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In memoriam Zafar H. Zaidi

THE PAKISTANI CONNECTION: domestication of wild-(type) protease inhibitor miniproteins from desert locust, Schistocerca gregaria.

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In memoriam Zafar H. Zaidi

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  1. THE PAKISTANI CONNECTION: domestication of wild-(type) protease inhibitor miniproteins from desert locust, Schistocerca gregaria László Gráf,Sumaira Amir, Zulfiquar Malik,Gábor Pál, Krisztián Fodor,Borbála Szenthe, András Perczel,Zoltán Gáspári,Veronika Harmat, Csaba Hetényi, József Antal,József Kardos, Gergely Katona andAndrás Patthy Departments of Biochemistry and Organic Chemistry, Eötvös Loránd University, Budapest

  2. In memoriam Zafar H. Zaidi

  3. %B 60 40 30 20 0 0.12 0,10 0,08 0.06 0.04 0.02 0.00 Absorbance at 220 nm 0 10 20 30 40 Time [min] SGCI SGTI Isolation and sequence determination of SGCI and SGTI SGCI EVTCEPGTTFKDKCNTCRCGSDGKSAACTLKACPQKSGTI EQECTPGQTKKQDCNTCNCTPTG-VWACTRKGCPPH

  4. %B 60 40 30 20 0 0.12 0,10 0,08 0.06 0.04 0.02 0.00 Absorbance at 220 nm SGCI 0 10 20 30 40 Time [min] natural synthetic Retention difference between natural and synthetic SGCI Retention difference suggests that the native inhibitor may be postranslationally modified

  5. %B 60 40 30 20 0 0.12 0,10 0,08 0.06 0.04 0.02 0.00 Tryptic map of synthetic SGCI #2 #1 #4 Absorbance at 220 nm #3 Tryptic map of natural SGCI 0 10 20 30 40 Time [min] EVTCEPGTTFKDKCNTCRCGSDGKSAACTLKACPQK SGCI • Localisation of disulphide bridges and the postranslational modification site by tryptic map • Two peaks resulted only Tryptic map of natural and synthetic SGCI Sequences of the peaks: #1;3: SAACTLK CNTCR ACPQ #2: EVTCEPGTTF CGSDGK #4: EVTCEPGT?FK CGSDGK • Identification of the moiety • Literature:LMCII:fucosilation • Mol. weight determination of SGCI forms by MS: • Mw [Da] • calculated: 3649,2 • natural : 3795,4 • synthetic : 3649,8 • difference: 145.6 • fucose: 164.2 The resulted structure is:

  6. protease binding loop C P1’ P1 b1 b2 b3 N

  7. Sequence alignment of inhibitors from the grasshopper family SGCI EVTCEP---GTTFKDKCNTCRCGSDGKSAACTLKA-CPQKSGTI EQECTP---GQTKKQDCNTCNCTPTG-VWACTRKG-CPPHBMPD9 AIVCVA---NRMFIKDCNTCWCNEDGTTFYCTRRV-CVPMCFPD3 GKICEP---GSTKKEDCNTCTCTPDGKNYMCTLMM-CGHHBMPD11 PATCVP---GSVYNQGCNVCRCTDEGRHATCTLMR-CPQEMCPD2 DKNCEP---GTIIQRDCNSCNCVP-GIGYACTKRA-CIKMPLD9 GGQCTE---GESWRQDCNMCSCS-TG-LRICSVKG-CPPTPLD3 ETECYGDPDTNRWRIECNWCRCV-NG-KGSCTRKG-CPQVMSPD1 EAECQP---GSEWESNCHSCKCSEAG-IAECLKQAACDKE Binding loop C,G Conserved amino acid residues Amino acids playing essential role in the stabilization of the structure

  8. Solution structure of the inhibitors (determined by NMR spectroscopy) Lys10 Trp25 Phe10 SGCI (PDB ID: 1KGM) SGTI (PDB ID: 1KJO) The residues and their interactions mainly responsible for structure stabilization is different in the two closely related molecules (Phe10 in SGCI and Lys10-Trp25 in SGTI) Gáspári et al., Eur. J. Biochem., (2002), 269: 527-537.

  9. S1 S1’ Inhibitor constants P1 P1’

  10. S1’ S1’ S1’ S1 S1 S1 Leu Lys Lys Leu Leu P1 P1’ P1’ P1 P1 Lys P1’ P1 P1 P1’ P1’ S1’ S1’ S1’ S1 S1 S1 Arg Arg Lys Lys P1 Arg P1’ P1 Lys P1’ S1’ S1’ S1’ S1 S1 S1 Arg Arg Met Met Arg Met P1’ P1 P1 P1’ Schemes of inhibitor docking Bovine chymotrypsin Bovine trypsin Crayfish trypsin SGCI (Leu-Lys) SGTI (Arg-Lys) SGCI (Arg-Met)

  11. Inhibitor constants

  12. Deciphering the structural background of taxon specificity • Disulfide bond stabilities in SGCI and SGTI • Proton-deuterium exchange studies • X-raycrystallography of crayfish trypsin-SGTI complex • Dynamics studies by NMR spectroscopy: • proton-deuterium exchange • 15N-relaxation measurements

  13. Disulfide bond stabilities in SGCI and SGTI Reduction and oxidation of SGTI and SGCI

  14. Proton-deuterium exchange studies :SGTI :SGCI H/D exchange of SGTI and SGCI by FT-IR

  15. Protein dynamics: time scales and experiments by NMR Time scaleexperiment ms ≥ H/D exchange (amide protons) rates depend on structure and slow dynamics ms-ms 15N-relaxation relevant parameter: Rex, the chemical exchange rate, a value of 0 means no significant motion on this time scale ps-ns 15N-relaxation relevant parameter: S2, general order parameter (0 < S2 < 1), 1 means that the corresponding N-H bond vector is fixed in the molecular reference frame, 0 means unrestricted motion

  16. 15N-relaxation experiments to probe protein dynamics • Relaxation parameters of 15N nuclei are dependent on the internal dynamics of the molecule • Parameteres to measure are T1 and T2 relaxation times of 15N nuclei as well as {1H}- 15N heteronuclear NOE • Requires 15N-labeled sample • In order to assign 15N resonances, 3D TOCSY-HSQC and NOES-HSQC spectra are needed

  17. Obtaining 15N-labeled SGCI and SGTI • Labeling can be achieved by biological expression (15NH4Cl as exclusive nitrogen source) • SGCI and SGTI are synthesized on one continous polypeptide chain as a”dimeric” precursor molecule (SGTCI) • After expressing the precursor, the individual inhibitors had to be separated: • 1, digesting with trypsinno cleavage • 2, engineered SGTCI, cleavage with BrCN SGTI + SGCI SGTCI: SGTI -KR- SGCI SGTCIMet: SGTI -M- SGCI

  18. Inhibition of Colorado potato beetle larvae by a locust proteinase inhibitor peptide expressed in potato Northern blot analysis of the Solanum tuberosum cv. Desiré lines transformed with the locust inhibitory peptide construct. C means non-transformed controls, the numbers indicate individual transgenic lines. Arrows show the lines selected for further experiments.

  19. Inhibition of Colorado potato beetle larvae by a locust proteinase inhibitor peptide expressed in potato • Average daily live weights of 10 potato beetle larvae fed on control Desiré (hollow columns) and of 10 with transgenic line LIP9 leaves (grey columns) • Dry weight of leaves and amount of LIP consumed daily by larvae.

  20. Internal dynamics of the inhibitors SGTI SGCI chem. exchange param. significant (>1 s-1) Rex order parameter increasing S2 (01)

  21. Thermal unfolding profiles of trypsins and their complexes with SGTI SGTI(───), crayfish trypsin; (·······), bovine trypsin; (­ ­ ­ ­), bovine trypsin-SGTI complex; (─ ─ ─), crayfish trypsin-SGTI complex

  22. Protein crystal from crayfish trypsin-SGTI complex

  23. Protein crystal from crayfish trypsin-SGTI complex

  24. Crystal structure of the crayfish trypsin - SGTI complex. BPTI SGTI CFT CFT BPTI SGTI Overall conformation of the crayfish trypsin -SGTI complex compared with bovine trypsin -BPTI complex. Conformations of the P3-P2' regions of the inhibitors in the complexes, with the catalytic triad. The conformation of the main chain is nearly identical.

  25. Hydrogen bond shortening during serine protease catalysis Hydrogen bond lengths in the CFT (green) : SGTI (yellow) complex and the elastase (cyan) : Ac-NPI (red) acyl-enzyme complex.

  26. Crystal structures of pacifastin complexes Overall conformation of the crayfish trypsin:SGTI complex Overall conformation of the chymotrypsin:PMP-C complex (PMP-C is an SGCI analouge)

  27. Important interaction surfaces SGTI PMP-C CFT CHY Important interaction surfaces in the CFT-SGPI-1 complex are marked with coloured boxes. The green box (shows the prime region of the inhibitors) indicates the possible molecular recognition site at the C-terminal of the inhibitor: the arthropod trypsin has an extended loop here (Loop37). The blue box shows the area of the canonical binding loop, while the red box (shows the non-prime region of the inhibitors) marks another interaction site in the area of helix173.

  28. Energetic analysis of binding SGTI by crayfish and bovine trypsin P4’-P5’ P3-P3’ P12-P4 B. The molecular surface of crayfish trypsin with the binding regions for P4'-P5', P5-P3' and P12-P6 of SGTI colored magenta, yellow and orange, respectively A. Intermolecular interaction energy values corresponding to each residue of SGTI in the X-ray structure of CFT-SGTI complex and energy-minimized structure of the bovine trypsin-SGTI complex.

  29. Structural change of SGTI upon its binding to crayfish trypsin X-ray NMR Shape adaptation upon binding of arthropod trypsin inhibitor SGTI to the surface of crayfish trypsin.

  30. The scheme of phage display Producing variants In vitro selection on immobilized target protein ≈1010 phage-protein clones Washing Binding Immobilizedtarget protein Gene variant Proteinvariant Elutionof functional clones, growing in E. coli, replication

  31. Selection for efficient folding and binding to bovine trypsin

  32. Selection for efficient folding and binding to crayfish trypsin

  33. Comparative Ki data of SGPI variants on bovine and crayfish trypsins

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