Reprogramming the Specificity of a Protein Interface by Computational and Data-Driven Design

•The specificity of a glutaminase subunit for synthase has been inverted•The switch was achieved by both computational and data-driven approach•The designed complexes were stable catalytically active The formation specific protein in cell is non-trivial problem given the co-existence thousands different polypeptide chains. A particularly difficult case are two glutamine amidotransferase (anthranilate [AS] aminodeoxychorismate [ADCS]), which composed homologous pairs subunits. We have attempted to identify discriminating interface residues TrpG from AS, responsible its interaction with TrpEx prevent binding closely related PabB ADCS. For this purpose, TrpG-specific grafted into PabA ADCS approaches, namely one. Both approaches resulted variants that bound higher affinity than PabB. Hence, we accomplished reprogramming protein-protein provides insights evolutionary adaptation interfaces. large fraction proteins found nature belongs compositionally well-defined homomeric or heteromeric complexes. example, survey oligomerization state Escherichia coli revealed only 20% polypeptides exist as monomers, whereas 80% form defined other proteins. Approximately these homo-oligomers hetero-oligomers (Goodsell Olson, 2000Goodsell D.S. Olson A.J. Structural symmetry function.Annu. Rev. Biophys. Biomol. Struct. 2000; 29: 105-153Crossref PubMed Scopus (648) Google Scholar; Levy et al., 2008Levy E.D. Boeri Erba E. Robinson C.V. Teichmann S.A. Assembly reflects evolution complexes.Nature. 2008; 453: 1262-1265Crossref (305) Scholar). Despite risk emergence millions non-physiological assemblies crowded environment cells, highly formed. This all more astonishing number geometries shown be restricted ~1,000 (Gao Skolnick, 2010Gao M. Skolnick J. space interfaces degenerate, close complete, connected.Proc. Natl. Acad. Sci. U S A. 2010; 107: 22517-22522Crossref (98) Garma 2012Garma L. Mukherjee S. Mitra P. Zhang Y. How many interactions types nature?.PLoS One. 2012; 7: e38913Crossref (26) Marsh Teichmann, 2015Marsh J.A. Structure, dynamics, assembly, complexes.Annu. Biochem. 2015; 84: 551-575Crossref (207) Therefore, it important understand how guaranteed crosstalk prevented under such constraints. avoidance non-productive most cases where share same fold possess similar geometries. To disclose driving forces confer one cases, exemplarily analyzed complex amidotransferases (GATases). GATases enzyme family whose members incorporation nitrogen within numerous metabolic pathways (Mouilleron Golinelli-Pimpaneau, 2007Mouilleron Golinelli-Pimpaneau B. Conformational changes ammonia-channeling amidotransferases.Curr. Opin. Biol. 2007; 17: 653-664Crossref (72) Raushel 1999Raushel F.M. Thoden J.B. Holden H.M. enzymes: molecular machines production delivery ammonia.Biochemistry. 1999; 38: 7891-7899Crossref (90) bienzyme complexes, consisting subunit. activities subunits mutually coupled 2-fold manner: (1) substrate allosterically induces hydrolysis glutamate nascent ammonia at subunit; (2) then transported through an intermolecular channel site reacts “waiting” reaction products. glutaminases can categorized classes according their folds compositions: class I show ??-hydrolase catalytic triad Cys-His-Glu (Ollis 1992Ollis D.L. Cheah Cygler Dijkstra Frolow F. Franken S.M. Harel Remington S.J. Silman I. Schrag al.The ?/? hydrolase fold.Protein Eng. 1992; 5: 197-211Crossref (1797) Scholar), II Ntn-hydrolase N-terminal Cys (Brannigan 1995Brannigan Dodson G. Duggleby H.J. Moody P.C. Smith J.L. Tomchick D.R. Murzin A.G. framework nucleophile capable self-activation.Nature. 1995; 378: 416-419Crossref (533) Compared glutaminases, various structurally quite diverse. Exceptions, however, 4-amino-4-deoxychorismate (ADCS) anthranilate (AS). enzymes use chorismate catalyze committed steps biosynthetic leading folate tryptophan, respectively. In AS not but also synthases homologs three-dimensional structures (Morollo Eck, 2001Morollo A.A. Eck M.J. Structure cooperative allosteric Salmonella typhimurium.Nat. 2001; 8: 243-247Crossref (77) Parsons 2002Parsons J.F. Jensen P.Y. Pachikara A.S. Howard Eisenstein Ladner J.E. synthase: architectural conservation diversity chorismate-utilizing enzymes.Biochemistry. 2002; 41: 2198-2208Crossref (56) Semmelmann 2019bSemmelmann Straub K. Nazet Rajendran C. Merkl R. Sterner Mapping communication network synthase.J. Mol. 2019; 431: 2718-2728Crossref (6) ensuring pair particular challenge. Remarkably, species, functionally interacts TrpE (Plach 2017Plach M.G. Busch Heizinger Wysocki V.H. Evolutionary diversification add-ons.Proc. 2017; 114: E8333-E8342Crossref (14) Consequently, tryptophan biosynthesis cannot regulated independently level. issue resolved some “modern” ?-proteobacteria evolving additional referred TrpG, specifically modified version refer (x stands extended) (Figure 1A). TrpEx, comparison PabB, contains insertion toward hypothesized “interface add-on” guarantees avoids (Figures 1B 1C). phylogenetic analysis indicated evolved gene duplication speciation course bona fide pathway mutations must occurred allowed time TrpE. retrace putative mutational events laboratory, replaced assumed interact add-on TrpEx. resulting variant PabA? formed strong while retained significant original partner 1D). Based on findings, goal study generate exclusively no longer able bind 1E). achieve conversion specificity, used design based Rosetta software suite and, alternatively, approach exploits differential TrpG. previously reconstruct generalist specialized generated functional properties made us presume structural element complementary required interface. should ensure exclusive suitable method introduce silico natural epitope grafting (Schreiber Fleishman, 2013Schreiber Fleishman Computational interactions.Curr. 2013; 23: 903-910Crossref (44) broad functionality well-proven (Rohl 2004Rohl C.A. Strauss C.E. Misura K.M. Baker D. Protein structure prediction using Rosetta.Methods Enzymol. 2004; 383: 66-93Crossref (1181) Scholar) assists user addressing state-of-the art research challenges means protocols. protocol supports “anchored design” (Lewis Kuhlman, 2011Lewis Kuhlman B.A. Anchored interfaces.PLoS 2011; 6: e20872Crossref (48) utilized Rosetta:MSFAnchoredDesign (Löffler 2017Löffler Schmitz Hupfeld Rosetta:MSF: modular multi-state design.PLoS Comput. 13: e1005600Crossref (27) because implementation design. opted considered representation alternative poses less susceptible modeling errors phase. Thus, applied combination anchor 25 successive Initially, chimeric sequence PabA-CA created contained complement residue positions 6–30 2). subjected genetic algorithm Rosetta:MSFAnchoredDesign, set 239 sequences improving fitness respect states, i.e., PabA:TrpEx configurations. design, contributing Rosetta. Whereas initial differed 13 ecPabA coli, PabA-CAD 17 (Table 1).Table 1Overview VariantsecPabAPabA?PabA-CAPabA-CADPabA?+PabA??DiffY8IKII?D9DA?Y16YAAYY?Q17DDDDDY18QQCQQ?F19LL?C20RRRRRE21TTTTL22NN?A24HH?D25NN?L27VV?V28VIVVVK29YYYYYR30RDR?D32QCHH?C54CCGGR95RQQ?K98EE?M100E?T104AA?T125A?The first column denotes wild-type PabA, columns denote corresponding listed (white unchanged residue). characterized bold hydrogen bond across modeled native PabA:PabB italic those taken Bold models bond, underlined ones salt bridge Interface possessing PabA?? “?” last column. Open table new tab localization mutated Figures 3A 3B . assess similarity superposed C? atoms relaxed conformations model (based PDB-ID: 6qur) typhimurium (PDB-ID: 1i1q). root-mean-square deviation value 0.7 Å. low confirms excellent match backbones consequence, arrangement finding, fully automated Rosetta, choose substitutions might change end, logo 487 32 in-house program 3C). Our amino acid occupancy significantly between Starting initially PabA?+: strictly conserved Y8 D32 abundant H, addition, PabA. It known mutation T125A causes 10-fold acceleration basal activity, likely due rearrangement near (Semmelmann PabA??, compared PabA?+ 3C; Table 1). 3B. genes coding introduced pET21a-BsaI via BsaI-cloning described (Rohweder 2018Rohweder Endres Standardized cloning vectors generation libraries coli.BioTechniques. 2018; 64: 24-26Crossref (8) expressed purified soluble extract immobilized metal ion chromatography preparative size-exclusion (SEC). ecPabB ecTrpEx ppTrpE Pseudomonas putida, well stTrpG typhimurium, instead ecTrpGD fusion protein, available previous studies 2019aSemmelmann fold-independent crucial signaling amidotransferases.Biochemistry. 58: 2584-2588Crossref ability PabB/TrpE tested qualitatively analytical SEC. Three SEC runs performed each interest: isolated subunit, equimolar mixture Complex appearance new, early eluting peak disappearance peaks + sample. As expected, observed partners 4A, left panel) right panel). No combinations 4B, These samples served positive negative controls, showed detectable incomplete ecTrpEx, free addition 5A 5B , upper panels). Moreover, neither nor 5B, middle panels) lower Taken together, results indicate partially converted present PabA-CAD. basis logo, ecTrpEx; slightly propensities: elution profile formation, small unbound 6A, contrast, combined quantitative 6B, Remarkably either 6A suggest extent included PabA??. reflected stimulation respective On basis, activity titrations obtain measure variants. rates monitored function varied concentrations 7). activation data determination apparent dissociation constants (KDapp) maximum turnover (kcatapp), 2. specificities variants, quotient KDapp(ecPabB)/KDapp(ecTrpEx) calculated. <<1 ecPabA, indicating high ecPabB, >>1 stTrpG, ecTrpEx. With exception PabA-CA, >1 Admittedly, preferences (quotient = 7.3), 9.1), 3.9) still preference displayed >> Nevertheless, newly moderately surpass 2.3) inverted << 1) 2), pointing switch. titration experiments presence saturating glutamine. obtained allow numbers (kcatapp) stimulated much stronger cognate non-cognate ones. All levels comparable glutaminase-synthase pairs. An PabA-CAD, kcatapp values thus 2).Table 2Stimulated Glutaminase Activities VariantsGlutaminaseecPabBecTrpExKDapp(ecPabB)/KDapp(ecTrpEx)KDapp (nM)kcatapp (s?1)KDapp (s?1)ecPabA2280.25>5,000n.d.<<1stTrpG>5,000n.d.1620.36>>1PabA?9290.0923960.292.3PabA-CA1,0360.162,6020.170.40PabA-CAD1,2000.0371650.0447.3PabA?+3,6690.154030.179.1PabA??1,7900.304530.183.9The KDapp derived hyperbolic fits Figure 7.n.d., determined. 7. n.d., gain distinguish stability our designs, PISA service EMBL-EBI. After upload, underlying determines, complex, parameters estimate macromolecular (Krissinel Henrick, 2007Krissinel Henrick Inference crystalline state.J. 372: 774-797Crossref (6209) concentrated potential bonds bridges substantial contribution stability. begin with, built experimentally confirmed (see STAR Methods): ecPabA:ecPabB five stTrpEx, closest relative structure. relax structures, they short dynamics simulation final stored PDB format. Finally, six files uploaded server involved identified. 1 shows four Y8, D9, Q17, C54 ecPabB. stTrpEx 8). PabA-CAD:stTrpEx forms G54 G259 another V28 E114, latter located stTrpEx. Two R20 A371; moreover, D17 R382. PabA??:stTrpEx even interactions: Residue G259; G380. Interestingly, V28, Y29, R30 N115, L111, P110, E98 R257 R382, suggests least assumption agreement biochemical characterization 2): range stTrpG:ecTrpEx. However, analysis, homology did explain differences detail. Cells contain proteins, principle myriad majority avoided, would presumably lead unproductive crosstalk. But does assure physiologically meaningful formed, limited quaternary topologies (Ahnert 2015Ahnert S.E. Hernandez H. Principles assembly reveal periodic complexes.Science. 350: aaa2245Crossref (130) Scholar)? seems serious when compete Keating, 2011Schreiber Keating A.E. versus promiscuity.Curr. 21: 50-61Crossref (167) Class GATases, well-characterized (Zalkin, 1993Zalkin amidotransferases.in: Meister Adv Enzymol Relat Areas Mol Wiley, 1993: 203-309Crossref systems studying cases. Each member wit