Affinity maturation of the RLIP76 Ral binding domain to inform the design of stapled peptides targeting the Ral GTPases
نویسندگان
چکیده
Ral GTPases have been implicated as critical drivers of cell growth and metastasis in numerous Ras-driven cancers. We previously reported stapled peptides, based on the effector RLIP76, that can disrupt signaling. Stapled peptides are short locked into their bioactive form using a synthetic brace. Here, an affinity maturation RLIP76 Ral-binding domain, we identified several sequence substitutions together improve binding to proteins by more than 20-fold. Hits from selection were rigorously analyzed determine contributions individual residues two 1.5 Å cocrystal structures tightest-binding mutants complex with RalB revealed key interactions. Insights gained this used design second-generation exhibited vastly improved selectivity for when compared first-generation lead peptide. The was quantified site peptide determined NMR. successfully competed multiple Ral–effector interactions cellular lysates. Our findings demonstrate how manipulation native partner assist rational inhibitors targeting protein–protein interaction. Ras (H-Ras, N-Ras, K-Ras) well known most commonly mutated oncoproteins human cancer, activating mutations found approximately 20% cancers higher incidences pancreatic (88%), colorectal (55%) lung (33%) (1Prior I.A. Hood F.E. Hartley J.L. frequency cancer.Cancer Res. 2020; 80: 2969-2974Crossref PubMed Scopus (125) Google Scholar). These small exist distinct conformations: inactive GDP-bound active GTP-bound which they bind signal through downstream proteins. Through mechanism ability act molecular switches. signaling is “switched on” Guanine Exchange Factors (GEFs) off” GTPase Activating Proteins (GAPs, reviewed (2Cherfils J. Zeghouf M. Regulation GEFs, GAPs, GDIs.Physiol. Rev. 2013; 93: 269-309Crossref (669) Scholar)). Most cancer render protein constitutively active, state. This leads deregulated pathways, resulting uncontrolled proliferation. 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Sci. 117: 7131-7139Crossref (9) emerged years promising tools PPIs mediated ?-helices. technique, developed Verdine involves introduction unnatural olefin-bearing acids i, i + 4/7 positions, covalently linked Grubbs’ ring closing metathesis (31Schafmeister C.E. Po G.L. An all-hydrocarbon cross-linking system enhancing helicity metabolic stability peptides.J. Am. Soc. 2000; 122: 5891-5892Crossref (723) chemical staples stabilize helical structure impart beneficial properties penetration, increased proteolytic 32Chu Q. Moellering R.E. Hilinski G.J. Kim Grossmann T. Towards understanding penetration peptides.Med. Commun. 6: 111-119Crossref applied (reviewed (33Hurd C.A. Mott H.R. Owen superfamily.Pept. 112: 1-14Crossref (4) use (34Thomas J.C. Cooper Clayton N.S. Abell approach.J. 291: 18310-18325Abstract (16) solution RBD interaction well-structured coiled-coil 80% ?2 helix (35Fenwick L.J. Rajasekar Prasannan Nietlispach RalB-RLIP76 reveals novel mode ral-effector interaction.Structure. 2010; 985-995Abstract (33) As common (36Campbell Peppa Crabtree Shafiq McGough Thermodynamic mapping interfaces RalB.Biochemistry. 54: 1380-1389Crossref (5) postulated produced series spanning staple lengths positions assessed work led identification RBD, interactions, enter autophagy, RalB-dependent process. selective over closely related R-Ras. tool guide A CIS display performed modified sequences alterations similar manner. Investigations previous issues relating poor solubility nonspecific addressed here, selections (37Odegrip Coomber Eldridge Hederer Kuhlman P.A. Ullman FitzGerald McGregor display: vitro libraries protein-DNA complexes.Proc. 2004; 101: 2806-2810Crossref (111) carried out changes within our sequence, (Fig. 1A). In display, variant library fused gene encoding RepA, captures DNA it translated upon recognition cis element, coupling genetic material library. Up 1014 method. utilized included conventional methods, (residues 393–446) model binding. During selections, only contained interface permitted alter 1B). Trp430 retained Biotinylated immobilized separate allow discriminated between After four rounds biopanning, enrichment relatively (38Tetley G.J.N. Murphy N.P. Bonetto Ivanova-Berndt Revell Cooley R.N. discovery biologics G Cdc42: bioblockade signalling.J. 295: 2866-2884Abstract (7) phenomenon may indicate parent already achieved close evolutionary maximum capacity proteinogenic acids. obtained largely involving RalB, reflecting Three consensus across 1C) represented enriched selections. reasoned that, amalgamation sequences, subtle cooperation might high-affinity Therefore, appeared highest each three major clusters recombinantly coli quantify Sequences containing K440P mutation (Cluster 3, Fig. precipitation during purification: likely helix-breaking Pro disrupts instability. lacking substitution (E426L/E427T/Q433T/R434L/T437R) His-tagged construct did scintillation proximity assays (SPAs) S1). Affinities measured competition SPAs 2 Table 1). six bound tightly wild-type RBD: all Cluster 1 tightest (HLR SMLR) gave least 20-fold improvement resembling 1C, WDASQSR, WNASELR WDASTAY) lower RBD. Competition SPA experiments however effector, possible binders occupied To ensure case, WDASQSR SPA; S2) circular dichroism (CD) disrupted S3).Table 1Affinities RBDs SPANameSequenceKd (nM)aStandard error curve fitting.RalA·GTPRalB·GTPWild-typeRLIP76 (393–422, C411S)-LSKEERLWEVQRILTALKRKLREA96 ± 17109 16HLR....H.....L......R......5 31 2SMLR....S.M...L......R......3 32 2DVLR....D.V...L......R......12 47 2WDASQSR...WD.A...SQ..S..R......3020 7206970 930WNASELR...WN.A...SE..L..R......2750 85010,350 2250WDASTAY...WD.A...ST..A..Y......530 701200 140a Standard fitting. Open table new tab investigate mechanism(s) driving proteins, set obtain data complexes. high-quality crystals RalB/RLIP76 HLR SMLR complexes, diffracted resolution 3A 2). highly RMSD less 0.1 formed identical, suggesting E427H/S R429M important binding.Table 2Data collection refinement statistics RalB·GMPPNP (6ZQT) (6ZRN)Protein complexRalB·GMPPNP:RLIP76 (HLR)aThe numbers parentheses represent values highest-resolution shell.RalB·GMPPNP:RLIP76 (SMLR)aThe shell.PDB identifier6ZQT6ZRNResolution (Å)50.4–1.51 (1.55–1.51)65.8–1.48 (1.56–1.48)Space groupP 21 1P 1Cell dimensions a,b,c (Å)47.5, 77.4, 66.447.2, 77.5, 65.8 ?,?,? (°)90, 90.3, 9090, 90.1, 90Total reflections569,182 (24,662)1,159,648 (176,059)Redundancy7.6 (4.5)14.8 (15.4)Completeness (%)99.7 (98.7)100.0 (100.0)I/?18.3 (1.2)8.0 (1.3)Wilson B-factor (Å2)24.519.3Refinement Rwork/Rfree (%)18.9/22.1 (33.6/36.3)20.1/23.9 (34.9/36.1) No. atoms36863725 ligand atoms7278 water molecules362244 length (Å)0.0050.006 angles (°)0.750.80 Ramachandran favored regions (%)97.196.9 allowed (%)2.93.1 Outliers (%)00 Mean (Å2)34.530.8a shell. delineate thermodynamic contribution variant, constructs one prepared triple double isothermal titration calorimetry (ITC) 3). ITC 2.7 ?M, agreement value 1.9 ?M affinities differed those competitions (approximately tenfold RBD); therefore, comparisons given technique made.Table 3Binding parameters titrated RalBNameSequenceKd (nM)aData mean independent standard deviation.N valueaData deviation.?H (kcal/mol)aData deviation.T?S deviation.Wild-typeRLIP76(393–422, C411S)-LSKEERLWEVQRILTALKRKLREA2720 6401.00 0.01?10.5 0.2?2.89 0.33HLR....H.....L......R......96.2 29.10.89 0.12?17.7 2.9?8.12 3.08LR..........L......R......132 460.81 0.00?21.0 1.3?11.6 1.5HL....H.....L.............224 940.83 0.15?19.5 2.3?10.3 2.0HR....H............R......4350 6300.99 0.06?9.05 0.16?1.72 0.08L..........L.............110 190.75 0.06?22.4 1.4?12.9 1.3a Data deviation. previously, driven favorable enthalpic term (?H), outweighs entropic cost (?S)
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ژورنال
عنوان ژورنال: Journal of Biological Chemistry
سال: 2021
ISSN: ['1083-351X', '0021-9258', '1067-8816']
DOI: https://doi.org/10.1074/jbc.ra120.015735