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• W2070958052 abstract "Complete folding is not a prerequisite for protein function, as disordered and partially folded states of proteins frequently perform essential biological functions. In order to understand their functions at the molecular level, we utilized diverse experimental measurements to calculate ensemble models of three nonhomologous, intrinsically disordered proteins: I-2, spinophilin, and DARPP-32, which bind to and regulate protein phosphatase 1 (PP1). The models demonstrate that these proteins have dissimilar propensities for secondary and tertiary structure in their unbound forms. Direct comparison of these ensemble models with recently determined PP1 complex structures suggests a significant role for transient, preformed structure in the interactions of these proteins with PP1. Finally, we generated an ensemble model of partially disordered I-2 bound to PP1 that provides insight into the relationship between flexibility and biological function in this dynamic complex. Complete folding is not a prerequisite for protein function, as disordered and partially folded states of proteins frequently perform essential biological functions. In order to understand their functions at the molecular level, we utilized diverse experimental measurements to calculate ensemble models of three nonhomologous, intrinsically disordered proteins: I-2, spinophilin, and DARPP-32, which bind to and regulate protein phosphatase 1 (PP1). The models demonstrate that these proteins have dissimilar propensities for secondary and tertiary structure in their unbound forms. Direct comparison of these ensemble models with recently determined PP1 complex structures suggests a significant role for transient, preformed structure in the interactions of these proteins with PP1. Finally, we generated an ensemble model of partially disordered I-2 bound to PP1 that provides insight into the relationship between flexibility and biological function in this dynamic complex. First ensemble comparison of three different IDPs that bind the same target, PP1 Ensemble models of unbound PP1 regulators show diverse transient 2° and 3° structure Free and bound state similarities suggest preformed structure is important Model of partially disordered PP1:I-2 complex provides insight into function In recent years, many exceedingly flexible proteins with important biological functions, known as intrinsically disordered proteins (IDPs), have been described (Dunker et al., 2001Dunker A.K. Lawson J.D. Brown C.J. Williams R.M. Romero P. Oh J.S. Oldfield C.J. Campen A.M. Ratliff C.M. Hipps K.W. et al.Intrinsically disordered protein.J. Mol. Graph. Model. 2001; 19: 26-59Crossref PubMed Scopus (1836) Google Scholar, Dunker et al., 2002Dunker A.K. Brown C.J. Lawson J.D. Iakoucheva L.M. Obradovic Z. Intrinsic disorder and protein function.Biochemistry. 2002; 41: 6573-6582Crossref PubMed Scopus (1486) Google Scholar, Dunker and Obradovic, 2001Dunker A.K. Obradovic Z. The protein trinity–linking function and disorder.Nat. Biotechnol. 2001; 19: 805-806Crossref PubMed Scopus (496) Google Scholar, Iakoucheva et al., 2002Iakoucheva L.M. Brown C.J. Lawson J.D. Obradovic Z. Dunker A.K. Intrinsic disorder in cell-signaling and cancer-associated proteins.J. Mol. Biol. 2002; 323: 573-584Crossref PubMed Scopus (971) Google Scholar, Tompa, 2002Tompa P. Intrinsically unstructured proteins.Trends Biochem. Sci. 2002; 27: 527-533Abstract Full Text Full Text PDF PubMed Scopus (1672) Google Scholar, Uversky et al., 2000Uversky V.N. Gillespie J.R. Fink A.L. Why are “natively unfolded” proteins unstructured under physiologic conditions?.Proteins. 2000; 41: 415-427Crossref PubMed Scopus (1765) Google Scholar, Uversky, 2002aUversky V.N. Natively unfolded proteins: a point where biology waits for physics.Protein Sci. 2002; 11: 739-756Crossref PubMed Scopus (1506) Google Scholar, Uversky, 2002bUversky V.N. What does it mean to be natively unfolded?.Eur. J. Biochem. 2002; 269: 2-12Crossref PubMed Scopus (815) Google Scholar, Wright and Dyson, 1999Wright P.E. Dyson H.J. Intrinsically unstructured proteins: re-assessing the protein structure-function paradigm.J. Mol. Biol. 1999; 293: 321-331Crossref PubMed Scopus (2330) Google Scholar). IDPs lack the typical hydrophobic cores that generally stabilize folded proteins, and their highly dynamic nature prevents their description as single, rigid structures. However, structural studies of a number of IDPs indicate that they are not random-coil polymers; rather, they often have preferences for transient secondary and tertiary structure elements. It has been proposed that preorganization and spatial restriction in IDPs expose primary contact sites to enable faster and more effective binding to target molecules (Cheng et al., 2007Cheng Y. Oldfield C.J. Meng J. Romero P. Uversky V.N. Dunker A.K. Mining alpha-helix-forming molecular recognition features with cross species sequence alignments.Biochemistry. 2007; 46: 13468-13477Crossref PubMed Scopus (267) Google Scholar, Csizmok et al., 2005Csizmok V. Bokor M. Banki P. Klement E. Medzihradszky K.F. Friedrich P. Tompa K. Tompa P. Primary contact sites in intrinsically unstructured proteins: the case of calpastatin and microtubule-associated protein 2.Biochemistry. 2005; 44: 3955-3964Crossref PubMed Scopus (88) Google Scholar, Mohan et al., 2006Mohan A. Oldfield C.J. Radivojac P. Vacic V. Cortese M.S. Dunker A.K. Uversky V.N. Analysis of molecular recognition features (MoRFs).J. Mol. Biol. 2006; 362: 1043-1059Crossref PubMed Scopus (583) Google Scholar, Oldfield et al., 2005Oldfield C.J. Cheng Y. Cortese M.S. Romero P. Uversky V.N. Dunker A.K. Coupled folding and binding with alpha-helix-forming molecular recognition elements.Biochemistry. 2005; 44: 12454-12470Crossref PubMed Scopus (531) Google Scholar, Tompa, 2005Tompa P. The interplay between structure and function in intrinsically unstructured proteins.FEBS Lett. 2005; 579: 3346-3354Abstract Full Text Full Text PDF PubMed Scopus (593) Google Scholar, Vacic et al., 2007Vacic V. Oldfield C.J. Mohan A. Radivojac P. Cortese M.S. Uversky V.N. Dunker A.K. Characterization of molecular recognition features, MoRFs, and their binding partners.J. Proteome Res. 2007; 6: 2351-2366Crossref PubMed Scopus (382) Google Scholar). Binding is often accompanied by stabilization of transient secondary structure or folding-upon-binding transitions (Dyson and Wright, 2002Dyson H.J. Wright P.E. Coupling of folding and binding for unstructured proteins.Curr. Opin. Struct. Biol. 2002; 12: 54-60Crossref PubMed Scopus (1125) Google Scholar). However, the abundance and diversity of transient structural elements in IDPs and their importance in binding interactions are still unclear. In addition, it is uncertain whether functionally related IDPs (e.g., IDPs that bind identical targets) show conservation of functional motifs with conserved structural features, as is commonly seen in folded proteins. Protein phosphatase 1 (PP1) is a major serine/threonine phosphatase with roles in diverse cellular processes such as muscle contraction and cell signaling (Cohen, 2002Cohen P.T. Protein phosphatase 1–targeted in many directions.J. Cell Sci. 2002; 115: 241-256Crossref PubMed Google Scholar). The activity of PP1 is controlled by two types of proteins: targeting proteins, which direct PP1 to specific subcellular locations and alter its substrate specificity, and inhibitor proteins. Nearly all PP1 regulators interact with PP1 via a common “RVxF” motif (Bollen, 2001Bollen M. Combinatorial control of protein phosphatase-1.Trends Biochem. Sci. 2001; 26: 426-431Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). However, this motif can be found in one third of all eukaryotic proteins, many of which do not interact with PP1, indicating that, although it is necessary for the interaction with PP1, it alone is not sufficient to provide specificity for PP1 binding (Wakula et al., 2003Wakula P. Beullens M. Ceulemans H. Stalmans W. Bollen M. Degeneracy and function of the ubiquitous RVXF motif that mediates binding to protein phosphatase-1.J. Biol. Chem. 2003; 278: 18817-18823Crossref PubMed Scopus (148) Google Scholar). Biochemical data (Bollen, 2001Bollen M. Combinatorial control of protein phosphatase-1.Trends Biochem. Sci. 2001; 26: 426-431Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, Cohen, 2002Cohen P.T. Protein phosphatase 1–targeted in many directions.J. Cell Sci. 2002; 115: 241-256Crossref PubMed Google Scholar) and structures of PP1 in complex with its targeting proteins MYPT1 (Terrak et al., 2004Terrak M. Kerff F. Langsetmo K. Tao T. Dominguez R. Structural basis of protein phosphatase 1 regulation.Nature. 2004; 429: 780-784Crossref PubMed Scopus (296) Google Scholar), spinophilin (Ragusa et al., 2010Ragusa M.J. Dancheck B. Critton D.A. Nairn A.C. Page R. Peti W. Spinophilin directs Protein Phosphatase 1 specificity by blocking substrate binding sites.Nat. Struct. Mol. Biol. 2010; 17: 459-464Crossref PubMed Scopus (133) Google Scholar), and the inhibitor protein inhibitor-2 (I-2) (Hurley et al., 2007Hurley T.D. Yang J. Zhang L. Goodwin K.D. Zou Q. Cortese M. Dunker A.K. DePaoli-Roach A.A. Structural basis for regulation of protein phosphatase 1 by inhibitor-2.J. Biol. Chem. 2007; 282: 28874-28883Crossref PubMed Scopus (151) Google Scholar) show that PP1 regulators interact with multiple sites on PP1. Furthermore, both types of PP1 regulators are known to contain intrinsically disordered regions (Dancheck et al., 2008Dancheck B. Nairn A.C. Peti W. Detailed structural characterization of unbound protein phosphatase 1 inhibitors.Biochemistry. 2008; 47: 12346-12356Crossref PubMed Scopus (59) Google Scholar, Ragusa et al., 2010Ragusa M.J. Dancheck B. Critton D.A. Nairn A.C. Page R. Peti W. Spinophilin directs Protein Phosphatase 1 specificity by blocking substrate binding sites.Nat. Struct. Mol. Biol. 2010; 17: 459-464Crossref PubMed Scopus (133) Google Scholar). The flexible nature of these IDPs allows them to wrap around PP1, forming contacts with distal sites and burying large surface areas (Ragusa et al., 2010Ragusa M.J. Dancheck B. Critton D.A. Nairn A.C. Page R. Peti W. Spinophilin directs Protein Phosphatase 1 specificity by blocking substrate binding sites.Nat. Struct. Mol. Biol. 2010; 17: 459-464Crossref PubMed Scopus (133) Google Scholar, Terrak et al., 2004Terrak M. Kerff F. Langsetmo K. Tao T. Dominguez R. Structural basis of protein phosphatase 1 regulation.Nature. 2004; 429: 780-784Crossref PubMed Scopus (296) Google Scholar). While multiple, distant interaction sites on PP1 provide some specificity in the interaction with its intrinsically disordered regulators, it is unclear whether specificity is enhanced by preformed structural motifs present in the IDPs. Here, we describe the comprehensive structural analysis of three PP1 regulators. I-2, one of the most ancient PP1 regulatory proteins, is ubiquitously expressed from yeast to humans (Ceulemans et al., 2002Ceulemans H. Stalmans W. Bollen M. Regulator-driven functional diversification of protein phosphatase-1 in eukaryotic evolution.Bioessays. 2002; 24: 371-381Crossref PubMed Scopus (124) Google Scholar, Li et al., 2007Li M. Satinover D.L. Brautigan D.L. Phosphorylation and functions of inhibitor-2 family of proteins.Biochemistry. 2007; 46: 2380-2389Crossref PubMed Scopus (44) Google Scholar) and is important for the proper regulation of cell division (Wang et al., 2008Wang W. Stukenberg P.T. Brautigan D.L. Phosphatase inhibitor-2 balances protein phosphatase 1 and aurora B kinase for chromosome segregation and cytokinesis in human retinal epithelial cells.Mol. Biol. Cell. 2008; 19: 4852-4862Crossref PubMed Scopus (48) Google Scholar). DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein with molecular weight of 32 kDa) is a pseudosubstrate inhibitor of PP1 that requires phosphorylation on Thr34 to become a nanomolar PP1 inhibitor. It has been established as an essential link between the neuronal dopaminergic and glutaminergic signaling pathways. In this position, it plays a critical regulatory role for numerous neurological diseases and a large number of drugs of abuse (Nairn et al., 2004Nairn A.C. Svenningsson P. Nishi A. Fisone G. Girault J.A. Greengard P. The role of DARPP-32 in the actions of drugs of abuse.Neuropharmacology. 2004; 47: 14-23Crossref PubMed Scopus (105) Google Scholar). Finally, spinophilin is a PP1-targeting protein (Sarrouilhe et al., 2006Sarrouilhe D. di Tommaso A. Metaye T. Ladeveze V. Spinophilin: from partners to functions.Biochimie. 2006; 88: 1099-1113Crossref PubMed Scopus (115) Google Scholar). The PP1:spinophilin holo-enzyme dephosphorylates Ser845 on the GluR1 AMPA receptor subunit and regulates the closed/open state of the AMPA receptor. It is therefore essential for the regulation of long-term potentiation (Hsieh-Wilson et al., 1999Hsieh-Wilson L.C. Allen P.B. Watanabe T. Nairn A.C. Greengard P. Characterization of the neuronal targeting protein spinophilin and its interactions with protein phosphatase-1.Biochemistry. 1999; 38: 4365-4373Crossref PubMed Scopus (105) Google Scholar, Morishita et al., 2001Morishita W. Connor J.H. Xia H. Quinlan E.M. Shenolikar S. Malenka R.C. Regulation of synaptic strength by protein phosphatase 1.Neuron. 2001; 32: 1133-1148Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, Watanabe et al., 2001Watanabe T. Huang H.B. Horiuchi A. da Cruze Silva E.F. Hsieh-Wilson L. Allen P.B. Shenolikar S. Greengard P. Nairn A.C. Protein phosphatase 1 regulation by inhibitors and targeting subunits.Proc. Natl. Acad. Sci. USA. 2001; 98: 3080-3085Crossref PubMed Scopus (60) Google Scholar). Furthermore, spinophilin facilitates dephosphorylation of doublecortin by PP1 to mediate microtubule bundling at the axonal wrist, playing an important role in the organization of the neuronal cytoskeleton (Bielas et al., 2007Bielas S.L. Serneo F.F. Chechlacz M. Deerinck T.J. Perkins G.A. Allen P.B. Ellisman M.H. Gleeson J.G. Spinophilin facilitates dephosphorylation of doublecortin by PP1 to mediate microtubule bundling at the axonal wrist.Cell. 2007; 129: 579-591Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Previously we reported nuclear magnetic resonance (NMR) spectroscopy data on the dynamics and transient secondary and tertiary structural preferences of I-2 and DARPP-32 (Dancheck et al., 2008Dancheck B. Nairn A.C. Peti W. Detailed structural characterization of unbound protein phosphatase 1 inhibitors.Biochemistry. 2008; 47: 12346-12356Crossref PubMed Scopus (59) Google Scholar) and the PP1-binding domain of spinophilin (Ragusa et al., 2010Ragusa M.J. Dancheck B. Critton D.A. Nairn A.C. Page R. Peti W. Spinophilin directs Protein Phosphatase 1 specificity by blocking substrate binding sites.Nat. Struct. Mol. Biol. 2010; 17: 459-464Crossref PubMed Scopus (133) Google Scholar). Here, we have investigated the relationship between transient structure and the literature reported function in these three PP1 regulators by calculating detailed ensemble models of them using the program ENSEMBLE (Choy and Forman-Kay, 2001Choy W.Y. Forman-Kay J.D. Calculation of ensembles of structures representing the unfolded state of an SH3 domain.J. Mol. Biol. 2001; 308: 1011-1032Crossref PubMed Scopus (194) Google Scholar, Marsh and Forman-Kay, 2009Marsh J.A. Forman-Kay J.D. Structure and disorder in an unfolded state under nondenaturing conditions from ensemble models consistent with a large number of experimental restraints.J. Mol. Biol. 2009; 391: 359-374Crossref PubMed Scopus (132) Google Scholar, Marsh et al., 2007Marsh J.A. Neale C. Jack F.E. Choy W.Y. Lee A.Y. Crowhurst K.A. Forman-Kay J.D. Improved structural characterizations of the drkN SH3 domain unfolded state suggest a compact ensemble with native-like and non-native structure.J. Mol. Biol. 2007; 367: 1494-1510Crossref PubMed Scopus (97) Google Scholar). These models demonstrate that functionally related IDPs can have diverse structural properties. In addition, the availability of PP1-bound crystal structures for I-2 and spinophilin (Hurley et al., 2007Hurley T.D. Yang J. Zhang L. Goodwin K.D. Zou Q. Cortese M. Dunker A.K. DePaoli-Roach A.A. Structural basis for regulation of protein phosphatase 1 by inhibitor-2.J. Biol. Chem. 2007; 282: 28874-28883Crossref PubMed Scopus (151) Google Scholar, Ragusa et al., 2010Ragusa M.J. Dancheck B. Critton D.A. Nairn A.C. Page R. Peti W. Spinophilin directs Protein Phosphatase 1 specificity by blocking substrate binding sites.Nat. Struct. Mol. Biol. 2010; 17: 459-464Crossref PubMed Scopus (133) Google Scholar) allows for the direct comparison of the free and bound states of these proteins. This has enabled us to investigate the role of preformed transient structure in the molecular recognition of PP1. Finally, we have calculated an ensemble model of the PP1:I-2 complex, in which 75% of I-2 remains disordered, providing insight into both the function of I-2 and the structural properties of highly dynamic complexes. ENSEMBLE was used to calculate models of I-2 residues 9–164 (I-29-164), the spinophilin PP1-binding domain residues 417–494 (spinophilin417-494) and DARPP-32 residues 1–118 (DARPP-321-118). Three independent ensembles were calculated for each system, with the final ensembles containing 10–24 structures each according to the simplest ensemble approach, in which the smallest number of conformers possible are fit to the experimental restraints (Marsh and Forman-Kay, 2009Marsh J.A. Forman-Kay J.D. Structure and disorder in an unfolded state under nondenaturing conditions from ensemble models consistent with a large number of experimental restraints.J. Mol. Biol. 2009; 391: 359-374Crossref PubMed Scopus (132) Google Scholar). The experimental restraints included NMR chemical shifts, distance restraints derived from PRE measurements, 15N R2 relaxation rates, and hydrodynamic radii (Rh) from dynamic light scattering (Dancheck et al., 2008Dancheck B. Nairn A.C. Peti W. Detailed structural characterization of unbound protein phosphatase 1 inhibitors.Biochemistry. 2008; 47: 12346-12356Crossref PubMed Scopus (59) Google Scholar, Ragusa et al., 2010Ragusa M.J. Dancheck B. Critton D.A. Nairn A.C. Page R. Peti W. Spinophilin directs Protein Phosphatase 1 specificity by blocking substrate binding sites.Nat. Struct. Mol. Biol. 2010; 17: 459-464Crossref PubMed Scopus (133) Google Scholar). These four different experimental restraint types report on diverse structural properties including secondary and tertiary structure and overall compaction. Table 1 shows the number of experimental restraints used for each system, the agreement between these experimental restraints and the values predicted from the final calculated ensembles. Table 2 shows the general structural properties of the ensembles compared to reference “coil” ensembles (Feldman and Hogue, 2000Feldman H.J. Hogue C.W. A fast method to sample real protein conformational space.Proteins. 2000; 39: 112-131Crossref PubMed Scopus (101) Google Scholar, Marsh and Forman-Kay, 2009Marsh J.A. Forman-Kay J.D. Structure and disorder in an unfolded state under nondenaturing conditions from ensemble models consistent with a large number of experimental restraints.J. Mol. Biol. 2009; 391: 359-374Crossref PubMed Scopus (132) Google Scholar). Plots comparing our experimental measurements to the ensemble-predicted values are shown in Figures S1–S7 (available online).Table 1List of Experimental Restraints Used in ENSEMBLE Calculations and Agreement between Experimental and Predicted ValuesRestraint TypeI-29-164Spinophilin417-494DARPP-321-118I-2:PP1 complex#Rmsd#Rmsd#Rmsd#Rmsd13Cα chemical shifts1510.35 ppm770.35 ppm1120.34 ppm1000.33 ppm13Cβ chemical shifts1430.38 ppm740.39 ppm1070.39 ppm940.33 ppm13C′ chemical shifts1510.41 ppm760.41 ppm1050.42 ppm1000.40 ppm1Hα chemical shifts1510.08 ppm750.08 ppm1120.08 ppm1000.08 ppm1HN chemical shifts1390.17 ppm670.17 ppm990.16 ppm930.17 ppmPREs6310.99 Å420.86 Å4370.70 Å51420.5 ÅaThe PRE restraints were utilized in a very different way in the I2:PP1 complex than for the unbound intrinsically disordered ensembles which accounts for this large rmsd value; see Experimental Procedures for more details.Rh11.9 Å11.9 Å11.8 Å0—15N R2bThe 15N R2 restraints are applied by enforcing a correlation between experimental values and contacts within the ensemble and thus these values represent a Pearson correlation, not an rmsd.1410.85670.621020.68920.68See also Figures S1–S7.a The PRE restraints were utilized in a very different way in the I2:PP1 complex than for the unbound intrinsically disordered ensembles which accounts for this large rmsd value; see Experimental Procedures for more details.b The 15N R2 restraints are applied by enforcing a correlation between experimental values and contacts within the ensemble and thus these values represent a Pearson correlation, not an rmsd. Open table in a new tab Table 2General Properties of Calculated Unbound Intrinsically Disordered Ensembles and TraDES “Coil” EnsemblesI-29-164Spinophilin417-494DARPP-321-118CalculatedCoilCalculatedCoilCalculatedCoilNumber of structures21.00 ± 1.6321 ± 010.33 ± 0.4711 ± 014.00 ± 0.8214 ± 0Radius of gyration (Å)34.59 ± 0.9836.34 ± 1.5716.39 ± 0.3325.71 ± 1.6828.28 ± 0.5231.94 ± 2.03Hydrodynamic radius (Å)33.09 ± 0.0834.16 ± 0.6319.92 ± 0.0524.89 ± 0.6328.09 ± 0.0630.15 ± 0.74Solvent accessible surface area (Å2)17,259 ± 13318,326 ± 2008267 ± 1559213 ± 14412,595 ± 5713,783 ± 213Average values and standard deviations for three independently calculated ensembles and 100 TraDES “Coil” ensembles are presented. See also Figures S1–S7. Open table in a new tab See also Figures S1–S7. Average values and standard deviations for three independently calculated ensembles and 100 TraDES “Coil” ensembles are presented. See also Figures S1–S7. The residue-specific secondary structure content of the ensembles, including the fraction of residues within the broad α, left-β, and right-β regions of Ramachandran space (previously defined) (Marsh and Forman-Kay, 2009Marsh J.A. Forman-Kay J.D. Structure and disorder in an unfolded state under nondenaturing conditions from ensemble models consistent with a large number of experimental restraints.J. Mol. Biol. 2009; 391: 359-374Crossref PubMed Scopus (132) Google Scholar) and the fraction of residues identified as α-helical by STRIDE (Frishman and Argos, 1995Frishman D. Argos P. Knowledge-based protein secondary structure assignment.Proteins. 1995; 23: 566-579Crossref PubMed Scopus (2037) Google Scholar) are presented in Figure 1. The left-β region includes ϕ angles < −100°, typically associated with β strand formation, while the right-β region includes ϕ angles ≥ −100°, referred to as the polyproline II (PPII) region. Secondary structure elements identified in the PP1-bound complexes of I-2 and spinophilin are shown at the top of the plots. The most notable secondary structure element observed in the three ensembles is the ∼70% populated α helix in I-29-164 spanning residues 130–142. This α helix corresponds directly to an α helix present in the PP1:I-2 complex, which folds over the PP1 active site and is important for PP1 inhibition. Interestingly, two other α helices (residues 47–54 and 153–164) seen in the PP1:I-2 complex are not extensively populated in the unbound I-29-164 ensembles, demonstrating that transient secondary structure formation in free states is not necessary for all helices observed in bound complexes. Conversely, two transient α helices are observed from residues 36–40 (∼20% populated) and 97–105 (∼35% populated) that are in regions of I-2 that are not visible in the PP1:I-2 complex structure. Spinophilin417-494 has less secondary structure than I-29-164. However, a ∼25% populated α helix (residues 477–487) corresponds perfectly to the only α helix present in the PP1-bound spinophilin structure. In addition to this helix, two β strands (residues 430–434 and 456–460) are also observed in PP1-bound spinophilin. Interestingly, a peak in the left-β (i.e., β strand region) plot of spinophilin (residues 456–461) corresponds very well with this second β strand. However, there is no significant peak in the left-β plot corresponding to the first β strand (residues 430–434). Thus, while the region corresponding to the second β strand tends to sample strand-like Ramachandran angles, there is no evidence for hydrogen-bonded β sheet formation in disordered spinophilin417-494. Finally, for DARPP-321-118, a ∼30% populated α helix from residues 22–29 and a 10% populated α helix near the C terminus (residues 97–114) were identified. However, the lack of a PP1:DARPP-32 complex structure prohibits further analysis of this data. The identification and interpretation of tertiary structure in IDPs is much more complicated than secondary structure. Although contact plots are commonly used to demonstrate tertiary structure in folded and disordered proteins, low populated contacts within a heterogeneous ensemble can be difficult to discern. Thus, to analyze tertiary structure in our ensembles, we used a clustering algorithm to divide the ensembles into clusters of structurally similar conformers (Kelley et al., 1996Kelley L.A. Gardner S.P. Sutcliffe M.J. An automated approach for clustering an ensemble of NMR-derived protein structures into conformationally related subfamilies.Protein Eng. 1996; 9: 1063-1065Crossref PubMed Scopus (417) Google Scholar). The tertiary structure of each cluster is presented in Figure 2 using fractional contact plots (Marsh and Forman-Kay, 2009Marsh J.A. Forman-Kay J.D. Structure and disorder in an unfolded state under nondenaturing conditions from ensemble models consistent with a large number of experimental restraints.J. Mol. Biol. 2009; 391: 359-374Crossref PubMed Scopus (132) Google Scholar) which show tertiary contacts within these clusters. Contact plots for the PP1-bound structures of I-2 and spinophilin are shown in the bottom halves of the plots. Populations and structural properties of the clusters are presented in Table S1. For both I-29-164 and DARPP-321-118, a large number of PRE distance restraints were utilized in the ENSEMBLE calculations (Table 1). However, the spinophilin PP1-binding domain is particularly difficult to work with as it is rapidly degraded by proteases. Thus, accurate data from only one spin-label site was available. Contact patterns observed in the three major clusters of I-29-164 were analyzed. Cluster 1 is highest populated (44%) and most compact. It is dominated by contacts involving the C-terminal region of I-29-164, particularly residues 120–164. Cluster 2 (17% populated) has significant tertiary contacts between regions around residues 50 and 90. Cluster 3 (14% populated) has contacts between its N terminus and the region around residue 80. Direct comparison of tertiary structure between I-29-164 clusters and PP1-bound I-2 is difficult as electron density for I-2 was only observed for three isolated segments which lack intramolecular contacts. Nevertheless, although none of these clusters resemble the bound state, it is interesting that I-2 regions that directly interact with PP1 in the crystal structure (residues 11–16, 42–54, and 128–167) participate in a substantial fraction of the contacts detected in unbound I-2. Upon binding to PP1, spinophilin417-494 undergoes a folding-upon-binding transition and forms secondary and tertiary structure elements including two β strands that extend a large β sheet in PP1 and an α helix. Spinophilin417-494 cluster 1 (68% populated) has a large number of tertiary contacts, particularly near the N and C termini; none of these contacts resemble the topology of PP1-bound spinophilin. Cluster 2 (18% populated), on the other hand, has tertiary interactions that clearly resemble the β strand contacts of the PP1-bound state, seen as contacts perpendicular to the diagonal in the contact plot. Although this cluster also contains contacts not seen in the bound state, this suggests that free spinophilin417-494 has a minor population in which contacts resembling the bound state are beginning to form. Notably, these contacts are close to the position of the spin label, providing confidence in their accuracy. Three clusters are identified for DARPP-321-118. Cluster 1 (50% populated) is very compact. Cluster 2 (29% populated) is dominated by contacts involving the C-terminal region. Finally, cluster 3 (12% populated) is extended and has only very few tertiary contacts. Although these results suggest interesting tertiary structure in unbound DARPP-321-118, further analysis is limited due to the lack of a PP1:DARPP-32 complex structure. Notably, all three IDPs have at least one cluster with tertiary structures involving the N and/or C termini. This is interesting, given the observation that contacts involving the termini should be more likely due to the excluded volume of the polymer chain (Chan and Dill, 1989Chan H.S. Dill K.A. Interchain loops in polymers: effects of excluded volume.J. Chem. Phys. 1989; 90: 492-509Crossref Scopus (186) Google Scholar). This was also previously noted in the unfolded state of the drkN SH3 domain (Marsh and Forman-Kay, 2009Marsh J.A. Forman-Kay J.D. Structure and disorder in an unfolded state under nondenaturing conditions from ensemble models consistent with a large number of experimental restraints.J. Mol. Biol. 2009; 391: 359-374Crossref PubMed Scopus (132) Google Scholar). Thus, it seems likely that contacts involving the termini are a common structural feature of IDPs. As noted earlier, electron density for I-2 in the PP1:I-2 complex was only detected for ∼25% of the protein and, thus, I-2 remains largely disordered, even upon binding to PP1 (Hurley et al., 2007" @default.
• W2070958052 created "2016-06-24" @default.
• W2070958052 creator A5002238592 @default.
• W2070958052 creator A5010249240 @default.
• W2070958052 creator A5032410016 @default.
• W2070958052 creator A5040013818 @default.
• W2070958052 creator A5067107586 @default.
• W2070958052 creator A5087683429 @default.
• W2070958052 date "2010-09-01" @default.
• W2070958052 modified "2023-10-16" @default.
• W2070958052 title "Structural Diversity in Free and Bound States of Intrinsically Disordered Protein Phosphatase 1 Regulators" @default.
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