Structural, biochemical and computational studies on PROPPINs, proteins important in autophagy

by Andreea Scacioc
Doctoral thesis
Date of Examination:2014-12-17
Date of issue:2015-12-16
Advisor:Dr. Karin Kühnel
Referee:Dr. Karin Kühnel
Referee:Prof. Dr. Michael Thumm
Persistent Address: http://dx.doi.org/10.53846/goediss-5434

 

 

Files in this item

Name:Andreea_Scacioc_PhD_thesis.pdf
Size:52.3Mb
Format:PDF

The following license files are associated with this item:

Abstract

English

PROPPINs (β-propellers that bind polyphosphoinositides) are conserved eukaryotic proteins that play an important role in autophagy. Yeast contains three PROPPINs: Atg18, Atg21 and Hsv2 (homologous with swollen vacuole phenotype 2), which specifically bind PI3P and PI(3,5)P2. Based on the crystal structure of Kluyveromyces lactis Hsv2, which was determined earlier in our group, I performed docking studies to characterize PI3P and PI(3,5)P2 binding in the two binding sites present in PROPPINs. Based on these docking studies I proposed a model where PROPPINs bind perpendicular to the membrane through their two phosphoinositide binding sites. In addition to the two phosphoinositide binding sites loop 6CD is also required for membrane targeting of PROPPINs. Using coarse-grained and atomistic molecular dynamics simulations I showed that loop 6CD inserts into the lipid bilayer and acts as an anchor for membrane binding of PROPPINs. I also determined the 1.8 Å resolution crystal structure of Pichia angusta Atg18, which represents the first high resolution PROPPIN structure. As Hsv2 it forms a seven bladed β-propeller with a non-velcro like propeller closure topology. In order to experimentally validate my proposed PROPPIN-membrane binding model I designed mutants based on the P. angusta Atg18 crystal structure and set-up a fluorescence based assay to measure their distances to the membrane. Initial measurements confirm this model. Through extensive through isothermal titration calorimetry measurements I quantified PI3P and PI(3,5)P2 binding of P. angusta Atg18, K. lactis Atg21 and S. cerevisiae Hsv2. These PROPPINs bind phosphoinositides with nanomolar and low-micromolar affinities and both Atg18 and Hsv2 bind tighter to PI(3,5)P2. Analysis of single binding site mutants further showed that the affinities of these mutants are 15- to 30-fold lower compared to the wild-type protein which explains that PROPPINs need two phosphinositide binding sites in order to achieve high affinity binding to membranes. Taken together by combining computational studies, X-ray crystallography and other biophysical methods I gained new insights how PROPPINs interact with phosphoinositides on a molecular level.
Keywords: autophagy; Atg18; Atg2; Hsv2; Atg21; PROPPIN; x-ray crystallography
 
Statistik