The Cryo-EM structure of AAV2 Rep68 in complex with ssDNA reveals a malleable AAA+ machine that can switch between oligomeric states

Abstract

The adeno-associated virus (AAV) non-structural Rep proteins catalyze all the DNA transactions required for virus viability including, DNA replication, transcription regulation, genome packaging, and during the latent phase, site-specific integration. Rep proteins contain two multifunctional domains: an Origin Binding Domain (OBD) and a SF3 helicase domain (HD). Studies have shown that Rep proteins have a dynamic oligomeric behavior where the nature of the DNA substrate molecule modulates its oligomeric state. In the presence of ssDNA, Rep68 forms a large double-octameric ring complex. To understand the mechanisms underlying AAV Rep function, we investigated the cryo-EM and X-ray structures of Rep68–ssDNA complexes. Surprisingly, Rep68 generates hybrid ring structures where the OBD forms octameric rings while the HD forms heptamers. Moreover, the binding to ATPγS promotes a large conformational change in the entire AAA+ domain that leads the HD to form both heptamer and hexamers. The HD oligomerization is driven by an interdomain linker region that acts as a latch to ‘catch’ the neighboring HD subunit and is flexible enough to permit the formation of different stoichiometric ring structures. Overall, our studies show the structural basis of AAV Rep's structural flexibility required to fulfill its multifunctional role during the AAV life cycle.

Fluoxetine targets an allosteric site in the enterovirus 2C AAA+ ATPase and stabilizes a ring-shaped hexameric complex

“…Here, we demonstrate that replacement of the N-terminal region of 2C with a hexamerization domain can produce a homogeneous complex amenable to cryo-EM. It remains possible that 2C can adopt multiple stoichiometries, as seen for the adeno-associated virus nonstructural Rep proteins ( 44 ). Nevertheless, the modular nature of our construct means that the cc-hex component can, in principle, be replaced with any other parallel coiled coil ( 45 ).…”

The AAA+ superfamily: a review of the structural and mechanistic principles of these molecular machines

“…It engages DNA through the use of Lys506, coordinating the ssDNA phosphate, the main-chain amide of His507 (both of which are found in the b-hairpin insert) and several other residues (Figure 6) (Enemark and Joshua-Tor 2006). Interestingly, the REP68 complex in adeno-associated virus forms a heptameric AAAþ ring structure (Santosh et al 2020). Under different conditions, depending on the presence of substrate and type of nucleotide, the AAAþ ring (referred to as the SF3 helicase domain) could transition into a hexameric form as well.…”

“…AAAþ domains loaded in tandem accomplish the unraveling of folded proteins and even the disassembly of protein complexes, such as ubiquitintagged proteins (clade 3) (DeLaBarre and Banerjee et al 2016;Bodnar et al 2018;Cooney et al 2019;Twomey et al 2019;Pan et al 2021) and SNARE protein complexes (Zhao et al 2015;White et al 2018), respectfully. AAAþ proteins are encoded within viral genomes to assist with their replication (clade 4) (Shen et al 2005;Enemark and Joshua-Tor 2006;Santosh et al 2020). AAAþ proteins can also assist in the untangling of protein aggregates prior to destruction by a proteasomal complex (Yedidi et al 2017).…”

Recent Advances in Molecular Biology of Human Bocavirus 1 and Its Applications

“…One loop and an alpha helix protrusion are sequence specific and specifically bind to Ori major and minor grooves. For AAVs, the interaction between Rep78/68 OBD and the Ori has been studied in detail by analyzing the structure of the Rep78/68 and Ori complex ( Hickman et al, 2004 ; Santosh et al, 2020 ). Rep78/68 oligomerizes and binds to sequence specific tetra-nucleotides repeats ( Hickman et al, 2004 ; Santosh et al, 2020 ).…”

“…For AAVs, the interaction between Rep78/68 OBD and the Ori has been studied in detail by analyzing the structure of the Rep78/68 and Ori complex ( Hickman et al, 2004 ; Santosh et al, 2020 ). Rep78/68 oligomerizes and binds to sequence specific tetra-nucleotides repeats ( Hickman et al, 2004 ; Santosh et al, 2020 ). In HBoV1, the two DNA binding regions of the HBoV1 OBD are positively charged, and the mutation of which greatly diminished viral genome replication ( Shen et al, 2016 ).…”