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Article: Mutational analysis of the complex of human RNase inhibitor and human eosinophil-derived neurotoxin (RNase 2)

TitleMutational analysis of the complex of human RNase inhibitor and human eosinophil-derived neurotoxin (RNase 2)
Authors
Issue Date2003
PublisherAmerican Chemical Society. The Journal's web site is located at http://pubs.acs.org/biochemistry
Citation
Biochemistry, 2003, v. 42 n. 6, p. 1451-1459 How to Cite?
AbstractRNase inhibitor (RI) binds diverse proteins in the pancreatic RNase superfamily with extremely high avidity. Previous studies showed that tight binding of RNase A and angiogenin (Ang) is achieved primarily through interactions of hot spot residues in the 434-460 C-terminal segment of RI with the enzymatic active site; Asp435 of RI forms key hydrogen bonds with the catalytic lysine in both complexes, whereas the other contacts are largely distinctive. Here we have investigated the structural basis for recognition of a third ligand, eosinophil-derived neurotoxin (EDN), by single-site and multisite mutagenesis. Surprisingly, Ala replacement of Asp435 decreases affinity for EDN only by 14-fold, as compared to the several hundred-fold decreases with RNase A and Ang, and individual mutations of three other hot spot residues - Tyr434, Tyr437, and Ser460 - have essentially no effect. Ala substitutions of nine additional residues, selected by examining a computational model of the RI-EDN complex, also have no marked impact. Overall, the losses in affinity for the single-residue variants examined account for only ∼25% of the free energy of binding for the complex. However, multisite mutagenesis of RI reveals strong superadditivity of mutational effects, indicating that part of this shortfall reflects negative cooperativity. Replacement of Tyr434 together with Asp435 or Tyr437 increases Ki by 540- and 290-fold, respectively. Thus, the C-terminal region of RI again plays an important role in ligand recognition, although probably smaller than for binding RNase A and Ang. Simultaneous substitutions of three neighboring tryptophans (261, 263, and 318) on RI attenuate affinity even more dramatically (by 4900-fold), indicating that the interactions of this RI region also contribute a considerable amount of the binding energy for the EDN complex. These findings highlight the potential importance of cooperativity in protein - protein interactions and the consequent limitations of single-site mutagenesis for assessing interface energetics.
Persistent Identifierhttp://hdl.handle.net/10722/157356
ISSN
2015 Impact Factor: 2.876
2015 SCImago Journal Rankings: 1.769
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorTeufel, DPen_US
dc.contributor.authorKao, RYTen_US
dc.contributor.authorAcharya, KRen_US
dc.contributor.authorShapiro, Ren_US
dc.date.accessioned2012-08-08T08:49:12Z-
dc.date.available2012-08-08T08:49:12Z-
dc.date.issued2003en_US
dc.identifier.citationBiochemistry, 2003, v. 42 n. 6, p. 1451-1459en_US
dc.identifier.issn0006-2960en_US
dc.identifier.urihttp://hdl.handle.net/10722/157356-
dc.description.abstractRNase inhibitor (RI) binds diverse proteins in the pancreatic RNase superfamily with extremely high avidity. Previous studies showed that tight binding of RNase A and angiogenin (Ang) is achieved primarily through interactions of hot spot residues in the 434-460 C-terminal segment of RI with the enzymatic active site; Asp435 of RI forms key hydrogen bonds with the catalytic lysine in both complexes, whereas the other contacts are largely distinctive. Here we have investigated the structural basis for recognition of a third ligand, eosinophil-derived neurotoxin (EDN), by single-site and multisite mutagenesis. Surprisingly, Ala replacement of Asp435 decreases affinity for EDN only by 14-fold, as compared to the several hundred-fold decreases with RNase A and Ang, and individual mutations of three other hot spot residues - Tyr434, Tyr437, and Ser460 - have essentially no effect. Ala substitutions of nine additional residues, selected by examining a computational model of the RI-EDN complex, also have no marked impact. Overall, the losses in affinity for the single-residue variants examined account for only ∼25% of the free energy of binding for the complex. However, multisite mutagenesis of RI reveals strong superadditivity of mutational effects, indicating that part of this shortfall reflects negative cooperativity. Replacement of Tyr434 together with Asp435 or Tyr437 increases Ki by 540- and 290-fold, respectively. Thus, the C-terminal region of RI again plays an important role in ligand recognition, although probably smaller than for binding RNase A and Ang. Simultaneous substitutions of three neighboring tryptophans (261, 263, and 318) on RI attenuate affinity even more dramatically (by 4900-fold), indicating that the interactions of this RI region also contribute a considerable amount of the binding energy for the EDN complex. These findings highlight the potential importance of cooperativity in protein - protein interactions and the consequent limitations of single-site mutagenesis for assessing interface energetics.en_US
dc.languageengen_US
dc.publisherAmerican Chemical Society. The Journal's web site is located at http://pubs.acs.org/biochemistryen_US
dc.relation.ispartofBiochemistryen_US
dc.subject.meshAlanine - Geneticsen_US
dc.subject.meshAmino Acid Substitution - Geneticsen_US
dc.subject.meshAngiotensins - Chemistryen_US
dc.subject.meshAspartic Acid - Geneticsen_US
dc.subject.meshCarrier Proteinsen_US
dc.subject.meshDna Mutational Analysisen_US
dc.subject.meshEosinophil-Derived Neurotoxinen_US
dc.subject.meshEosinophils - Enzymologyen_US
dc.subject.meshHumansen_US
dc.subject.meshKineticsen_US
dc.subject.meshMacromolecular Substancesen_US
dc.subject.meshModels, Molecularen_US
dc.subject.meshMutagenesis, Site-Directeden_US
dc.subject.meshProtein Binding - Geneticsen_US
dc.subject.meshProteins - Chemistry - Geneticsen_US
dc.subject.meshRibonucleases - Chemistry - Geneticsen_US
dc.subject.meshSequence Deletionen_US
dc.subject.meshTyrosine - Geneticsen_US
dc.titleMutational analysis of the complex of human RNase inhibitor and human eosinophil-derived neurotoxin (RNase 2)en_US
dc.typeArticleen_US
dc.identifier.emailKao, RYT:rytkao@hkucc.hku.hken_US
dc.identifier.authorityKao, RYT=rp00481en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.doi10.1021/bi026852oen_US
dc.identifier.pmid12578357-
dc.identifier.scopuseid_2-s2.0-0037452522en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0037452522&selection=ref&src=s&origin=recordpageen_US
dc.identifier.volume42en_US
dc.identifier.issue6en_US
dc.identifier.spage1451en_US
dc.identifier.epage1459en_US
dc.identifier.isiWOS:000180955900011-
dc.publisher.placeUnited Statesen_US
dc.identifier.scopusauthoridTeufel, DP=16425048000en_US
dc.identifier.scopusauthoridKao, RYT=7101675499en_US
dc.identifier.scopusauthoridAcharya, KR=35518435100en_US
dc.identifier.scopusauthoridShapiro, R=7403082810en_US

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