Oxometalates as artificial proteases: sequence versus region selectivity

1. Oxometalates as artificial proteases: • sequence versus region selectivity Karen Stroobants

2. Inorganicchemistry « Whyisproteinhydrolysis important? » • Oxometalates as artificial proteases: • sequence versus region selectivity « Whatiswrongwithtrypsin? » • limitedsolvent compatibility • unreactivetowardIDPs • small fragments Proteinchemistry

3. Need for complementary and versatile cleavage agents “The development of alternativecleavage strategies would greatly facilitate the study of protein structure and function.” Grant 2006 Oftenproposed solution: The use of artificialmetallopeptidases  metal ions as hydrolytic active centers Currentproblems:  Lowreactivityatphysiological pH and temperature  Non specific interaction / Non selectivecleavage

4. Conceptuallynewartificialcleavageagents IsoOxometalates “Sarcoplasmicreticulum calcium ATPaseinteractionswithdecaniobate, decavanadate, vanadate, tungstate and molybdate” “Functional and structural interactions of Nb, V, Mo and W oxometalates with the sarcoplasmic reticulum Ca2(+) -ATPase reveal new insights into inhibition processes” (Aureliano et al.) Hetero Polyoxometalates “Polyoxometalate Binding to Human Serum Albumin: A Thermodynamic and Spectroscopic Approach”“A multitechnique study of europium decatungstateand human serum albuminmolecularinteraction” (Nadjo et al.)

5. Conceptuallynewartificialcleavageagents IsoOxometalates “Sarcoplasmicreticulum calcium ATPaseinteractionswithdecaniobate, decavanadate, vanadate, tungstate and molybdate” “Functional and structural interactions of Nb, V, Mo and W oxometalates with the sarcoplasmic reticulum Ca2(+) -ATPase reveal new insights into inhibition processes” (Aureliano et al.) Hetero Polyoxometalates “Polyoxometalate Binding to Human Serum Albumin: A Thermodynamic and Spectroscopic Approach”“A multitechnique study of europium decatungstateand human serum albuminmolecularinteraction” (Nadjo et al.)

6. Molybdate(IV) hydrolyzes HEWL SDS-PAGE & Edmandegradation: Asp18-Asn19 Asp48-Gly49 Asp52-Trp53 Asp101-Gly102 pH 5.0 / 60 °C + Ho et al. (2011) Inorg. Chem. Stroobants et al. (2013) submitted to J. Inorg. Biochem.

7. Sequencespecifichydrolysis at Asp-Xbonds Interactionoccurs at manypositions and thus is notselective ReactiononlyoccurswhenanAsp-X bond is accesible and thus is sequenceselective

8. Side chainassistencemechanism Polarization of the carbonyl due to [MoO4]2- binding & Internal nucleophilic attack of the Asp side chain

9. Side chainassistencemechanism Appearanceof Gly Disappearanceof Asp-Gly Polarization of the carbonyl due to [MoO4]2- binding & Internal nucleophilic attack of the Asp side chain ppm

10. Conceptuallynewartificialcleavageagents IsoOxometalates “Sarcoplasmicreticulum calcium ATPaseinteractionswithdecaniobate, decavanadate, vanadate, tungstate and molybdate” “Functional and structural interactions of Nb, V, Mo and W oxometalates with the sarcoplasmic reticulum Ca2(+) -ATPase reveal new insights into inhibition processes” (Aureliano et al.) Hetero Polyoxometalates “Polyoxometalate Binding to Human Serum Albumin: A Thermodynamic and Spectroscopic Approach”“A multitechnique study of europium decatungstateand human serum albuminmolecularinteraction” (Nadjo et al.)

11. Polyoxotungstates as more tunablealternative Polyoxotungstateligand SPECIFIC INTERACTION Lewis active metal HYDROLYTIC ACTIVITY • Higherstability (vs. Oxomolybdate) • Knownregionspecificinteractionwithprotein surfaces • Notreactive as such!!! • Oxophilicity • Coordinationnumber- Ligand exchange kinetics •  Ce(IV) ion

12. Zr(IV)-substitutedPOMshydrolyze HSA …NLPArg114//Leu115VRP… …DDRAla257//Asp258LAK… …KQNCys392//Glu393LFE… Stroobants et al. (2013) Chem. Eur. J. …VESLys313//Asp314VCK…

13. Zr(IV) and Wells Dawson POM driveninteractions lacunaryWells Dawson Zr(IV) substitutedWells Dawson 1:1 interaction Ka~ 106M-1 1:1 interaction / 1:3 interaction Ka~ 108M-1/ Ka ~ 105M-1

14. Zr(IV)-Wells Dawson exhibitsregionselectiveinteraction lacunaryWells Dawson Zr(IV) substitutedWells Dawson 1:1 interaction Ka~ 106M-1 1:1 interaction / 1:3 interaction Ka~ 108M-1/ Ka ~ 105M-1 * Arg114-Leu115 known POM binding positiveregionNadjo et al. * Ala257-Asp258 * Lys313-Asp314 * Cys392-Glu393 regions of mixed charge, hydrolysis downstream fromacidicresidue

15. Additionalfragmentsuponprolongedreaction time Edmandegradation …DDRAla257//Asp258LAK… …VESLys313//Asp314VCK…

16. Sameselectivityforsecondaryfragmentation

17. Sameselectivityforsecondaryfragmentation

18. Is X-Asp/Gluhydrolysismechanisticallyrelatedto Asp-Xcleavage? Proposedassistencemechanism - does onlydominate < pH 6 - does onlyoccurforAsp (notforGlu) * Ala257-Asp258 * Lys313-Asp314 * Cys392-Glu393 regions of mixed charge, hydrolysis downstream fromacidicresidue • Reactionoccurs • at pH 7.4 • at Asp and Glu • residues

19. Different mechanismsforoxometalate versus metal-substituted POM reactivity Non-specific binding Sequenceselectivehydrolysis, via sidechainassistencemechanism Regionselectiveinteraction Hydrolysis without sidechainassistance, presumably via external water nucleophiledelivery

20. (Poly)oxoanionsholdpromise as artificialproteases Evidencefor* SequenceselectiveAsp-Xproteinhydrolysis in the presence of oxomolybdate & * Regionselectiveproteincleavage in the presence of metal-substitutedpolyoxotungstates Futureperspective « Hydrolysis of membrane proteins and IDPs possible? »

21. Manythanks to… My promotor – Prof. Parac-Vogt Tatjana POM post-doc – Dr. AbsillisGregory Edmandegradation – Prof. Proost Paul & Dr. Moelants Eva ITC – Dr. Bruylants Gilles NMR technician – Karel Deurinckx Masterstudents – LaureBaeyens, DouniaSaadallah, Anja Bourgois, Vincent Goovaerts & Jeroen Lannoeye All the colleagues of lab LBC / LIC / ULB FWO forfinancial support & youforyourattention!