title: Structural studies of Vps4 protein complexes
Creators: name: Ahmed, Ishtiaq
Publisher: name: Griffith University
Year: 2022
Subjects: subject: Vps4p
subject: Vps2p
subject: Transmission Electron Microscopy (TEM)
subject: proteins γ- glutamyl kinase (Pro1p)
subject: serine/threonine-specific protein kinase (Sak1p)
subject: adenosine triphosphate (ATP)

description: ESCRT (Endosomal sorting complex required for transport) machinery drives different cellular processes such as endosomal sorting, organelle biogenesis, vesicular trafficking, maintenance of plasma membrane integrity, membrane fission during cytokinesis and enveloped virus budding. The normal cycle of assembly and disassembly of some ESCRT complexes at the membrane requires the AAA-ATPase vacuolar protein sorting 4 (Vps4). A number of ESCRT proteins are hijacked by clinically significant enveloped viruses including Ebola, and Human Immunodeficiency Virus (HIV) to enable enveloped virus budding and Vps4p provides energy for the disassembly and recycling of these ESCRT proteins. Vps4, a member of the AAA-family (ATPase Associated with a variety of cellular Activities) of proteins is present in all eukaryotes where it mediates endosomal membrane trafficking. Comprehensive evaluation of genomic databases validated the existence of three isoforms in animals, Vps4 -a, -b, and -c. However, yeast has only one Vps4 representative, while two isoforms Vps4a and Vps4b exist in mammals. Of particular interest to this study was the protein Vps4 structure and its binding properties. Vps4 protein possesses an MIT (Microtubule-Interacting and Trafficking) domain at the N-terminus, separated by a long linker from a central AAA-domain, which includes the ATPase catalytic site, and then a small domain comprising beta-sheet (β-domain). At the C-terminus of Vps4 is an α-helix (C-terminal helix). The various domains of Vps4 protein play vital roles by mediating the interaction of Vps4 with a number of key partner proteins. Vps4p has been proposed to use the energy of ATP hydrolysis to break protein-protein interactions on the surface of endosomes. The N-terminal coiled-coil domain of Vps4p appeared to be responsible for binding substrate endosomal coiled-coil domain proteins while ATP hydrolysis by the AAA domain supplies energy to disrupt coiled-coil interactions. The present project yielded single particle analysis of the Vps4p-Vps2p co-complex as the foundation of future structural biology studies to obtain greater resolution which is critical to the future design of low molecular weight compounds that will fit into this interaction interface and perturb Vps4p-Vps2p interaction. Thus, the drugs that perturb Vps4p-Vps2p interaction would be anticipated to interfere with Vps4 function and to have a broad-spectrum antiviral activity. Saccharomyces cerevisiae was used as an alternative to the high usage of animals and animal cell lines in antiviral drug development. This study was aimed to identify the structure of the Vps4p-Vps2p interacting complex using Transmission Electron Microscopy (TEM). In this study, we also investigated the functional role of the novel Vps4p-interacting proteins γ-glutamyl kinase (Pro1p) and a serine/threonine-specific protein kinase (Sak1p). Both Pro1p and Sak1p interacted in the yeast two-hybrid system with the N-terminal coiled-coil domain of Vps4p and form complexes with Vps4p in vitro. The interaction of Pro1p with Vps4p is direct and is broken upon hydrolysis of adenosine triphosphate (ATP). In contrast the interaction of Sak1p with Vps4p appears to require other factors. We identified two highly conserved sequence motifs within the N-terminal domain of Vps4p which are essential for interaction of Vps4p with both Pro1p and Sak1p. The current technical advancements in microscopes are driving visualisation of biological samples at the micro- and nano-scales. The single particle analysis of macromolecular complexes in combination with negatively-stained biological samples were elucidated by the use of the TEM. The negatively-stained samples typically result in lower resolution images and that limited visualisation of 3D structural details. We believe that an understanding of the structure of Vps4 interacting with its binding partners will lead to clear targets for future antiviral treatments.