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• W3156632669 abstract "Speckle-type POZ protein (SPOP) is a ubiquitin ligase adaptor that binds substrate proteins and facilitates their proteasomal degradation. Most SPOP substrates present multiple SPOP-binding (SB) motifs and undergo liquid–liquid phase separation with SPOP. Pancreatic and duodenal homeobox 1 (Pdx1), an insulin transcription factor, is downregulated by interaction with SPOP. Unlike other substrates, only one SB motif has previously been reported within the Pdx1 C-terminal intrinsically disordered region (Pdx1-C). Given this difference, we aimed to determine the specific mode of interaction of Pdx1 with SPOP and how it is similar or different to that of other SPOP substrates. Here, we identify a second SB motif in Pdx1-C, but still find that the resulting moderate valency is insufficient to support phase separation with SPOP in cells. Although Pdx1 does not phase separate with SPOP, Pdx1 and SPOP interaction prompts SPOP relocalization from nuclear speckles to the diffuse nucleoplasm. Accordingly, we find that SPOP-mediated ubiquitination activity of Pdx1 occurs in the nucleoplasm and that highly efficient Pdx1 turnover requires both SB motifs. Our results suggest that the subnuclear localization of SPOP–substrate interactions and substrate ubiquitination may be directed by the properties of the substrate itself. Speckle-type POZ protein (SPOP) is a ubiquitin ligase adaptor that binds substrate proteins and facilitates their proteasomal degradation. Most SPOP substrates present multiple SPOP-binding (SB) motifs and undergo liquid–liquid phase separation with SPOP. Pancreatic and duodenal homeobox 1 (Pdx1), an insulin transcription factor, is downregulated by interaction with SPOP. Unlike other substrates, only one SB motif has previously been reported within the Pdx1 C-terminal intrinsically disordered region (Pdx1-C). Given this difference, we aimed to determine the specific mode of interaction of Pdx1 with SPOP and how it is similar or different to that of other SPOP substrates. Here, we identify a second SB motif in Pdx1-C, but still find that the resulting moderate valency is insufficient to support phase separation with SPOP in cells. Although Pdx1 does not phase separate with SPOP, Pdx1 and SPOP interaction prompts SPOP relocalization from nuclear speckles to the diffuse nucleoplasm. Accordingly, we find that SPOP-mediated ubiquitination activity of Pdx1 occurs in the nucleoplasm and that highly efficient Pdx1 turnover requires both SB motifs. Our results suggest that the subnuclear localization of SPOP–substrate interactions and substrate ubiquitination may be directed by the properties of the substrate itself. Regulation of protein stability is a critical determinant of cellular health and function. In pancreatic β cells, the transcription factor pancreatic and duodenal homeobox 1 (Pdx1; also known as glucose-sensitive factor (1Marshak S. Totary H. Cerasi E. Melloul D. Purification of the beta-cell glucose-sensitive factor that transactivates the insulin gene differentially in normal and transformed islet cells.Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15057-15062Crossref PubMed Scopus (151) Google Scholar), insulin promoter factor 1 (2Ohlsson H. Karlsson K. Edlund T. IPF1, a homeodomain-containing transactivator of the insulin gene.EMBO J. 1993; 12: 4251-4259Crossref PubMed Scopus (745) Google Scholar), insulin upstream factor 1 (3Boam D.S. Docherty K. A tissue-specific nuclear factor binds to multiple sites in the human insulin-gene enhancer.Biochem. J. 1989; 264: 233-239Crossref PubMed Scopus (65) Google Scholar), and islet/duodenum homeobox 1 (4Miller C.P. McGehee Jr., R.E. Habener J.F. IDX-1: A new homeodomain transcription factor expressed in rat pancreatic islets and duodenum that transactivates the somatostatin gene.EMBO J. 1994; 13: 1145-1156Crossref PubMed Scopus (370) Google Scholar)) modulates insulin production in response to blood-glucose levels (5MacFarlane W.M. Read M.L. Gilligan M. Bujalska I. Docherty K. Glucose modulates the binding activity of the beta-cell transcription factor IUF1 in a phosphorylation-dependent manner.Biochem. J. 1994; 303: 625-631Crossref PubMed Scopus (121) Google Scholar, 6Docherty H.M. Hay C.W. Ferguson L.A. Barrow J. Durward E. Docherty K. Relative contribution of PDX-1, MafA and E47/beta2 to the regulation of the human insulin promoter.Biochem. J. 2005; 389: 813-820Crossref PubMed Scopus (68) Google Scholar). In addition to its role in maintaining glucose homeostasis, Pdx1 is also critical to pancreatic development (7Habener J.F. Kemp D.M. Thomas M.K. Minireview: Transcriptional regulation in pancreatic development.Endocrinology. 2005; 146: 1025-1034Crossref PubMed Scopus (331) Google Scholar, 8Ahlgren U. Jonsson J. Jonsson L. Simu K. Edlund H. beta-cell-specific inactivation of the mouse Ipf1/Pdx1 gene results in loss of the beta-cell phenotype and maturity onset diabetes.Genes Dev. 1998; 12: 1763-1768Crossref PubMed Scopus (736) Google Scholar, 9Jonsson J. Carlsson L. Edlund T. Edlund H. Insulin-promoter-factor 1 is required for pancreas development in mice.Nature. 1994; 371: 606-609Crossref PubMed Scopus (1512) Google Scholar) and β cell differentiation (10Wilding L. Gannon M. The role of pdx1 and HNF6 in proliferation and differentiation of endocrine precursors.Diabetes Metab. Res. Rev. 2004; 20: 114-123Crossref PubMed Scopus (20) Google Scholar). Given the critical roles of Pdx1 in overall pancreatic health, Pdx1 mutation and dysregulation is predictably associated with diabetic phenotypes (7Habener J.F. Kemp D.M. Thomas M.K. Minireview: Transcriptional regulation in pancreatic development.Endocrinology. 2005; 146: 1025-1034Crossref PubMed Scopus (331) Google Scholar, 11Stoffers D.A. Ferrer J. Clarke W.L. Habener J.F. Early-onset type-II diabetes mellitus (MODY4) linked to IPF1.Nat. Genet. 1997; 17: 138-139Crossref PubMed Scopus (8) Google Scholar). Among the regulatory signals that control Pdx1 stability and function is a degradation pathway wherein Pdx1 associates with a ubiquitin ligase adaptor, speckle-type POZ protein (SPOP). SPOP recognizes the disordered C terminus of Pdx1 in conditions of low glucose in the β cell (12Claiborn K.C. Sachdeva M.M. Cannon C.E. Groff D.N. Singer J.D. Stoffers D.A. Pcif1 modulates Pdx1 protein stability and pancreatic beta cell function and survival in mice.J. Clin. Invest. 2010; 120: 3713-3721Crossref PubMed Scopus (46) Google Scholar, 13Liu A. Desai B.M. Stoffers D.A. Identification of PCIF1, a POZ domain protein that inhibits PDX-1 (MODY4) transcriptional activity.Mol. Cell. Biol. 2004; 24: 4372-4383Crossref PubMed Scopus (43) Google Scholar). Unlike Pdx1, which functions almost exclusively in β cells, SPOP is found across many tissue types and interacts with numerous substrates (14Cuneo M.J. Mittag T. The ubiquitin ligase adaptor SPOP in cancer.FEBS J. 2019; 286: 3946-3958Crossref PubMed Scopus (12) Google Scholar). SPOP recruits the Cullin3-RING ubiquitin ligase, which facilitates the polyubiquination of SPOP-bound substrates (15Kwon J.E. La M. Oh K.H. Oh Y.M. Kim G.R. Seol J.H. Baek S.H. Chiba T. Tanaka K. Bang O.S. Joe C.O. Chung C.H. BTB domain-containing speckle-type POZ protein (SPOP) serves as an adaptor of Daxx for ubiquitination by Cul3-based ubiquitin ligase.J. Biol. Chem. 2006; 281: 12664-12672Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Upon binding to the SPOP–Cullin3-RING ligase complex, Pdx1 is ubiquitinated and degraded (12Claiborn K.C. Sachdeva M.M. Cannon C.E. Groff D.N. Singer J.D. Stoffers D.A. Pcif1 modulates Pdx1 protein stability and pancreatic beta cell function and survival in mice.J. Clin. Invest. 2010; 120: 3713-3721Crossref PubMed Scopus (46) Google Scholar) and thus cannot activate transcription. Of note, mutations in SPOP that affect substrate binding are associated with prostate and endometrial cancers, among others (16Janouskova H. El Tekle G. Bellini E. Udeshi N.D. Rinaldi A. Ulbricht A. Bernasocchi T. Civenni G. Losa M. Svinkina T. Bielski C.M. Kryukov G.V. Cascione L. Napoli S. Enchev R.I. et al.Opposing effects of cancer-type-specific SPOP mutants on BET protein degradation and sensitivity to BET inhibitors.Nat. Med. 2017; 23: 1046-1054Crossref PubMed Scopus (80) Google Scholar, 17Blattner M. Liu D. Robinson B.D. Huang D. Poliakov A. Gao D. Nataraj S. Deonarine L.D. Augello M.A. Sailer V. Ponnala L. Ittmann M. Chinnaiyan A.M. Sboner A. Chen Y. et al.SPOP mutation drives prostate tumorigenesis in vivo through coordinate regulation of PI3K/mTOR and AR signaling.Cancer Cell. 2017; 31: 436-451Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 18Dai X. Gan W. Li X. Wang S. Zhang W. Huang L. Liu S. Zhong Q. Guo J. Zhang J. Chen T. Shimizu K. Beca F. Blattner M. Vasudevan D. et al.Prostate cancer-associated SPOP mutations confer resistance to BET inhibitors through stabilization of BRD4.Nat. Med. 2017; 23: 1063-1071Crossref PubMed Scopus (127) Google Scholar, 19Gao K. Jin X. Tang Y. Ma J. Peng J. Yu L. Zhang P. Wang C. Tumor suppressor SPOP mediates the proteasomal degradation of progesterone receptors (PRs) in breast cancer cells.Am. J. Cancer Res. 2015; 5: 3210-3220PubMed Google Scholar), and so the study of SPOP and its interacting partners has broad implications across many fields. In addition to a DNA-binding domain, Pdx1 contains two intrinsically disordered regions (IDRs) that mediate many protein–protein interactions (Fig. 1A). Notably, many documented diabetes-linked mutations exist within the Pdx1 IDRs, not the DNA-binding domain (Table S1) (11Stoffers D.A. Ferrer J. Clarke W.L. Habener J.F. Early-onset type-II diabetes mellitus (MODY4) linked to IPF1.Nat. Genet. 1997; 17: 138-139Crossref PubMed Scopus (8) Google Scholar, 20Macfarlane W.M. Frayling T.M. Ellard S. Evans J.C. Allen L.I. Bulman M.P. Ayres S. Shepherd M. Clark P. Millward A. Demaine A. Wilkin T. Docherty K. Hattersley A.T. Missense mutations in the insulin promoter factor-1 gene predispose to type 2 diabetes.J. Clin. Invest. 1999; 104: R33-R39Crossref PubMed Scopus (213) Google Scholar). SPOP is composed of three domains: the meprin and tumor necrosis factor receptor-associated factor homology (MATH) substrate-interaction domain and two dimerization domains, bric à brac, tramtrack, broad complex and bric à brac, tramtrack, broad complex and C-terminal Kelch (Fig. 1B). SPOP oligomerizes through sequential dimerization events (21Marzahn M.R. Marada S. Lee J. Nourse A. Kenrick S. Zhao H. Ben-Nissan G. Kolaitis R.M. Peters J.L. Pounds S. Errington W.J. Prive G.G. Taylor J.P. Sharon M. Schuck P. et al.Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles.EMBO J. 2016; 35: 1254-1275Crossref PubMed Scopus (89) Google Scholar), which serves to enhance apparent affinity for substrates by the display of multiple MATH domains (Fig. 1B) (22Pierce W.K. Grace C.R. Lee J. Nourse A. Marzahn M.R. Watson E.R. High A.A. Peng J. Schulman B.A. Mittag T. Multiple weak linear motifs enhance recruitment and processivity in SPOP-mediated substrate ubiquitination.J. Mol. Biol. 2016; 428: 1256-1271Crossref PubMed Scopus (20) Google Scholar). To this end, SPOP substrates tend to present multiple binding motifs that individually have relatively weak affinities for SPOP, yet several motifs in a single substrate contribute to a substantially strengthened binding interaction to oligomeric SPOP (Fig. 1C) (14Cuneo M.J. Mittag T. The ubiquitin ligase adaptor SPOP in cancer.FEBS J. 2019; 286: 3946-3958Crossref PubMed Scopus (12) Google Scholar). This phenomenon has been demonstrated for SPOP substrates transcriptional activator GLI3, androgen receptor (AR), and death domain-associated protein 6 (DAXX) (21Marzahn M.R. Marada S. Lee J. Nourse A. Kenrick S. Zhao H. Ben-Nissan G. Kolaitis R.M. Peters J.L. Pounds S. Errington W.J. Prive G.G. Taylor J.P. Sharon M. Schuck P. et al.Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles.EMBO J. 2016; 35: 1254-1275Crossref PubMed Scopus (89) Google Scholar, 22Pierce W.K. Grace C.R. Lee J. Nourse A. Marzahn M.R. Watson E.R. High A.A. Peng J. Schulman B.A. Mittag T. Multiple weak linear motifs enhance recruitment and processivity in SPOP-mediated substrate ubiquitination.J. Mol. Biol. 2016; 428: 1256-1271Crossref PubMed Scopus (20) Google Scholar, 23Bouchard J.J. Otero J.H. Scott D.C. Szulc E. Martin E.W. Sabri N. Granata D. Marzahn M.R. Lindorff-Larsen K. Salvatella X. Schulman B.A. Mittag T. Cancer mutations of the tumor suppressor SPOP disrupt the formation of active, phase-separated compartments.Mol. Cell. 2018; 72: 19-36.e18Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Given the numerous and diverse substrates under regulatory control by SPOP, including Pdx1, we must determine whether SPOP recognizes all substrates by the same mechanism or whether substrate-encoded differences result in their prioritization by SPOP. SPOP has recently been reported to recruit substrates via phase separation, which requires multivalent interaction (21Marzahn M.R. Marada S. Lee J. Nourse A. Kenrick S. Zhao H. Ben-Nissan G. Kolaitis R.M. Peters J.L. Pounds S. Errington W.J. Prive G.G. Taylor J.P. Sharon M. Schuck P. et al.Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles.EMBO J. 2016; 35: 1254-1275Crossref PubMed Scopus (89) Google Scholar, 23Bouchard J.J. Otero J.H. Scott D.C. Szulc E. Martin E.W. Sabri N. Granata D. Marzahn M.R. Lindorff-Larsen K. Salvatella X. Schulman B.A. Mittag T. Cancer mutations of the tumor suppressor SPOP disrupt the formation of active, phase-separated compartments.Mol. Cell. 2018; 72: 19-36.e18Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 24Nagai Y. Kojima T. Muro Y. Hachiya T. Nishizawa Y. Wakabayashi T. Hagiwara M. Identification of a novel nuclear speckle-type protein, SPOP.FEBS Lett. 1997; 418: 23-26Crossref PubMed Scopus (86) Google Scholar). Given that only one SPOP-binding (SB) motif has been identified in Pdx1, we tested whether Pdx1 and SPOP engage and function in phase-separated compartments in cells and found that the two do not phase separate together. Instead, Pdx1 relocalizes SPOP to the nucleoplasm. To test whether this recruitment is indeed the result of the substrate valence for SPOP, we characterized the interaction of Pdx1-C with SPOP in vitro and found evidence for a second SB motif in Pdx1 that contributes to high-affinity binding but no additional motifs that would mediate the formation of three-dimensional networks of complexes. Notably, neither SB motif in Pdx1 conforms to the established SB consensus sequence (25Zhuang M. Calabrese M.F. Liu J. Waddell M.B. Nourse A. Hammel M. Miller D.J. Walden H. Duda D.M. Seyedin S.N. Hoggard T. Harper J.W. White K.P. Schulman B.A. Structures of SPOP-substrate complexes: Insights into molecular architectures of BTB-Cul3 ubiquitin ligases.Mol. Cell. 2009; 36: 39-50Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar), but high-resolution structural characterization suggests that the Pdx1 SB motifs share a binding mode with other substrates. Finally, we find that the second SB motif is not required to draw SPOP out of nuclear speckles but that it does contribute to Pdx1 ubiquitination levels. Together, our results provide insight into Pdx1 turnover via SPOP and present the first example of a non–phase-separating SPOP substrate and the implications of such biophysical behavior for substrate prioritization. SPOP derives its name from the observation of SPOP localization to membraneless organelles within the nucleus. Specifically, SPOP colocalizes with nuclear speckles but has also been reported in other subnuclear compartments (24Nagai Y. Kojima T. Muro Y. Hachiya T. Nishizawa Y. Wakabayashi T. Hagiwara M. Identification of a novel nuclear speckle-type protein, SPOP.FEBS Lett. 1997; 418: 23-26Crossref PubMed Scopus (86) Google Scholar, 26Boysen G. Barbieri C.E. Prandi D. Blattner M. Chae S.S. Dahija A. Nataraj S. Huang D. Marotz C. Xu L. Huang J. Lecca P. Chhangawala S. Liu D. Zhou P. et al.SPOP mutation leads to genomic instability in prostate cancer.Elife. 2015; 4: 1-18Crossref Scopus (94) Google Scholar). The nuclear speckles that house SPOP in the absence of high concentrations of substrate are liquid-like in nature (21Marzahn M.R. Marada S. Lee J. Nourse A. Kenrick S. Zhao H. Ben-Nissan G. Kolaitis R.M. Peters J.L. Pounds S. Errington W.J. Prive G.G. Taylor J.P. Sharon M. Schuck P. et al.Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles.EMBO J. 2016; 35: 1254-1275Crossref PubMed Scopus (89) Google Scholar). The SPOP substrate DAXX typically localizes to promyelocytic leukemia bodies (27Li H. Leo C. Zhu J. Wu X. O'Neil J. Park E.J. Chen J.D. Sequestration and inhibition of Daxx-mediated transcriptional repression by PML.Mol. Cell. Biol. 2000; 20: 1784-1796Crossref PubMed Scopus (297) Google Scholar). When coexpressed in cells, however, SPOP and DAXX relocalize to liquid-like bodies that are distinct from nuclear speckles and promyelocytic bodies. Said new SPOP-DAXX bodies serve as the sites of ubiquitination activity (23Bouchard J.J. Otero J.H. Scott D.C. Szulc E. Martin E.W. Sabri N. Granata D. Marzahn M.R. Lindorff-Larsen K. Salvatella X. Schulman B.A. Mittag T. Cancer mutations of the tumor suppressor SPOP disrupt the formation of active, phase-separated compartments.Mol. Cell. 2018; 72: 19-36.e18Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Consistent with the observation of SPOP-mediated substrate ubiquitination within liquid-like bodies, liquid–liquid phase separation (LLPS) is gaining traction as a mechanism underlying compartmentalization of biological activities, dysregulation of which can result in disease pathologies (28Boeynaems S. Alberti S. Fawzi N.L. Mittag T. Polymenidou M. Rousseau F. Schymkowitz J. Shorter J. Wolozin B. Van Den Bosch L. Tompa P. Fuxreiter M. Protein phase separation: A new phase in cell biology.Trends Cell Biol. 2018; 28: 420-435Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar, 29Shin Y. Brangwynne C.P. Liquid phase condensation in cell physiology and disease.Science. 2017; 357: 1-11Crossref Scopus (957) Google Scholar). Multivalent protein–protein interactions mediate LLPS, and increasing valence within each protein enhances the driving force for phase separation (30Li P. Banjade S. Cheng H.C. Kim S. Chen B. Guo L. Llaguno M. Hollingsworth J.V. King D.S. Banani S.F. Russo P.S. Jiang Q.X. Nixon B.T. Rosen M.K. Phase transitions in the assembly of multivalent signalling proteins.Nature. 2012; 483: 336-340Crossref PubMed Scopus (951) Google Scholar). Higher-order oligomerization of SPOP and the associated multivalency for its substrates, which themselves present several SB motifs, also drive SPOP–substrate phase separation. Given that only one SB motif was known in Pdx1, we set out to assess whether Pdx1 and SPOP undergo phase separation in cells. We overexpressed GFP–Pdx1 and V5-tagged SPOP in HeLa cells, performed immunostaining, and observed the cellular localization of the proteins by confocal fluorescence microscopy. In contrast to other SPOP substrates (23Bouchard J.J. Otero J.H. Scott D.C. Szulc E. Martin E.W. Sabri N. Granata D. Marzahn M.R. Lindorff-Larsen K. Salvatella X. Schulman B.A. Mittag T. Cancer mutations of the tumor suppressor SPOP disrupt the formation of active, phase-separated compartments.Mol. Cell. 2018; 72: 19-36.e18Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), we find that GFP–Pdx1 does not localize to punctate structures in cells (Fig. 2A, top panel). As expected, V5-SPOP localizes to nuclear speckles, which are marked by staining for a nuclear speckle scaffold protein (serine/arginine repetitive matrix protein 2) using the SC-35 antibody (31Ilik I.A. Malszycki M. Lubke A.K. Schade C. Meierhofer D. Aktas T. SON and SRRM2 are essential for nuclear speckle formation.Elife. 2020; 9: 1-24Crossref Scopus (9) Google Scholar) (Fig. 2A, lower panel). However, when GFP–Pdx1–WT and V5-SPOP were coexpressed, SPOP relocalized from nuclear speckles into the diffuse nucleoplasm where GFP–Pdx1 resides. Importantly, Pdx1 did not undergo observable partitioning to any nuclear body (Fig. 2B). Furthermore, SPOP relocalization appears to depend on the concentration of GFP–Pdx1. In cells expressing GFP–Pdx1 at high levels (as assessed by high GFP intensity), SPOP is diffuse, whereas in cells that express GFP–Pdx1 at low levels, SPOP localizes to nuclear speckles (Fig. 2B). This result suggests that there exists a substrate concentration threshold above which SPOP is bound and relocalized by the substrate protein. To ensure that SPOP relocalization is dependent on interaction with Pdx1, we repeated the protein localization experiments using a construct of GFP–Pdx1 lacking its C terminus (GFP–Pdx1ΔC). Indeed, we found that a Pdx1 construct that is incapable of interacting with SPOP is also incapable of drawing SPOP out of nuclear speckles (Fig. 2B). To our knowledge, this is the first demonstration of SPOP redistribution from nuclear speckles toward interaction with a substrate diffusely in the nucleus instead of in a nuclear body. Our results suggested that the valence of one Pdx1 for SPOP was not sufficient to mediate phase separation. However, given that SB motifs can have weak affinities and some sequence variability, we set out to identify potential additional SB motifs in Pdx1 that might point to a different mechanism of SPOP redistribution. Given a single motif in the Pdx1 C terminus (32Liu A. Oliver-Krasinski J. Stoffers D.A. Two conserved domains in PCIF1 mediate interaction with pancreatic transcription factor PDX-1.FEBS Lett. 2006; 580: 6701-6706Crossref PubMed Scopus (15) Google Scholar, 33Ostertag M.S. Messias A.C. Sattler M. Popowicz G.M. The structure of the SPOP-Pdx1 interface reveals insights into the phosphorylation-dependent binding regulation.Structure. 2019; 27: 327-334.e323Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar), we probed this interaction by NMR spectroscopy. To identify specific residues on Pdx1 that may interact with SPOP, we used 13C direct-detect NMR experiments tailored to the biophysical characterization of IDRs. Such methods allow enhanced resolution over traditional proton-detect methods for cases, such as Pdx1-C, wherein spectral crowding and degeneracy in unique chemical shifts are barriers to data interpretation (34Gibbs E.B. Cook E.C. Showalter S.A. Application of NMR to studies of intrinsically disordered proteins.Arch. Biochem. Biophys. 2017; 628: 57-70Crossref PubMed Scopus (40) Google Scholar, 35Cook E.C. Usher G.A. Showalter S.A. The use of 13C direct-detect NMR to characterize flexible and disordered proteins.Methods Enzymol. 2018; 611: 81-100Crossref PubMed Scopus (7) Google Scholar). We collected (HACA)CON spectra of 13C, 15N-Pdx1-C (residues 204–283) with and without SPOP-MATH (residues 28–166) (Fig. 3A and Fig. S1, A and B). The addition of SPOP led to marked resonance intensity loss in two specific locations: amino acids 220 to 235 (highlighted in teal) and amino acids 265 to 275 (highlighted in purple, Fig. 3B). The teal region maps to the fragment of Pdx1 that was already determined by pulldown and crystallography to interact with SPOP, and we interpret the disappearance of resonances here and at the second cluster (purple) as binding to SPOP-MATH. We are confident that the spectral changes are due to direct interaction with SPOP because Pdx1-C shows no evidence of any intramolecular interactions that would otherwise explain the observed resonance intensity changes (36Cook E.C. Sahu D. Bastidas M. Showalter S.A. Solution ensemble of the C-terminal domain from the transcription factor Pdx1 resembles an excluded volume polymer.J. Phys. Chem. B. 2019; 123: 106-116Crossref PubMed Scopus (5) Google Scholar). Importantly, the amino acids in both regions of interest in Pdx1-C are highly conserved across several species, suggesting an evolutionary pressure to retain such motifs (Fig. S1C). Thus, we propose that Pdx1-C contains two distinct SB motifs, SB motif 1 (SBM1) and SB motif 2 (SBM2), the sequences of which are shown in alignment with other SPOP substrates in Figure 3C. We did not see evidence for any additional SB motifs. Addition of individual SBM1 (Pdx1 residues 224–236) and SBM2 (Pdx1 residues 265–283) peptides into 15N-SPOP-MATH showed that the resulting spectral changes are primarily localized to the substrate-binding groove (Fig. 4, A and B and Fig. S1, D and E), excluding the possibility that one SB motif preferentially interacts with another face of SPOP-MATH. Similarly, when SBM1 and SBM2 peptides were added to 15N-SPOP-MATH in equimolar ratios, the spectral changes were localized to the substrate-binding groove and largely resembled those observed with the SBM1 peptide alone (Fig. S1F). This suggests a higher SB affinity of Pdx1–SBM1 than Pdx1–SBM2 (as mentioned previously). The spectral changes correspond very well with the location of the Pdx1–SBM1 peptide in the published cocrystal structure (Fig. 4A) (33Ostertag M.S. Messias A.C. Sattler M. Popowicz G.M. The structure of the SPOP-Pdx1 interface reveals insights into the phosphorylation-dependent binding regulation.Structure. 2019; 27: 327-334.e323Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Furthermore, we posit that the intensity changes on the SPOP surface that extend beyond the binding groove may be explained by transient contacts with SPOP that could confer additional stability and/or affinity. Upon identification of the novel binding site for SPOP within Pdx1-C, we sought to understand the molecular basis for its interaction with SPOP. Owing to unfavorable intermediate exchange kinetics and the resulting line broadening, solution NMR was not a viable strategy for structure determination. We therefore pursued crystallography of the complex between the Pdx1 peptide (human protein residues 265–283) and SPOP-MATH (Fig. 4C). We also present, to our knowledge, the first X-ray structure of SPOP-MATH without bound substrate. X-ray data collection and refinement statistics may be found in Table S2. The original consensus SB motif (Fig. 3C) was established based on the substrate sequence conservation and structural features of a substrate-binding groove that spans one of the central β-sheets in SPOP-MATH. One side is largely nonpolar with a small cavity to accommodate the aliphatic (Φ) consensus residue. On the other side, the groove is lined with polar side chain and backbone functional groups. Many of the latter arise from unsatisfied H-bonding groups in the top strand in the central β-sheet in SPOP-MATH. In the structures of puckered protein (Puc), core histone Macro-H2A, cubitus interruptus, or DAXX peptides in complex with SPOP, the peptide side chains in sites 3 to 5 of the consensus motif (conserved as Ser or Thr in non-Pdx1 substrates characterized previously) are highly complementary to the polar end of the groove (25Zhuang M. Calabrese M.F. Liu J. Waddell M.B. Nourse A. Hammel M. Miller D.J. Walden H. Duda D.M. Seyedin S.N. Hoggard T. Harper J.W. White K.P. Schulman B.A. Structures of SPOP-substrate complexes: Insights into molecular architectures of BTB-Cul3 ubiquitin ligases.Mol. Cell. 2009; 36: 39-50Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Most of these side chains H-bond—either directly or via water-mediated contacts—to SPOP. However, many other sequence-independent substrate backbone contacts are also present, and these may confer the ability to bind nonstandard sequences, such as those found in Pdx1 SBM1 and SBM2. The recent X-ray structure of Pdx1–SBM1 bound to SPOP-MATH provided the first atomic-level characterization of an SPOP substrate with two deviations from the consensus SB motif (33Ostertag M.S. Messias A.C. Sattler M. Popowicz G.M. The structure of the SPOP-Pdx1 interface reveals insights into the phosphorylation-dependent binding regulation.Structure. 2019; 27: 327-334.e323Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar) (Fig. 3C). Despite these differences, the SBM1 peptide lies in the deep substrate-binding groove of SPOP, and this binding mode is stabilized by a network of direct and water-mediated hydrogen bonds. Pdx1–SBM1 forms ten H-bonds to SPOP: six via its backbone and four via its side chains (Fig. 4D). Notably, only two of the side chain–mediated hydrogen bonds involve residues within the consensus binding motif, T230 and S231, positions 2 and 3, respectively. The other two intermolecular side chain H-bonds lie upstream of the consensus SB motif (Table S3). The 1.7 Å resolution cocrystal structure of Pdx1–SBM2 bound to SPOP–MATH (Fig. 4B) reported here places the Pdx1 peptide in the canonical substrate-binding site and is consistent with NMR titration data (Fig. S1F) (25Zhuang M. Calabrese M.F. Liu J. Waddell M.B. Nourse A. Hammel M. Miller D.J. Walden H. Duda D.M. Seyedin S.N. Hoggard T. Harper J.W. White K.P. Schulman B.A. Structures of SPOP-substrate complexes: Insights into molecular architectures of BTB-Cul3 ubiquitin ligases.Mol. Cell. 2009; 36: 39-50Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 33Ostertag M.S. Messias A.C. Sattler M. Popowicz G.M. The structure of the SPOP-Pdx1 interface reveals insights into the phosphorylation-dependent binding regulation.Structure. 2019; 27: 327-334.e323Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 37Ostertag M.S. Hutwelker W. Plettenburg O. Sattler M. Popowicz G.M. Structural insights into BET client recognition of endometrial and prostate cancer-associated SPOP mutants.J. Mol. Biol. 2019; 431: 2213-2221Crossref PubMed Scopus (3) Google Scholar, 38Li G. Ci W. Karmakar S. Chen K. Dhar" @default.
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• W3156632669 title "Intrinsically disordered substrates dictate SPOP subnuclear localization and ubiquitination activity" @default.
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