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• W2912276490 abstract "Protein–protein interactions (PPIs) are ubiquitous in almost all biological processes and are often corrupted in diseased states. A detailed understanding of PPIs is therefore key to understanding cellular physiology and can yield attractive therapeutic targets. Here, we describe the development and structural characterization of novel Escherichia coli CueO multi-copper oxidase variants engineered to recapitulate protein–protein interactions with commensurate modulation of their enzymatic activities. The fully integrated single-protein sensors were developed through modular grafting of ligand-specific peptides into a highly compliant and flexible methionine-rich loop of CueO. Sensitive detection of diverse ligand classes exemplified by antibodies, an E3 ligase, MDM2 proto-oncogene (MDM2), and protease (SplB from Staphylococcus aureus) was achieved in a simple mix and measure homogeneous format with visually observable colorimetric readouts. Therapeutic antagonism of MDM2 by small molecules and peptides in clinical development for treatment of cancer patients was assayed using the MDM2-binding CueO enzyme. Structural characterization of the free and MDM2-bound CueO variant provided functional insight into signal-transducing mechanisms of the engineered enzymes and highlighted the robustness of CueO as a stable and compliant scaffold for multiple applications. Protein–protein interactions (PPIs) are ubiquitous in almost all biological processes and are often corrupted in diseased states. A detailed understanding of PPIs is therefore key to understanding cellular physiology and can yield attractive therapeutic targets. Here, we describe the development and structural characterization of novel Escherichia coli CueO multi-copper oxidase variants engineered to recapitulate protein–protein interactions with commensurate modulation of their enzymatic activities. The fully integrated single-protein sensors were developed through modular grafting of ligand-specific peptides into a highly compliant and flexible methionine-rich loop of CueO. Sensitive detection of diverse ligand classes exemplified by antibodies, an E3 ligase, MDM2 proto-oncogene (MDM2), and protease (SplB from Staphylococcus aureus) was achieved in a simple mix and measure homogeneous format with visually observable colorimetric readouts. Therapeutic antagonism of MDM2 by small molecules and peptides in clinical development for treatment of cancer patients was assayed using the MDM2-binding CueO enzyme. Structural characterization of the free and MDM2-bound CueO variant provided functional insight into signal-transducing mechanisms of the engineered enzymes and highlighted the robustness of CueO as a stable and compliant scaffold for multiple applications. Our present understanding of modular architectures evident in diverse classes of proteins has enabled design of engineered proteins with novel sensing properties. Whereas functional outcomes can be impacted by lesser understood, and often interrelated phenomena such as protein stability, allostery, and epistasis, several enzymes have been modified to integrate analyte detection and signal generation components within the same host protein. A common engineering approach entails insertional mutagenesis, whereby xeno-peptides/proteins are incorporated into host proteins to generate hybrid entities with novel sensing functions (1Ferraz R.M. Vera A. Arís A. Villaverde A. Insertional protein engineering for analytical molecular sensing.Microb. Cell Fact. 2006; 5 (16584558): 1510.1186/1475-2859-5-15Crossref PubMed Scopus (20) Google Scholar). Insertion of a protease substrate peptide into enzyme scaffolds such as β-galactosidase can convert these to protease sensors, with target protease engagement typically destabilizing the enzyme and resulting in measurable loss of activity (2Baum E.Z. Bebernitz G.A. Gluzman Y. β-Galactosidase containing a human immunodeficiency virus protease cleavage site is cleaved and inactivated by human immunodeficiency virus protease.Proc. Natl. Acad. Sci. U.S.A. 1990; 87 (2124694): 10023-1002710.1073/pnas.87.24.10023Crossref PubMed Scopus (39) Google Scholar, 3Vera A. Arís A. Daura X. Martínez M.A. Villaverde A. Engineering the E. coli beta-galactosidase for the screening of antiviral protease inhibitors.Biochem. Biophys. Res. Commun. 2005; 329 (15737608): 453-45610.1016/j.bbrc.2005.01.147Crossref PubMed Scopus (3) Google Scholar). A further iteration comprises a host reporter protein inhibited in cis by fusion to an inhibitory domain. Proteolytic cleavage releases the inhibitory domain, resulting in measurable signal turn-on as described using β-lactamase, RNase A, p53, NIa, and NS3 reporter proteins (4Geddie M.L. O'Loughlin T.L. Woods K.K. Matsumura I. Rational design of p53, an intrinsically unstructured protein, for the fabrication of novel molecular sensors.J. Biol. Chem. 2005; 280 (16118206): 35641-3564610.1074/jbc.M508149200Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar5Nirantar S.R. Li X. Siau J.W. Ghadessy F.J. Rapid screening of protein-protein interaction inhibitors using the protease exclusion assay.Biosens. Bioelectron. 2014; 56 (24508816): 250-25710.1016/j.bios.2013.12.060Crossref PubMed Scopus (10) Google Scholar, 6Stein V. Nabi M. Alexandrov K. Ultrasensitive scaffold-dependent protease sensors with large dynamic range.ACS Synth. Biol. 2017; 6 (28291337): 1337-134210.1021/acssynbio.6b00370Crossref PubMed Scopus (26) Google Scholar7Plainkum P. Fuchs S.M. Wiyakrutta S. Raines R.T. Creation of a zymogen.Nat. Struct. Biol. 2003; 10 (12496934): 115-11910.1038/nsb884Crossref PubMed Scopus (35) Google Scholar). Incorporation of antigenic peptides can also discern binding by specific antibodies. Using this approach, engineered β-galactosidase, alkaline phosphatase, and β-lactamase variants have been described with activities modulated by antibody binding (8Legendre D. Soumillion P. Fastrez J. Engineering a regulatable enzyme for homogeneous immunoassays.Nat. Biotechnol. 1999; 17 (9920272): 67-7210.1038/5243Crossref PubMed Scopus (70) Google Scholar, 9Benito A. Feliu J.X. Villaverde A. β-Galactosidase enzymatic activity as a molecular probe to detect specific antibodies.J. Biol. Chem. 1996; 271 (8702899): 21251-2125610.1074/jbc.271.35.21251Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar10Brennan C. Christianson K. Surowy T. Mandecki W. Modulation of enzyme activity by antibody binding to an alkaline phosphatase-epitope hybrid protein.Protein Eng. 1994; 7 (7518083): 509-51410.1093/protein/7.4.509Crossref PubMed Scopus (36) Google Scholar). A reciprocal approach utilizing GFP–antibody hybrids further enables intracellular detection of antigenic peptides (11Wongso D. Dong J. Ueda H. Kitaguchi T. Flashbody: a next generation fluobody with fluorescence intensity enhanced by antigen binding.Anal. Chem. 2017; 89 (28534613): 6719-672510.1021/acs.analchem.7b00959Crossref PubMed Scopus (23) Google Scholar). In this case, fluorescence readout of the hybrid protein is enhanced by peptide binding. Exposed loop regions gleaned from a priori structural data are typically exploited as peptide insertion sites. Random insertion coupled to selection has also been described for β-lactamase variants that bind and sense anti-prostate-specific antigen antibodies (8Legendre D. Soumillion P. Fastrez J. Engineering a regulatable enzyme for homogeneous immunoassays.Nat. Biotechnol. 1999; 17 (9920272): 67-7210.1038/5243Crossref PubMed Scopus (70) Google Scholar). Larger protein domains have been inserted into the β-lactamase, maltose-binding protein, GFP, calmodulin, and dihydrofolate reductase hosts via rational or random approaches to yield allosteric biosensing chimeras recognizing small-molecule and metal analytes (12Choi J.H. Xiong T. Ostermeier M. The interplay between effector binding and allostery in an engineered protein switch.Protein Sci. 2016; 25 (27272021): 1605-161610.1002/pro.2962Crossref PubMed Scopus (4) Google Scholar13Edwards W.R. Busse K. Allemann R.K. Jones D.D. Linking the functions of unrelated proteins using a novel directed evolution domain insertion method.Nucleic Acids Res. 2008; 36 (18559359): e7810.1093/nar/gkn363Crossref PubMed Scopus (69) Google Scholar, 14Doi N. Yanagawa H. Design of generic biosensors based on green fluorescent proteins with allosteric sites by directed evolution.FEBS Lett. 1999; 453 (10405165): 305-30710.1016/S0014-5793(99)00732-2Crossref PubMed Scopus (102) Google Scholar, 15Kohn J.E. Plaxco K.W. Engineering a signal transduction mechanism for protein-based biosensors.Proc. Natl. Acad. Sci. U.S.A. 2005; 102 (16046542): 10841-1084510.1073/pnas.0503055102Crossref PubMed Scopus (57) Google Scholar, 16Baird G.S. Zacharias D.A. Tsien R.Y. Circular permutation and receptor insertion within green fluorescent proteins.Proc. Natl. Acad. Sci. U.S.A. 1999; 96 (10500161): 11241-1124610.1073/pnas.96.20.11241Crossref PubMed Scopus (737) Google Scholar, 17Nagai T. Sawano A. Park E.S. Miyawaki A. Circularly permuted green fluorescent proteins engineered to sense Ca2+.Proc. Natl. Acad. Sci. U.S.A. 2001; 98 (11248055): 3197-320210.1073/pnas.051636098Crossref PubMed Scopus (806) Google Scholar, 18Nadler D.C. Morgan S.A. Flamholz A. Kortright K.E. Savage D.F. Rapid construction of metabolite biosensors using domain-insertion profiling.Nat. Commun. 2016; 7 (27470466)1226610.1038/ncomms12266Crossref PubMed Scopus (66) Google Scholar19Marvin J.S. Schreiter E.R. Echevarría I.M. Looger L.L. A genetically encoded, high-signal-to-noise maltose sensor.Proteins. 2011; 79 (21989929): 3025-303610.1002/prot.23118Crossref PubMed Scopus (68) Google Scholar). Desirable properties of an ideal host protein are known structure, insertional tolerance proximal to active site, simple enzymatic readout, elevated thermostability, and ease of recombinant production. The Escherichia coli multi-copper oxidase CueO displays many of these criteria but has not been validated as a host scaffold. CueO plays an important role in copper homeostasis by oxidation of toxic cuprous ions to cupric ions (20Djoko K.Y. Chong L.X. Wedd A.G. Xiao Z. Reaction mechanisms of the multicopper oxidase CueO from Escherichia coli support its functional role as a cuprous oxidase.J. Am. Chem. Soc. 2010; 132 (20088522): 2005-201510.1021/ja9091903Crossref PubMed Scopus (81) Google Scholar21Kim C. Lorenz W.W. Hoopes J.T. Dean J.F. Oxidation of phenolate siderophores by the multicopper oxidase encoded by the Escherichia coli yacK gene.J. Bacteriol. 2001; 183 (11466290): 4866-487510.1128/JB.183.16.4866-4875.2001Crossref PubMed Scopus (125) Google Scholar, 22Outten F.W. Huffman D.L. Hale J.A. O'Halloran T.V. The independent cue and cus systems confer copper tolerance during aerobic and anaerobic growth in Escherichia coli.J. Biol. Chem. 2001; 276 (11399769): 30670-3067710.1074/jbc.M104122200Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar23Grass G. Rensing C. Genes involved in copper homeostasis in Escherichia coli.J. Bacteriol. 2001; 183 (11222619): 2145-214710.1128/JB.183.6.2145-2147.2001Crossref PubMed Scopus (188) Google Scholar). As with all multi-copper oxidases, it contains four copper atoms distributed within one type 1 (T1) 2The abbreviations used are: T1T2, T3, and T4, type 1, 2, 3, and 4 copper sites, respectivelysCusubstrate copperMRSmethionine-rich segmentABTS2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)HAhemagglutininIVTin vitro transcription/translationMWCOmolecular weight cutoff. copper site and a trinuclear cluster comprising the T2 and T3 copper sites. A further Cu(I)-binding site, termed the substrate copper (sCu) site or T4 lies proximal to T1, and its occupancy is linked to oxidation of proximally bound polyphenols, metal ions, and aromatic polyamines (24Roberts S.A. Wildner G.F. Grass G. Weichsel A. Ambrus A. Rensing C. Montfort W.R. A labile regulatory copper ion lies near the T1 copper site in the multicopper oxidase CueO.J. Biol. Chem. 2003; 278 (12794077): 31958-3196310.1074/jbc.M302963200Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). A four-electron transfer between these sites couples substrate oxidation to reduction of dioxygen bound to the trinuclear site, with commensurate production of water. A distinguishing feature of CueO is a partially structured 45-amino acid segment (residues 356–404) capping the entrance to the T1/sCu copper-binding sites (25Roberts S.A. Weichsel A. Grass G. Thakali K. Hazzard J.T. Tollin G. Rensing C. Montfort W.R. Crystal structure and electron transfer kinetics of CueO, a multicopper oxidase required for copper homeostasis in Escherichia coli.Proc. Natl. Acad. Sci. U.S.A. 2002; 99 (11867755): 2766-277110.1073/pnas.052710499Crossref PubMed Scopus (283) Google Scholar). Mutagenesis studies indicate this methionine-rich segment (MRS) to be important for both Cu(I) binding and regulation of substrate specificity (26Singh S.K. Roberts S.A. McDevitt S.F. Weichsel A. Wildner G.F. Grass G.B. Rensing C. Montfort W.R. Crystal structures of multicopper oxidase CueO bound to copper(I) and silver(I): functional role of a methionine-rich sequence.J. Biol. Chem. 2011; 286 (21903583): 37849-3785710.1074/jbc.M111.293589Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Notably, complete deletion of the MRS (with replacement by a minimal dipeptide linker) does not abrogate function, instead leading to emergence of altered/novel substrate specificities (27Kataoka K. Komori H. Ueki Y. Konno Y. Kamitaka Y. Kurose S. Tsujimura S. Higuchi Y. Kano K. Seo D. Sakurai T. Structure and function of the engineered multicopper oxidase CueO from Escherichia coli—deletion of the methionine-rich helical region covering the substrate-binding site.J. Mol. Biol. 2007; 373 (17804014): 141-15210.1016/j.jmb.2007.07.041Crossref PubMed Scopus (95) Google Scholar). Both the inherent plasticity and substrate-binding site proximity of the MRS make CueO an attractive host for comprehensive engineering. T2, T3, and T4, type 1, 2, 3, and 4 copper sites, respectively substrate copper methionine-rich segment 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) hemagglutinin in vitro transcription/translation molecular weight cutoff. The goal of the current study was to engineer the highly compliant MRS such that CueO activity would be modulated by engagement of a partner protein with a scaffolded peptide. We first inserted peptide motifs derived from p53 that bind the N-terminal domain of the E3 ligase MDM2, a key negative regulator of the p53 tumor suppressor and therapeutic target (28Momand J. Zambetti G.P. Olson D.C. George D. Levine A.J. The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation.Cell. 1992; 69 (1535557): 1237-124510.1016/0092-8674(92)90644-RAbstract Full Text PDF PubMed Scopus (2787) Google Scholar29Haupt Y. Maya R. Kazaz A. Oren M. Mdm2 promotes the rapid degradation of p53.Nature. 1997; 387 (9153395): 296-29910.1038/387296a0Crossref PubMed Scopus (3705) Google Scholar, 30Kubbutat M.H. Jones S.N. Vousden K.H. Regulation of p53 stability by Mdm2.Nature. 1997; 387 (9153396): 299-30310.1038/387299a0Crossref PubMed Scopus (2839) Google Scholar, 31Honda R. Tanaka H. Yasuda H. Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53.FEBS Lett. 1997; 420 (9450543): 25-2710.1016/S0014-5793(97)01480-4Crossref PubMed Scopus (1597) Google Scholar, 32Pazgier M. Liu M. Zou G. Yuan W. Li C. Li C. Li J. Monbo J. Zella D. Tarasov S.G. Lu W. Structural basis for high-affinity peptide inhibition of p53 interactions with MDM2 and MDMX.Proc. Natl. Acad. Sci. U.S.A. 2009; 106 (19255450): 4665-467010.1073/pnas.0900947106Crossref PubMed Scopus (276) Google Scholar, 33Popowicz G.M. Dömling A. Holak T.A. The structure-based design of Mdm2/Mdmx-p53 inhibitors gets serious.Angew. Chem. Int. Ed. Engl. 2011; 50 (21341346): 2680-268810.1002/anie.201003863Crossref PubMed Scopus (133) Google Scholar34Roxburgh P. Hock A.K. Dickens M.P. Mezna M. Fischer P.M. Vousden K.H. Small molecules that bind the Mdm2 RING stabilize and activate p53.Carcinogenesis. 2012; 33 (22301280): 791-79810.1093/carcin/bgs092Crossref PubMed Scopus (34) Google Scholar). MDM2 engagement with the scaffolded peptides resulted in an increase in enzyme activity that could be abrogated by small-molecule and peptidic MDM2 inhibitors. Insertion of antigenic peptides resulted in an antibody-dependent abrogation of enzymatic activity. To help rationalize these opposing analyte-dependent phenotypes, we solved the structures of free and MDM2 (residues 6–125)-bound CueO. Our results validate CueO as robust host protein for use in biosensing and drug-screening applications. A panel of CueO variants was generated with differing modifications in the MRS (Fig. 1A). These included insertion of the parental MDM2-binding peptide sequence present in the N-terminal domain of p53 along with a higher-affinity derivative (peptide 12.1) (35Böttger V. Böttger A. Howard S.F. Picksley S.M. Chène P. Garcia-Echeverria C. Hochkeppel H.K. Lane D.P. Identification of novel mdm2 binding peptides by phage display.Oncogene. 1996; 13 (8950981): 2141-2147PubMed Google Scholar) into the MRS α5 helix to generate CueO-p53 and CueO-12.1-α5, respectively. C-terminal residues in the MRS were further deleted in the latter construct to generate CueO-12.1Δ. These MDM2-binding peptides comprise obligate Phe, Trp, and Leu residues (underlined) essential for high affinity binding (36Kussie P.H. Gorina S. Marechal V. Elenbaas B. Moreau J. Levine A.J. Pavletich N.P. Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain.Science. 1996; 274 (8875929): 948-95310.1126/science.274.5289.948Crossref PubMed Scopus (1782) Google Scholar). A further triple point mutant (CueO-FWL) was constructed with these residues introduced into the α5 helix in the same register observed for the MDM2-binding peptides. Peptide 12.1 and a control peptide with the Phe, Trp, and Leu residues mutated to alanine were also inserted in the intrinsically disordered region of the MRS (linking helices α6 and α7) to yield variants CueO-12.1 and CueO-12.1CON, respectively. Mutational tolerance was assayed by in vitro translation coupled to a rapid colorimetric readout of oxidase activity using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) substrate. All variants displayed readily observable enzymatic activity, highlighting the robustness of the CueO scaffold (Fig. 1B). We next assayed the engineered CueO panel by pulldown assay, using MDM2 as bait and measuring residual bead-bound enzyme activity. The results showed interaction with MDM2 only for the CueO-12.1 variant, where the 12.1 peptide replaced residues 384–394 in the intrinsically disordered region of the MRS (Fig. 1C). Having delineated the MRS subregion suitable for introduction of peptide sequences, we produced two additional constructs (CueO-PMI and CueO-PM2) (Fig. 1A) using previously identified higher-affinity MDM2 binding peptides (32Pazgier M. Liu M. Zou G. Yuan W. Li C. Li C. Li J. Monbo J. Zella D. Tarasov S.G. Lu W. Structural basis for high-affinity peptide inhibition of p53 interactions with MDM2 and MDMX.Proc. Natl. Acad. Sci. U.S.A. 2009; 106 (19255450): 4665-467010.1073/pnas.0900947106Crossref PubMed Scopus (276) Google Scholar, 37Brown C.J. Quah S.T. Jong J. Goh A.M. Chiam P.C. Khoo K.H. Choong M.L. Lee M.A. Yurlova L. Zolghadr K. Joseph T.L. Verma C.S. Lane D.P. Stapled peptides with improved potency and specificity that activate p53.ACS Chem. Biol. 2013; 8 (23214419): 506-51210.1021/cb3005148Crossref PubMed Scopus (167) Google Scholar). The affinity of these variants for recombinant MDM2 (residues 6–125 comprising the p53 binding domain) was first measured by fluorescence polarization. Both variants showed high affinity to MDM2 (apparent Kd 28 ± 1 and 25 ± 1.5 nm, respectively), comparable with affinities of their unmodified linear and stapled versions (Table 1) (Fig. S1). Binding of the higher-affinity CueO-PMI to full-length MDM2 was also clearly observed by visual readout in the pulldown assay (Fig. 1D).Table 1Apparent binding affinities of CueO and indicated variants for MDM2(6–125)ConstructAffinityFree peptideStapled peptidenmCueO44,280 ± 12,690CueO-12.1722 ± 123240 ± 54aMeasured by ITC (54).CueO-12.1CONNo bindingCueO-PMI28 ± 147 ± 7bMeasured by FP (37).87bMeasured by FP (37).CueO-PM225 ± 228 ± 1bMeasured by FP (37).34 ± 2bMeasured by FP (37).a Measured by ITC (54Brown C.J. Dastidar S.G. Quah S.T. Lim A. Chia B. Verma C.S. C-terminal substitution of MDM2 interacting peptides modulates binding affinity by distinctive mechanisms.PLoS One. 2011; 6 (21904608)e2412210.1371/journal.pone.0024122Crossref PubMed Scopus (24) Google Scholar).b Measured by FP (37Brown C.J. Quah S.T. Jong J. Goh A.M. Chiam P.C. Khoo K.H. Choong M.L. Lee M.A. Yurlova L. Zolghadr K. Joseph T.L. Verma C.S. Lane D.P. Stapled peptides with improved potency and specificity that activate p53.ACS Chem. Biol. 2013; 8 (23214419): 506-51210.1021/cb3005148Crossref PubMed Scopus (167) Google Scholar). Open table in a new tab Enzymatic activity of CueO-PM2 after incubation with MDM2 (10 μm) was next assayed at varying concentrations of syringaldazine substrate (12.5–100 μm). Clear MDM2-dependent potentiation of CueO-PM2 activity was observed, with maximal signal differentiation (with or without MDM2) observed visually using 25 μm syringaldazine (Fig. 2A). This concentration was used for all subsequent experiments. Titration of MDM2 indicated dose-responsive enhancement of CueO-PM2 activity, with spectroscopic and visual limits of detection around 750 nm and 3 μm, respectively (Fig. 2, B and C). No activation was seen using BSA control at the same concentrations. To further demonstrate specificity, the assay was repeated in the presence of competitive inhibitors. PM2 stapled peptide (37Brown C.J. Quah S.T. Jong J. Goh A.M. Chiam P.C. Khoo K.H. Choong M.L. Lee M.A. Yurlova L. Zolghadr K. Joseph T.L. Verma C.S. Lane D.P. Stapled peptides with improved potency and specificity that activate p53.ACS Chem. Biol. 2013; 8 (23214419): 506-51210.1021/cb3005148Crossref PubMed Scopus (167) Google Scholar) competed with CueO-PM2 for MDM2 binding (Fig. 3A), resulting in signal attenuation that was not observed for a control stapled peptide (PM2-CON) (Fig. 3B). The small-molecule MDM2 inhibitors RG7112 and AMG232 (38Vu B. Wovkulich P. Pizzolato G. Lovey A. Ding Q. Jiang N. Liu J.J. Zhao C. Glenn K. Wen Y. Tovar C. Packman K. Vassilev L. Graves B. Discovery of RG7112: a small-molecule MDM2 inhibitor in clinical development.ACS Med. Chem. Lett. 2013; 4 (24900694): 466-46910.1021/ml4000657Crossref PubMed Scopus (232) Google Scholar, 39Sun D. Li Z. Rew Y. Gribble M. Bartberger M.D. Beck H.P. Canon J. Chen A. Chen X. Chow D. Deignan J. Duquette J. Eksterowicz J. Fisher B. Fox B.M. et al.Discovery of AMG 232, a potent, selective, and orally bioavailable MDM2-p53 inhibitor in clinical development.J. Med. Chem. 2014; 57 (24456472): 1454-147210.1021/jm401753eCrossref PubMed Scopus (180) Google Scholar), both presently in clinical trials for treatment of leukemia, also showed dose-responsive attenuation of signal (Fig. 3, C and D).Figure 3Detection of pharmaceutical MDM2 antagonism. A, CueO-PM2 (1.5 μm) was incubated with 7.5 μm MDM2(6–125) and the indicated concentrations of PM2 stapled peptide. After incubation (16 °C, 1 h) syringaldazine substrate was added, and enzyme activity was measured over 10 min. B, as in A, with PM2 control stapled peptide. C, as in A, with small-molecule MDM2 inhibitor RG7112. D, as in A, with AMG232 small-molecule inhibitor. Values represent the average of three independent experiments ± S.D.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Antibodies commonly recognize short linear peptide sequences in target proteins. We therefore focused on recapitulating antibody–peptide interactions in the context of a CueO-scaffolded antigenic peptide. The human influenza hemagglutinin (HA) epitope was incorporated into the MRS and CueO-HA activity assayed in the presence of anti-HA or nonspecific (anti-Myc) antibody (Fig. 4, A and B). Only specific antibody resulted in clear dose-responsive reduction of enzyme activity, with an ∼60 nm limit of detection. Incorporation of the FLAG epitope (CueO-FLAG) led to detection of anti-FLAG antibody (Fig. 4C), further highlighting modularity of the CueO scaffold. To understand the opposing signal readouts generated upon MDM2 and antibody binding, we compared enzymatic activities of CueO, CueO-PM2, and CueO-HA in the absence of protein analytes. Whereas the activities of CueO and CueO-HA were similar, CueO-PM2 showed ∼2-fold reduced activity (Fig. 5A), indicating auto-inhibition by the PM2 peptide that is relieved upon MDM2 interaction. Kinetics data indicated that insertions into the MRS primarily affected kcat for syringaldazine oxidation, with no marked influence on affinity (Table 2). This was most pronounced for the 12.1 and PM2 peptide insertions, reducing kcat ∼2.2- and 2.8-fold, respectively. In the case of CueO-PM2, co-incubation with MDM2 resulted in ∼1.6-fold increased kcat (and no change in Km). Notably, the kcat value for CueO-12.1 was ∼1.6-fold lower than the control CueO-12.1CON, implicating one or more of the Phe, Trp, and Leu residues present in 12.1 (and also PMI/PM2) as being responsible for the inhibitory phenotype. No marked difference in affinity for syringaldazine was observed between these two proteins. Perturbation of copper (Cu) binding at the T1 site could account for the observed kcat deficits in the absence of analyte binding. In agreement, Cu occupancy at the T1 site as measured by absorbance at 610 nm (40Kataoka K. Hirota S. Maeda Y. Kogi H. Shinohara N. Sekimoto M. Sakurai T. Enhancement of laccase activity through the construction and breakdown of a hydrogen bond at the type I copper center in Escherichia coli CueO and the deletion mutant Δα5–7 CueO.Biochemistry. 2011; 50 (21142169): 558-56510.1021/bi101107cCrossref PubMed Scopus (28) Google Scholar) was reduced in the CueO-PM2/12.1 constructs compared with CueO, CueO-HA, and the control CueO-12.1CON (Fig. 5B).Table 2Kinetic parameters of engineered CueO variantsConstructVmaxKmkcatkcat/KmRelative efficiencyμm min−1μmmin−1min−1 μm−1%CueO-WT14.37 ± 0.78293.18 ± 23.08151.3 ± 8.170.5298CueO (M358I)12.78 ± 1.08256.09 ± 9.59134.5 ± 11.420.53100CueO-HA8.51 ± 0.86286.06 ± 22.8989.6 ± 9.050.3158.49CueO-HA + anti-HA2.68 ± 0.19175.37 ± 11.6528.18 ± 2.050.1630.19CueO-PM24.63 ± 0.16284.77 ± 25.5048.69 ± 1.730.1732.07CueO-PM2 + MDM27.29 ± 0.64251.12 ± 17.4876.69 ± 6.720.3158.49CueO-12.15.86 ± 0.35290.65 ± 44.3361.73 ± 3.680.2139.62CueO12.1CON9.18 ± 1.36234.51 ± 41.9996.67 ± 14.290.4177.36 Open table in a new tab To gain further mechanistic insights, we determined crystal structures of both the free and MDM2(6–125)-bound forms of CueO-PM2 along with CueO-12.1. The asymmetric unit for the MDM2-bound form comprised a single binary complex with the scaffolded PM2 peptide adopting its prototypical α-helical conformation (Fig. 6A). The three key signature residues (Phe, Trp, and Leu) projecting from the outer face of the helix are accommodated by discrete pockets in a prolonged hydrophobic cleft of MDM2. Comparison with structures of unscaffolded PMI, a related stapled derivate (MO6, differing by one amino acid), and the parental p53 peptide bound to MDM2 (32Pazgier M. Liu M. Zou G. Yuan W. Li C. Li C. Li J. Monbo J. Zella D. Tarasov S.G. Lu W. Structural basis for high-affinity peptide inhibition of p53 interactions with MDM2 and MDMX.Proc. Natl. Acad. Sci. U.S.A. 2009; 106 (19255450): 4665-467010.1073/pnas.0900947106Crossref PubMed Scopus (276) Google Scholar, 36Kussie P.H. Gorina S. Marechal V. Elenbaas B. Moreau J. Levine A.J. Pavletich N.P. Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain.Science. 1996; 274 (8875929): 948-95310.1126/science.274.5289.948Crossref PubMed Scopus (1782) Google Scholar, 41Chee S.M. Wongsantichon J. Soo Tng Q. Robinson R. Joseph T.L. Verma C. Lane D.P. Brown C.J. Ghadessy F.J. Structure of a stapled peptide antagonist bound to nutlin-resistant Mdm2.PLoS One. 2014; 9 (25115702)e10491410.1371/journal.pone.0104914Crossref PubMed Scopus (32) Google Scholar) shows highly similar side chain conformations of these residues (Fig. 6B). The adjacent MRS residues Met-396 and Asn-397 extend the PM2 α-helix to more fully occupy the MDM2 binding groove. The PM2 conformation is further stabilized by hydrophobic interactions between residues on its inner face (Leu-393 and Tyr-390) and Ile-358 and Met-361 on the α5 helix. In addition to a polar contact between side chains of Lys-94 of MDM2 and Gln-365 in the α5 helix, these interactions likely drive ordering of the MRS linker residues abutting PM2 that are highly mobile in native CueO (25Roberts S.A. Weichsel A. Grass G. Thakali K. Hazzard J.T. Tollin G. Rensing C. Montfort W.R. Crystal structure and electron transfer kinetics of CueO, a multicopper oxidase required for copper homeostasis in Escherichia coli.Proc. Natl. Acad. Sci. U.S.A. 2002; 99 (11867755): 2766-277110.1073/pnas.052710499Crossref PubMed Scopus (283) Google Scholar). The MDM2-bound PM2 helix is positioned well away from the sCu and T1 copper-binding sites, permitting unhindered access to Cu and syringaldazine substrates and resulting in the observed MDM2-dependent activity gains. The reduced activity of CueO-PM2 compared with CueO and CueO-HA a" @default.
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• W2912276490 title "Development and structural characterization of an engineered multi-copper oxidase reporter of protein–protein interactions" @default.
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