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• W3094960678 abstract "Aminopeptidase N (APN, CD13) is a transmembrane ectopeptidase involved in many crucial cellular functions. Besides its role as a peptidase, APN also mediates signal transduction and is involved in the activation of matrix metalloproteinases (MMPs). MMPs function in tissue remodeling within the extracellular space and are therefore involved in many human diseases, such as fibrosis, rheumatoid arthritis, tumor angiogenesis, and metastasis, as well as viral infections. However, the exact mechanism that leads to APN-driven MMP activation is unclear. It was previously shown that extracellular 14-3-3 adapter proteins bind to APN and thereby induce the transcription of MMPs. As a first step, we sought to identify potential 14-3-3–binding sites in the APN sequence. We constructed a set of phosphorylated peptides derived from APN to probe for interactions. We identified and characterized a canonical 14-3-3–binding site (site 1) within the flexible, structurally unresolved N-terminal APN region using direct binding fluorescence polarization assays and thermodynamic analysis. In addition, we identified a secondary, noncanonical binding site (site 2), which enhances the binding affinity in combination with site 1 by many orders of magnitude. Finally, we solved crystal structures of 14-3-3σ bound to mono- and bis-phosphorylated APN-derived peptides, which revealed atomic details of the binding mode of mono- and bivalent 14-3-3 interactions. Therefore, our findings shed some light on the first steps of APN-mediated MMP activation and open the field for further investigation of this important signaling pathway. Aminopeptidase N (APN, CD13) is a transmembrane ectopeptidase involved in many crucial cellular functions. Besides its role as a peptidase, APN also mediates signal transduction and is involved in the activation of matrix metalloproteinases (MMPs). MMPs function in tissue remodeling within the extracellular space and are therefore involved in many human diseases, such as fibrosis, rheumatoid arthritis, tumor angiogenesis, and metastasis, as well as viral infections. However, the exact mechanism that leads to APN-driven MMP activation is unclear. It was previously shown that extracellular 14-3-3 adapter proteins bind to APN and thereby induce the transcription of MMPs. As a first step, we sought to identify potential 14-3-3–binding sites in the APN sequence. We constructed a set of phosphorylated peptides derived from APN to probe for interactions. We identified and characterized a canonical 14-3-3–binding site (site 1) within the flexible, structurally unresolved N-terminal APN region using direct binding fluorescence polarization assays and thermodynamic analysis. In addition, we identified a secondary, noncanonical binding site (site 2), which enhances the binding affinity in combination with site 1 by many orders of magnitude. Finally, we solved crystal structures of 14-3-3σ bound to mono- and bis-phosphorylated APN-derived peptides, which revealed atomic details of the binding mode of mono- and bivalent 14-3-3 interactions. Therefore, our findings shed some light on the first steps of APN-mediated MMP activation and open the field for further investigation of this important signaling pathway. Aminopeptidase N (APN, CD13) is a zinc-dependent ectopeptidase of the M1 family. It is a type II integral membrane protein and is located on the surface of many mammalian cells like fibroblasts, epithelial and myeloid cells (1Luan Y. Xu W. The structure and main functions of aminopeptidase N.Curr. Med. Chem. 2007; 14 (17346152): 639-64710.2174/092986707780059571Crossref PubMed Scopus (155) Google Scholar, 2Wickström M. Larsson R. Nygren P. Gullbo J. Aminopeptidase N (CD13) as a target for cancer chemotherapy.Cancer Sci. 2011; 102 (21205077): 501-50810.1111/j.1349-7006.2010.01826.xCrossref PubMed Scopus (225) Google Scholar). APN consists of 967 amino acids (aa), which can be divided into three regions. A short N-terminal region is located in the cytoplasm (aa 1–9), followed by a single-helix transmembrane domain (aa 10–27) and a large extracellular region (aa 28–967) (3Wong A.H.M. Zhou D. Rini J.M. The x-ray crystal structure of human aminopeptidase N reveals a novel dimer and the basis for peptide processing.J. Biol. Chem. 2012; 287 (22932899): 36804-3681310.1074/jbc.M112.398842Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). APN is involved in multiple processes. It is most widely known for its protease activity in the renin-angiotensin system, where it proteolytically converts angiotensin III to IV (4Danziger R.S. Aminopeptidase N in arterial hypertension.Heart Fail. Rev. 2008; 13 (18008160): 293-29810.1007/s10741-007-9061-yCrossref PubMed Scopus (44) Google Scholar). In addition to its enzymatic activity, it functions as a receptor for coronaviruses and has been proposed to participate in the endocytosis of cholesterol (5Nomura R. Kiyota A. Suzaki E. Kataoka K. Ohe Y. Miyamoto K. Senda T. Fujimoto T. Human coronavirus 229E binds to CD13 in rafts and enters the cell through caveolae.J. Virol. 2004; 78 (15280478): 8701-870810.1128/JVI.78.16.8701-8708.2004Crossref PubMed Scopus (120) Google Scholar, 6Mina-Osorio P. The moonlighting enzyme CD13: old and new functions to target.Trends Mol. Med. 2008; 14 (18603472): 361-37110.1016/j.molmed.2008.06.003Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 7Kolb A.F. Hegyi A. Maile J. Heister A. Hagemann M. Siddell S.G. Molecular analysis of the coronavirus-receptor function of aminopeptidase N.Adv. Exp. Med. Biol. 1998; 440 (9782265): 61-6710.1007/978-1-4615-5331-1_8Crossref PubMed Scopus (28) Google Scholar). Some of the functions of APN are mediated by protein-protein interactions. Binding of extracellular 14-3-3 proteins, for instance, was shown to induce transcription of various matrix-metalloproteinases (MMPs) via p38 MAPK signaling (8Ghahary A. Marcoux Y. Karimi-Busheri F. Li Y. Tredget E.E. Kilani R.T. Lam E. Weinfeld M. Differentiated keratinocyte-releasable stratifin (14-3-3 Sigma) stimulates MMP-1 expression in dermal fibroblasts.J. Invest. Dermatol. 2005; 124 (15654971): 170-17710.1111/j.0022-202X.2004.23521.xAbstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 9Asdaghi N. Kilani R.T. Hosseini-Tabatabaei A. Odemuyiwa S.O. Hackett T.L. Knight D.A. Ghahary A. Moqbel R. Extracellular 14-3-3 from human lung epithelial cells enhances MMP-1 expression.Mol. Cell. Biochem. 2012; 360 (21948273): 261-27010.1007/s11010-011-1065-1Crossref PubMed Scopus (16) Google Scholar, 10Ghahary A. Karimi-Busheri F. Marcoux Y. Li Y. Tredget E.E. Kilani R.T. Li L. Zheng J. Karami A. Keller B.O. Weinfeld M. Keratinocyte-releasable stratifin functions as a potent collagenase-stimulating factor in fibroblasts.J. Invest. Dermatol. 2004; 122 (15140222): 1188-119710.1111/j.0022-202X.2004.22519.xAbstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 11Ghaffari A. Li Y. Kilani R.T. Ghahary A. 14-3-3σ associates with cell surface aminopeptidase N in the regulation of matrix metalloproteinase-1.J. Cell Sci. 2010; 123 (20699358): 2996-300510.1242/jcs.069484Crossref PubMed Scopus (26) Google Scholar, 12Eun K.L. Youn S.L. Lee H. Cheol Y.C. Seok H.P. 14-3-3ε protein increases matrix metalloproteinase-2 gene expression via p38 MAPK signaling in NIH3T3 fibroblast cells.Exp. Mol. Med. 2009; 41 (19322035): 453-46110.3858/emm.2009.41.7.050Crossref PubMed Scopus (11) Google Scholar). MMPs act in tissue remodeling by rearranging the extracellular matrix (13Birkedal-Hansen H. Moore W.G.I. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Matrix metalloproteinases: a review.Crit. Rev. Oral Biol. Med. 1993; 4 (8435466): 197-25010.1177/10454411930040020401Crossref PubMed Scopus (2577) Google Scholar). They are involved in several human diseases, such as fibrosis and rheumatoid arthritis (14Maksymowych W.P. van der Heijde D. Allaart C.F. Landewé R. Boire G. Tak P.P. Gui Y. Ghahary A. Kilani R. Marotta A. 14-3-3η is a novel mediator associated with the pathogenesis of rheumatoid arthritis and joint damage.Arthritis Res. Ther. 2014; 16 (24751211): R9910.1186/ar4547Crossref PubMed Scopus (48) Google Scholar, 15Ghaffari A. Li Y. Karami A. Ghaffari M. Tredget E.E. Ghahary A. Fibroblast extracellular matrix gene expression in response to keratinocyte-releasable stratifin.J. Cell. Biochem. 2006; 98 (16440305): 383-39310.1002/jcb.20782Crossref PubMed Scopus (55) Google Scholar). MMPs play also important roles in diverse types of cancers by promoting angiogenesis and metastasis (16Egeblad M. Werb Z. New functions for the matrix metalloproteinases in cancer progression.Nat. Rev. Cancer. 2002; 2 (11990853): 161-17410.1038/nrc745Crossref PubMed Scopus (4846) Google Scholar, 17Stetler-Stevenson W.G. Matrix metalloproteinases in angiogenesis: a moving target for therapeutic intervention.J. Clin. Invest. 1999; 103 (10225966): 1237-124110.1172/JCI6870Crossref PubMed Scopus (675) Google Scholar). The family of 14-3-3 proteins are highly conserved eukaryotic adapter proteins, which are involved in several hundred protein-protein interactions and therefore a plethora of cellular functions. Seven homologs are present in human (β/α, η, σ, ζ, τ, ε, and γ) with a molecular mass of ∼30 kDa (18Aitken A. Collinge D.B. van Heusden B.P.H. Isobe T. Roseboom P.H. Rosenfeld G. Soll J. 14-3-3 proteins: a highly conserved, widespread family of eukaryotic proteins.Trends Biochem. Sci. 1992; 17 (1471260): 498-50110.1016/0968-0004(92)90339-BAbstract Full Text PDF PubMed Scopus (418) Google Scholar). 14-3-3 consists of nine α-helices and forms via its N-terminal dimerization region homo- and heterodimers in solution (19Yaffe M.B. How do 14-3-3 proteins work?—Gatekeeper phosphorylation and the molecular anvil hypothesis.FEBS Lett. 2002; 513 (11911880): 53-5710.1016/S0014-5793(01)03288-4Crossref PubMed Scopus (531) Google Scholar, 20Tzivion G. Shen Y.H. Zhu J. 14-3-3 proteins; bringing new definitions to scaffolding.Oncogen. 2001; 20 (11607836): 6331-633810.1038/sj.onc.1204777Crossref PubMed Scopus (248) Google Scholar). Each of the two protomers possesses an amphipathic binding groove to interact with their partner. 14-3-3 binding occurs usually in a phosphorylation-dependent manner, in which a serine or threonine of the target protein is phosphorylated and subsequently able to bind to a conserved basic patch within 14-3-3 (21Petosa C. Masters S.C. Bankston L.A. Pohl J. Wang B. Fu H. Liddington R.C. 14-3-3ζ binds a phosphorylated Raf peptide and an unphosphorylated peptide via its conserved amphipathic groove.J. Biol. Chem. 1998; 273 (9632691): 16305-1631010.1074/jbc.273.26.16305Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 22Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. The structural basis for 14-3-3: phosphopeptide binding specificity.Cell. 1997; 91 (9428519): 961-97110.1016/S0092-8674(00)80487-0Abstract Full Text Full Text PDF PubMed Scopus (1275) Google Scholar). Due to its dimeric nature, each 14-3-3 dimer harbors two of these binding grooves, and several studies have shown bivalent binding between 14-3-3 dimers and their interaction partner (23Kalabova D. Smidova A. Petrvalska O. Alblova M. Kosek D. Man P. Obsil T. Obsilova V. Human procaspase-2 phosphorylation at both S139 and S164 is required for 14-3-3 binding.Biochem. Biophys. Res. Commun. 2017; 493 (28943433): 940-94510.1016/j.bbrc.2017.09.116Crossref PubMed Scopus (8) Google Scholar, 24Kast D.J. Dominguez R. Mechanism of IRSp53 inhibition by 14-3-3.Nat. Commun. 2019; 10 (30696821): 48310.1038/s41467-019-08317-8Crossref PubMed Scopus (16) Google Scholar, 25Kostelecky B. Saurin A.T. Purkiss A. Parker P.J. McDonald N.Q. Recognition of an intra-chain tandem 14-3-3 binding site within PKCε.EMBO Rep. 2009; 10 (19662078): 983-98910.1038/embor.2009.150Crossref PubMed Scopus (71) Google Scholar, 26Stevers L.M. De Vries R.M.J.M. Doveston R.G. Milroy L.G. Brunsveld L. Ottmann C. Structural interface between LRRK2 and 14-3-3 protein.Biochem. J. 2017; 474 (28202711): 1273-128710.1042/BCJ20161078Crossref PubMed Scopus (29) Google Scholar). Throughout the last decade, despite its widespread intracellular roles, some 14-3-3 homologs (e.g. β/α, η, σ (also known as stratifin (SFN)), ζ, and ε) were shown to be secreted and are also present in the extracellular space. Up to now, extracellular 14-3-3 could be linked to functions, among others, in collagenase expression or rheumatoid arthritis or as an anti-fibrogenic factor (10Ghahary A. Karimi-Busheri F. Marcoux Y. Li Y. Tredget E.E. Kilani R.T. Li L. Zheng J. Karami A. Keller B.O. Weinfeld M. Keratinocyte-releasable stratifin functions as a potent collagenase-stimulating factor in fibroblasts.J. Invest. Dermatol. 2004; 122 (15140222): 1188-119710.1111/j.0022-202X.2004.22519.xAbstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 14Maksymowych W.P. van der Heijde D. Allaart C.F. Landewé R. Boire G. Tak P.P. Gui Y. Ghahary A. Kilani R. Marotta A. 14-3-3η is a novel mediator associated with the pathogenesis of rheumatoid arthritis and joint damage.Arthritis Res. Ther. 2014; 16 (24751211): R9910.1186/ar4547Crossref PubMed Scopus (48) Google Scholar, 15Ghaffari A. Li Y. Karami A. Ghaffari M. Tredget E.E. Ghahary A. Fibroblast extracellular matrix gene expression in response to keratinocyte-releasable stratifin.J. Cell. Biochem. 2006; 98 (16440305): 383-39310.1002/jcb.20782Crossref PubMed Scopus (55) Google Scholar, 27Chavez-Muñoz C. Hartwell R. Jalili R.B. Jafarnejad S.M. Lai A. Nabai L. Ghaffari A. Hojabrpour P. Kanaan N. Duronio V. Guns E. Cherkasov A. Ghahary A. SPARC/SFN interaction, suppresses type I collagen in dermal fibroblasts.J. Cell. Biochem. 2012; 113 (22422640): 2622-263210.1002/jcb.24137Crossref PubMed Scopus (8) Google Scholar, 28Rahmani-Neishaboor E. Jallili R. Hartwell R. Leung V. Carr N. Ghahary A. Topical application of a film-forming emulgel dressing that controls the release of stratifin and acetylsalicylic acid and improves/prevents hypertrophic scarring.Wound Repair Regen. 2013; 21 (23126516): 55-6510.1111/j.1524-475X.2012.00857.xCrossref PubMed Scopus (15) Google Scholar). In contrast to the intracellular signal transduction that induces MMPs via the p38 MAPK pathway, the extracellular stimulation of APN by 14-3-3 is less understood. Extracellular 14-3-3 binds directly to APN in a phosphorylation-dependent manner (11Ghaffari A. Li Y. Kilani R.T. Ghahary A. 14-3-3σ associates with cell surface aminopeptidase N in the regulation of matrix metalloproteinase-1.J. Cell Sci. 2010; 123 (20699358): 2996-300510.1242/jcs.069484Crossref PubMed Scopus (26) Google Scholar, 29Nefla M. Sudre L. Denat G. Priam S. Andre-Leroux G. Berenbaum F. Jacques C. The pro-inflammatory cytokine 14-3-3ε is a ligand of CD13 in cartilage.J. Cell Sci. 2015; 128 (26208633): 3250-326210.1242/jcs.169573Crossref PubMed Scopus (18) Google Scholar). This interaction can be suppressed by blocking the 14-3-3 binding groove either by small-molecule inhibitors (phosphonates) or peptidomimetics (30Thiel P. Röglin L. Meissner N. Hennig S. Kohlbacher O. Ottmann C. Virtual screening and experimental validation reveal novel small-molecule inhibitors of 14-3-3 protein–protein interactions.Chem. Commun. 2013; 49 (23939230): 8468-847010.1039/c3cc44612cCrossref PubMed Scopus (35) Google Scholar, 31Krüger D.M. Glas A. Bier D. Pospiech N. Wallraven K. Dietrich L. Ottmann C. Koch O. Hennig S. Grossmann T.N. Structure-based design of non-natural macrocyclic peptides that inhibit protein-protein interactions.J. Med. Chem. 2017; 60 (29028171): 8982-898810.1021/acs.jmedchem.7b01221Crossref PubMed Scopus (26) Google Scholar). Also, an APN-derived peptide containing a phosphorylated tyrosine was shown to inhibit the 14-3-3–mediated MMP induction (29Nefla M. Sudre L. Denat G. Priam S. Andre-Leroux G. Berenbaum F. Jacques C. The pro-inflammatory cytokine 14-3-3ε is a ligand of CD13 in cartilage.J. Cell Sci. 2015; 128 (26208633): 3250-326210.1242/jcs.169573Crossref PubMed Scopus (18) Google Scholar). To identify the region of direct interaction between APN and 14-3-3, we showed the detailed analysis of potential binding epitopes. Using phosphorylated peptides, we identified, validated (direct fluorescence polarization (FP)-binding assay), and in-depth characterized (by isothermal titration calorimetry (ITC)) potential APN phosphorylated serine- and threonine-based 14-3-3–binding sites. We showed that a second noncanonical 14-3-3–binding site increases the affinity and therefore implicates a bivalent interaction mode. Additionally, we solved the crystal structure of mono- and bis-phosphorylated APN stretches in complex with 14-3-3σ. Therefore, we contribute a detailed analysis of a bivalent 14-3-3 interaction partner and highlight the importance of secondary, noncanonical 14-3-3–binding sites. For a detailed analysis of APN binding to 14-3-3, we aimed for the characterization of its exact binding mode. As often phosphorylated Ser and Thr residues are found in 14-3-3–binding sites (32Obsil T. Obsilova V. Structural basis of 14-3-3 protein functions.Semin. Cell Dev. Biol. 2011; 22 (21920446): 663-67210.1016/j.semcdb.2011.09.001Crossref PubMed Scopus (176) Google Scholar, 33Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine.Cell. 1996; 84 (8601312): 889-89710.1016/S0092-8674(00)81067-3Abstract Full Text Full Text PDF PubMed Scopus (1140) Google Scholar, 34Mhawech P. 14-3-3 proteins—an update.Cell Res. 2005; 15 (15857577): 228-23610.1038/sj.cr.7290291Crossref PubMed Scopus (231) Google Scholar, 35Dougherty M.K. Morrison D.K. Unlocking the code of 14-3-3.J. Cell Sci. 2004; 117 (15090593): 1875-188410.1242/jcs.01171Crossref PubMed Scopus (373) Google Scholar), we performed an analysis of all 138 extracellular Ser and Thr residues of APN using three selection criteria (Fig. 1A). First, we analyzed the potential of each Ser/Thr-containing sequence motif for 14-3-3 binding (14-3-3Pred (36Madeira F. Tinti M. Murugesan G. Berrett E. Stafford M. Toth R. Cole C. MacKintosh C. Barton G.J. 14-3-3-Pred: improved methods to predict 14-3-3-binding phosphopeptides.Bioinformatics. 2015; 31 (25735772): 2276-228310.1093/bioinformatics/btv133Crossref PubMed Scopus (81) Google Scholar)). From the three 14-3-3Pred classifiers, we defined that at least one threshold needed to be met. This resulted in 20 potential 14-3-3–binding regions within APN (Table S1). Second, we reasoned that the according Ser/Thr residue should be surface accessible (≥20%, Swiss PDB Viewer (37Guex N. Peitsch M.C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling.Electrophoresis. 1997; 18 (9504803): 2714-272310.1002/elps.1150181505Crossref PubMed Scopus (9111) Google Scholar)), which narrowed the number of candidates down to seven potential 14-3-3–binding sites. Two of these (Ser43 and Thr57) are located in the structurally nonresolved N-terminal region of APN (PDB entry 4FYQ (3Wong A.H.M. Zhou D. Rini J.M. The x-ray crystal structure of human aminopeptidase N reveals a novel dimer and the basis for peptide processing.J. Biol. Chem. 2012; 287 (22932899): 36804-3681310.1074/jbc.M112.398842Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar)). Finally, the potential binding motifs needed to be located in accessible, flexible secondary structural elements (the define secondary structure of proteins algorithm (DSSP) via MRS, Fig. 1B (38Joosten R.P. Te Beek T.A.H. Krieger E. Hekkelman M.L. Hooft R.W.W. Schneider R. Sander C. Vriend G. A series of PDB related databases for everyday needs.Nucleic Acids Res. 2011; 39: 411-419Crossref PubMed Scopus (449) Google Scholar, 39Kabsch W. Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features.Biopolymers. 1983; 22 (6667333): 2577-263710.1002/bip.360221211Crossref PubMed Scopus (11505) Google Scholar, 40Hekkelman M.L. Vriend G. MRS: A fast and compact retrieval system for biological data.Nucleic Acids Res. 2005; 33: 766-769Crossref Scopus (25) Google Scholar)), which resulted in five potential 14-3-3–binding sites (Fig. 1C). Our sequence and structural assessment of potential 14-3-3–binding motifs within APN revealed five potential candidates. To validate these motifs, we synthesized peptides 1–5 (Fig. 1C) via standard solid-phase peptide synthesis (SPPS). Each peptide was composed of five N-terminal (–5) and three C-terminal (+3) amino acids relative to the pSer/pThr position and an N-terminal FITC attached via a PEG2 linker. The well-established 14-3-3–binding sequence of RAF1 (RQRSTpSTPN) was used as a positive control and synthesized similarly. Purified peptides were tested for direct binding toward 14-3-3σ in an FP assay, and their affinities were quantified (Fig. 2A). 1 was identified as the best binder (KD = 1.7 ± 0.1 μm). In addition, Ser43 is predicted to be potentially phosphorylated (PhosphoNET and NetPhos3.1 (41Blom N. Gammeltoft S. Brunak S. Sequence and structure-based prediction of eukaryotic protein phosphorylation sites.J. Mol. Biol. 1999; 294 (10600390): 1351-136210.1006/jmbi.1999.3310Crossref PubMed Scopus (2377) Google Scholar, 42Pelech S. PhosphoNET. Kinexus Bioinformatics Corp., Vancouver, Canada2017Google Scholar)). Interestingly, this peptide contained a serine residue (Ser44) in the +1 position (relative to pSer43). To test sequence specificity and the robustness of our filtering process, the according APN 39–47 pSer44 peptide (6, KNANSpSPVA) was synthesized and analyzed in our FP assay. In contrast to 1, 6 did not show any binding toward 14-3-3σ (Fig. 2A), indicating a high sequence specificity for the amino acids surrounding the phosphorylation site. To test the influence of the used fluorophore, label-free peptides 1 and 2 were titrated against a 14-3-3σ–bound FITC-labeled RAF1 probe (FP competition assay; Fig. 2B). As expected from our direct FP measurements (Fig. 2A), 2 was not able to compete with FITC-labeled RAF1 (Fig. 2B). In contrast, 1 was able to compete with similar potency as the unlabeled RAF1 peptide (1, IC50 = 18.8 ± 1.5 μm; RAF1, IC50 = 13.4 ± 0.7 μm; Fig. 2B), which underlines that APN-derived motif 1 is a direct 14-3-3σ binder. As only some homologs of 14-3-3 are currently described to be also located in the extracellular space (43Kaplan A. Bueno M. Fournier A.E. Extracellular functions of 14-3-3 adaptor proteins.Cell. Signal. 2017; 31 (27993556): 26-3010.1016/j.cellsig.2016.12.007Crossref PubMed Scopus (22) Google Scholar), we wanted to know whether 1 is preferably bound by a subset of 14-3-3 homologs. We performed an FP assay using all human 14-3-3 homologs (β/α, γ, ε, ζ, η, and θ) and determined their relative affinities to the previously characterized 14-3-3σ. All tested homologs demonstrated an increased binding affinity, with 14-3-3β/α (5.7-fold) and 14-3-3η (18.6-fold) showing the highest preference (Fig. 2C and Fig. S1). To analyze the interaction of peptide 1 with 14-3-3 proteins in an orthogonal manner, we performed a pulldown assay. To this end, we used biotinylated peptides 1 and 6 immobilized on streptavidin-agarose beads and lysates of His6-14-3-3β/α– or His6-14-3-3η–overexpressing Escherichia coli cells. The interacting proteins were analyzed via SDS-PAGE. All samples of the Coomassie-stained gel showed a band occurring at the expected molecular mass of His6-14-3-3η (∼30 kDa, Fig. 2D) or His6-14-3-3β/α (Fig. S2A) with the highest intensity for the sample containing immobilized peptide 1. The His6 tag–specific NTA-Atto488 stain (Fig. 2E and Fig. S2B) showed that, in contrast to the negative control (6Mina-Osorio P. The moonlighting enzyme CD13: old and new functions to target.Trends Mol. Med. 2008; 14 (18603472): 361-37110.1016/j.molmed.2008.06.003Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar), only immobilized peptide 1 binds specifically to 14-3-3. All other bands of weaker intensities are nonspecific contaminants. Furthermore, acetylated, soluble peptide 1 was able to compete 14-3-3 proteins off the beads (Fig. 2D and Fig. S2), showing the specificity and the reversibility of the binding. To investigate binding of peptide 1 to 14-3-3 in more detail, we co-crystallized unlabeled peptide 1 and 14-3-3σ ΔC (aa 1–231). The collected data set was used up to a resolution of 1.60 Å (Table 1, PDB entry 6XWD), and the structure was solved in space group C2221 by molecular replacement (phaser (44Winn M.D. Ballard C.C. Cowtan K.D. Dodson E.J. Emsley P. Evans P.R. Keegan R.M. Krissinel E.B. Leslie A.G.W. McCoy A. McNicholas S.J. Murshudov G.N. Pannu N.S. Potterton E.A. Powell H.R. et al.Overview of the CCP4 suite and current developments.Acta Crystallogr. D Biol. Crystallogr. 2011; 67 (21460441): 235-24210.1107/S0907444910045749Crossref PubMed Scopus (8188) Google Scholar, 45McCoy A.J. Grosse-Kunstleve R.W. Adams P.D. Winn M.D. Storoni L.C. Read R.J. Phaser crystallographic software.J. Appl. Crystallogr. 2007; 40 (19461840): 658-67410.1107/S0021889807021206Crossref PubMed Scopus (12940) Google Scholar)) using a previously solved 14-3-3σ structure as search model (PDB entry 3MHR, chain A (46Schumacher B. Skwarczynska M. Rose R. Ottmann C. Structure of a 14-3-3σ-YAP phosphopeptide complex at 1.15 Å resolution.Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2010; 66 (20823509): 978-98410.1107/S1744309110025479Crossref PubMed Scopus (48) Google Scholar)). Iterative rounds of model building and refinement (Coot, Phenix (47Emsley P. Lohkamp B. Scott W.G. Cowtan K. Features and development of Coot.Acta Crystallogr. D Biol. Crystallogr. 2010; 66 (20383002): 486-50110.1107/S0907444910007493Crossref PubMed Scopus (14339) Google Scholar, 48Liebschner D. Afonine P.V. Baker M.L. Bunkóczi G. Chen V.B. Croll T.I. Hintze B. Hung L.W. Jain S. McCoy A.J. Moriarty N.W. Oeffner R.D. Poon B.K. Prisant M.G. Read R.J. et al.Macromolecular structure determination using X-rays, neutrons and electrons: Recent developments in Phenix.Acta Crystallogr. D Struct. Biol. 2019; 75 (31588918): 861-87710.1107/S2059798319011471Crossref PubMed Scopus (830) Google Scholar)) led to the final structural model (PDB entry 6XWD). An unbiased composite-omit electron density map clearly showed the exact location and orientation of peptide 1 within the binding groove of 14-3-3 (Fig. 3A). As the structure revealed one 14-3-3 monomer per asymmetric unit, the biological dimer is generated via the 2-fold crystallographic symmetry. Both binding grooves of the dimer were occupied with one peptide each.Table 1Crystal data of structure 6XWD and 7AEWPDB entry6XWD7AEWCrystal data Space groupC2221C2221 Cell dimensions a, b, c (Å)82.0, 111.6, 62.482.2, 112.1, 62.6 α, β, γ (degrees)90.0, 90.0, 90.090.0, 90.0, 90.0 Molecules/asymmetric unit11 Wavelength (Å)0.9116500.911650 Resolution limits (Å)45.37–1.60 (1.70–1.60)45.52–1.20 (1.30–1.20) Unique reflections38,136 (6243)86,519 (16,344) Completeness (%)100.00 (100.00)95.8 (85.8) Multiplicity13.45 (13.65)13.41 (10.16) I/σI15.06 (2.66)19.82 (2.27) CC½99.9 (87.9)100.0 (85.4) Robs (%)10.7 (75.4)5.8 (81.9) Rmeas (%)11.1 (78.4)6.0 (85.6)Refinement Resolution limits (Å)45.37–1.60 (1.70–1.60)45.52–1.20 (1.30–1.20) Rwork/Rfree (%)16.76/19.6216.18/18.36 Root mean square deviation Bond length (Å)0.0060.015 Bond angle (degrees)0.7781.876 B-factor (Å3)19.9717.76 No. of atoms Protein20042103 Peptide67102 Ligand/ion55 Water419426 Ramachandran (%) Favored98.397.3 Allowed1.72.7 Outliers0.00.0 Open table in a new tab All residues for APN 39-46 could be modeled into the density. A superimposition of 14-3-3 phosphopeptide complex structures revealed that peptide 1 was bound to the common phospho-binding site with the identical N- to C-terminal orientation (Fig. 3B). Knowing the exact three-dimensional positioning of APN 39–46 bound to 14-3-3, we investigated the amino acid sequence up- and downstream of the phosphorylation position (pSer43). We were interested in whether the linker region (APN 28–67), between the plasma membrane and the globular domain of APN (aa 68–967), exposes enough space for 14-3-3 binding. Therefore, we prepared a geometrical arrangement of our 14-3-3 (PDB entry 6XWD) and the APN crystal structure (PDB entry 4FYQ (3Wong A.H.M. Zhou D. Rini J.M. The x-ray crystal structure of human aminopeptidase N reveals a novel dimer and the basis for peptide processing.J. Biol. Chem. 2012; 287 (22932899): 36804-3681310.1074/jbc.M112.398842Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar)) and calculated the linker lengths based on the number of amino acids within the flexible linker region (Fig. 3C). Thereby, we could estimate the distance between the core domain and the plasma membrane to be about 130 Å. This possesses enough space for a 14-3-3 protein dimer of ∼65 Å to bind (Fig. 3C). The 14-3-3 dimer can either bind to a second APN molecule via the same pSer43 site or to the same APN molecule via an adjacent secondary APN-binding motif. We investigated whether the Ser/Thr-rich residual unstructured patches of 11 N-terminal and 21 C-terminal residues of the APN linker region (Fig. 3C) might harbor additional noncanonical binding sites. In this region, only Thr57 (peptide 5, Fig. 1) was identified as a potential canonical 14-3-3–binding site in our initial prediction analysis but did not show any relevant affinity (Fig. 2A). We decided to test this and all other spatial relevant potential Ser/Thr phosphorylation sites in combination with pSer43, to see whether bis-phosphorylated peptides show any change in bindi" @default.
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• W3094960678 date "2020-12-01" @default.
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• W3094960678 title "MMP activation–associated aminopeptidase N reveals a bivalent 14-3-3 binding motif" @default.
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• W3094960678 doi "https://doi.org/10.1074/jbc.ra120.014708" @default.
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