. Standard errors from multiple independent experiments are given. See also Figure S5 for individual binding curves. Phosphorylated designs are indicated by “p”. To investigate structural differences between wild-type and S47, the mutant that showed the greatest rate of phosphorylation, we acquired HSQC spectra for it as well as wild-type Erbin in the unbound form (Figure 4A). Although assignments were previously determined for wild-type Erbin bound to peptide TGWETWV (Skelton et al., 2003Skelton N.J. Koehler M.F. Zobel K. Wong W.L. Yeh S. Pisabarro M.T. Yin J.P. Lasky L.A. Sidhu S.S. Origins of PDZ domain ligand specificity. Structure determination and mutagenesis of the Erbin PDZ domain.J. Biol. Chem. 2003; 278: 7645-7654Crossref PubMed Scopus (126) Google Scholar), there were significant differences between it and the free form, preventing assignment transfer. Therefore, we obtained de novo 1H, 15N, 13CA, and 13CB resonance assignments for both WT and S47 in the unbound form. In Figure 4A, peaks for equivalent residues in WT and S47 are connected by lines. Also, as indicated, the spectrum for S47 had two additional 1H-15N peaks because of the P44R and P47S mutations. Figure 4B shows atom-specific chemical shift differences, as well as the overall chemical shift difference normalized by the standard deviation (σ) of all resonances found in a database of protein chemical shifts (Ulrich et al., 2008Ulrich E.L. Akutsu H. Doreleijers J.F. Harano Y. Ioannidis Y.E. Lin J. Livny M. Mading S. Maziuk D. Miller Z. et al.BioMagResBank.Nucleic Acids Res. 2008; 36: D402-D408PubMed Google Scholar). As expected, the most extensive chemical shift changes (up to 1σ) were for residues 42–52, which are next to the mutated residues in sequence. Interestingly, a segment of residues adjacent in the tertiary structure, 7–17, also had large shift differences of up to 0.7σ (Figure 4C), and a more distant segment, 82–85, had smaller differences up to 0.3σ. These differences suggest that there was a change in structure and/or dynamics that was more than would be expected for a mutation of several solvent-exposed residues. To accommodate recognition by PKA, there is likely some degree of local or global structural opening/unfolding that is either a transient or stable feature of the solution ensemble. We reasoned that such opening is likely associated with an increased solvent exchange of amide hydrogens. Using CLEANEX hydrogen exchange experiments (Hwang et al., 1998Hwang T.L. van Zijl P.C. Mori S. Accurate quantitation of water-amide proton exchange rates using the phase-modulated CLEAN chemical EXchange (CLEANEX-PM) approach with a Fast-HSQC (FHSQC) detection scheme.J. Biomol. NMR. 1998; 11: 221-226Crossref PubMed Scopus (296) Google Scholar) with mixing times from 10–100 ms, we observed residues in all three segments mentioned above having greater hydrogen exchange in S47 than in WT (Figure 5). These changes in solvent exchange are among the most significant observed over the whole protein structure. Assuming the hydrogen exchange profiles of the other nonphosphorylatable designs are similar to WT, this supports a model in which S47 is shifted toward a more open conformation at equilibrium, possibly contributing to recognition and phosphorylation by PKA. After noting the chemical shift differences in segments 7–17 and 42–52 of S47, we hypothesized that the structural changes enabling S47 to be phosphorylated might also rescue phosphorylation at other sites located in the altered regions. Furthermore, the increase in S47 hydrogen exchange at residues 15, 45, and 46 suggested that the changes might be related to structural opening. Therefore, we designed a different template protein, A47, which included the arginine mutations of S47 (P44R and E45R) but incorporated an alanine at position 47 to prevent phosphorylation. Using that template, we reintroduced the mutations for the previous S13, S14, S43, and S45 designs (Figure 6A). These candidate phosphosites were chosen because they were located on the structurally perturbed loops and in regions near the peptide that were more likely to affect binding affinity. Both radiolabeled phosphotransfer assays and mass spectrometry indicated that A47 did not phosphorylate (Figure 6B), as expected, showing that phosphorylation of S47 was specific to serine 47 and that the A47 construct was suitable for addition of other sites. The same assays showed that three out of the four constructs (S13, S14, and S43) could now be phosphorylated in the A47 background (Figure 6B). Determination of the thermostability using circular dichroism showed that A47 was more stable than S47 but still significantly less stable than WT. All of the secondary phosphosite designs were less stable than the A47 construct on which they were based, and the three phosphorylatable designs were less stable than S47. Beyond stability (Table 2 lists apparent melting temperatures) other sequence-based factors could play a role in the failure of the other designs. S33, S45–A47, and S53 had apparent melting temperatures in the 48°C–50°C range that were close to the other phosphorylatable designs. However, although all have an arginine residue two sequence positions before the serine, none of them have an arginine three positions before, likely reducing their recognition by PKA, which strongly prefers two upstream arginines at the −3 and −2 positions (Kemp et al., 1977Kemp B.E. Graves D.J. Benjamini E. Krebs E.G. Role of multiple basic residues in determining the substrate specificity of cyclic AMP-dependent protein kinase.J. Biol. Chem. 1977; 252: 4888-4894Abstract Full Text PDF PubMed Google Scholar; Hutti et al., 2004Hutti J.E. Jarrell E.T. Chang J.D. Abbott D.W. Storz P. Toker A. Cantley L.C. Turk B.E. A rapid method for determining protein kinase phosphorylation specificity.Nat. Methods. 2004; 1: 27-29Crossref Pub" @default.
• W2068361985 created "2016-06-24" @default.
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• W2068361985 date "2013-01-01" @default.
• W2068361985 modified "2023-09-30" @default.
• W2068361985 title "Design of a Phosphorylatable PDZ Domain with Peptide-Specific Affinity Changes" @default.
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