Biology subjects

Cook, A. S. I.

Publications and source records attributed to Cook, A. S. I..

3 recordsLinked to original sources

bioRxiv2025

Reconstitution of multistep recruitment of ULK1 to membranes in autophagy

The Human ULK1 autophagy-initiating complex consists of ULK1, FIP200, and the HORMA domain heterodimer ATG13:ATG101. PI3P is essential to recruit ULK1C to membranes for ULK1, but ULK1C subunits do not contain PI3P-binding domains. Here we show that the ATG13:ATG101 dimer forms a complex with the PI3P-binding protein WIPI3, as well as WIPI2. Bound to WIPI2-3, ATG13:ATG101 inserts its Trp-Phe (WF) finger into the membrane. MD simulations show that WIPIs and the WF finger cooperatively stabilizes the complex on membranes. Biochemical reconstitution and cell-based assays show that WIPI3:ATG13 engagement promotes ATG16L1 phosphorylation, autophagy and mitophagy. A kinase domain (KD)-proximal PVP motif in the ULK1 IDR docks onto the ATG13:ATG101 HORMA dimer brings the ULK1 KD close to the membrane. The PVP motif is essential for in vitro ULK1 phosphorylation of ATG16L1 and important for autophagy and mitophagy. These data establish a stepwise pathway for recruitment of the ULK1 KD to the vicinity of the membrane surface.

cell biology↗

bioRxiv2023

Structural pathway for class III PI 3-kinase activation by the myristoylated GTP-binding pseudokinase VPS15

The class III phosphatidylinositol (PI) 3-kinase complexes I and II (PI3KC3-C1 and -C2) are central to the initiation of macroautophagy and endosomal maturation, respectively. Through three-dimensional classification of a large cryo-EM dataset of human PI3KC3-C1 bound to the small GTPase RAB1A, we were able to map the structural pathway of enzyme activation. The inactive conformation is stabilized by an N-myristoyl modification of the pseudokinase (PK) subunit VPS15. The N-myristate is sequestered in the N-lobe of the VPS15 PK domain, which stabilizes a series of interactions whereby VPS15 sequesters and blocks the catalytic and membrane binding units of the VPS34 lipid kinase. In the activated conformation, the N-myristate and the VPS34 lipid kinase domain are liberated to interact with membranes and catalyze PI3P formation. The VPS15 PK domain contains a unique Arg at the gatekeeper position and binds tightly to GTP. GTP binding structurally stabilizes the N-myristate "in" conformation, which promotes the inactive conformation. This pathway provides a general mechanism for PI3KC3 activation in autophagy and endosome biogenesis and a roadmap for their pharmacological upregulation.

biochemistry↗

bioRxiv2023

Structure and activation of the human autophagy-initiating ULK1C:PI3KC3-C1 supercomplex

The unc-51-like kinase protein kinase complex (ULK1C) is the most upstream and central player in the initiation of macroautophagy in mammals. Here, the cryo-EM structure of the human ULK1C core was determined at amino acid residue-level resolution. A moderate resolution structure of the ULK1C core in complex with another autophagy core complex, the class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) was also determined. The two complexes co-assemble through extensive contacts between the FIP200 scaffold subunit of ULK1C and the VPS15, ATG14, and BECN1 subunits of PI3KC3-C1. The FIP200:ATG13:ULK1 core of ULK1C undergoes a rearrangement from 2:1:1 to 2:2:2 stoichiometry in the presence of PI3KC3-C1. This suggests a structural mechanism for the initiation of autophagy through formation of a ULK1C:PI3KC3-C1 supercomplex and dimerization of ULK1 on the FIP200 scaffold.

biochemistry↗