PhD Scientific Days 2018

Budapest, April 19–20, 2018

Role and structure of scaffolds in canonical serine protease inhibitor variants: an NMR spectroscopic view

Sebák, Fanni

Fanni Sebák1, Eszter Boros2, Gábor Pál2, Andrea Bodor1
1 Laboratory of Structural Chemistry and Biology, Institute of Chemistry, Eötvös Loránd University, Budapest
2 Department of Biochemistry, Eötvös Loránd University, Budapest

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Text of the abstract

Introduction: Our research is focused on canonical serine protease inhibitors. Structurally each of these proteins contains a binding loop with a common main chain conformation, which is stabilized by the scaffold – that differs for each inhibitor family apart. According to the Laskowski-modell, same binding loop is ideal for all inhibitors. Our studies are intended to prove that this affirmation does not always hold. For this purpose the chymotrypsine binding loop in P4-P4’ positions were evolved by directed evolution, phage display in case of two inhibitors possessing different scaffolds: SPINK1 and SGPI-2.

Aims: Our goal is to understand the structural changes on atomic level caused by the loop exchange on the different scaffolds for SPINK1 and SGPI-2 systems. In order to achieve this we used solution state NMR spectroscopy.

Method: NMR spectra were recorded on a Bruker Avance III 700 spectrometer equipped with a 5mm TCI Prodigy probehead. Peak assignments and sequential connectivities for unlabeled SGPI-2 variants were determined from homonuclear 2D (TOCSY, NOESY) measurements. For 15N and 13C/15N labeled SPINK1 variants 2D 13C-HSQC, 15N-HSQC, 3D HSQC-TOCSY, HSQC-NOESY, and BEST-type 3D HNCO, HNCA, HNCACB spectra were recorded.

Results: We found that loop exchange causes structural changes for SGPI-2 variants: while the wild-type and the chymotrypsine inhibition variant have similar structures with three β-strands, in the loop swapped variant the C-terminal β-strand becomes unstructured.
For the SPINK1 variants the scaffold structure is similar, containing one α-helix, three short β-strands and one disordered binding loop. However, for this case the loop exchange does not cause major structural changes in the scaffold.

Conclusion: we tested different ideal binding loops in the two inhibitors, and proved that the loop swap the loss of inhibition in both proteins is due to a structural and hydrophobic change that affects either the loop region solely or the scaffold as well.

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Doctoral School: Pharmaceutical Sciences
Program: Modern Trends in Pharmaceutical Scientific Research
Supervisor: Andrea Bodor
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