Biology subjects

Escalante, C. R.

Publications and source records attributed to Escalante, C. R..

4 recordsLinked to original sources

bioRxiv2025

Insights into the AAV packaging mechanism: Cryo-EM Structure of the AAV2 Rep-Capsid Packaging Complex

The Adeno-associated virus (AAV) has become the most used viral vector for gene therapy applications to treat monogenic diseases, with over 250 clinical trials and six FDA-approved biologics. AAV has a single-stranded DNA genome encapsulated in a 60-subunit icosahedral protein shell. Assembly of empty capsids occurs in the nucleus, and in a subsequent step, the genome is packaged by the motor activity of the AAV Rep proteins. The translocation of ssDNA has been suggested to occur through one of the twelve channels at the fivefold symmetry axis. While there is substantial evidence that Rep proteins directly interact with the capsid, the specific molecular determinants, stoichiometry, and translocation mechanism remain unknown. To understand how Rep proteins assemble on the capsid, we examined Rep-capsid complexes using cryo-electron microscopy with single-particle reconstruction. Our results show that Rep proteins can assemble into the capsid fivefold pore as either pentameric or hexameric rings. The different ring complexes dock into the pore similarly, using the post-sensor1 {beta}-hairpin motif (pos1{beta}h2) as a capsid interaction module. This interaction induces the folding of a segment in the capsid HI loop, resulting in an expansion of the pos1{beta}h2 {beta}-sheet. Our structures show that any of the AAV Rep proteins can interact with the capsid, and this mode of interaction may be a conserved mechanism across all parvoviruses. Collectively, our results provide insights into the mechanism of AAV genome encapsidation and could inform strategies to improve recombinant AAV packaging efficiency.

biochemistry↗

bioRxiv2024

Full-length structure and heme binding in the transcriptional regulator HcpR

HcpR is a CRP-family transcriptional regulator found in many Gram-negative anaerobic bacteria. In the perio-pathogen Porphyromonas gingivalis, HcpR is crucial for the response to reactive nitrogen species such as nitric oxide (NO). Binding of NO to the heme group of HcpR leads to transcription of the redox enzyme Hcp. However, the molecular mechanisms of heme binding to HcpR remain unknown. In this study we present the 2.3[A] structure of the P. gingivalis HcpR. Interdomain interactions present in the structure help to form a hydrophobic pocket in the N-terminal sensing domain. A comparison analysis with other CRP-family members reveals that the molecular mechanisms of HcpR-mediated regulation may be distinct from other family members. Using docking studies, we identify a putative heme binding site in the sensing domain. In vitro complementation and mutagenesis studies verify Met68 as an important residue in activation of HcpR. Finally, heme binding studies with purified forms of recombinant HcpR support Met68 and His149 residues as important for proper heme coordination in HcpR.

microbiology↗

bioRxiv2024

Cryo-EM Structure of AAV2 Rep68 bound to integration site AAVS1: Insights into the mechanism of DNA melting

The Rep68 protein from Adeno-Associated Virus (AAV) is a multifunctional SF3 helicase that performs most of the DNA transactions required for the viral life cycle. During AAV DNA replication, Rep68 assembles at the origin and catalyzes the DNA melting and nicking reactions during the hairpin rolling replication process to complete the second-strand synthesis of the AAV genome. Here, we report the Cryo-EM structures of Rep68 bound to double-stranded DNA (dsDNA) containing the sequence of the AAVS1 integration site in different nucleotide-bound states. In the apo state, Rep68 forms a heptameric complex around DNA, with three Origin Binding Domains (OBDs) bound to the Rep Binding Site (RBS) sequence and three other OBDs forming transient dimers with them. The AAA+ domains form an open ring with no interactions between subunits and with DNA. We hypothesize the heptameric quaternary structure is necessary to load onto dsDNA. In the ATP{gamma}S-bound state, a subset of three subunits binds the nucleotide, undergoing a large conformational change, inducing the formation of intersubunit interactions interaction and interaction with three consecutive DNA phosphate groups. Moreover, the induced conformational change positions three phenylalanine residues to come in close contact with the DNA backbone, producing a distortion in the DNA. We propose that the phenylalanine residues can potentially act as a hydrophobic wedge in the DNA melting process.

biochemistry↗

bioRxiv2020

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

The adeno-associated virus (AAV) Rep proteins use a unique AAA+ domain to catalyze DNA replication, transcription regulation, and genome packaging. Also, they mediate site-specific integration during a latent phase. 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 Origin-Binding-Domain (OBD) forms octameric rings while the helicase domain (HD) forms heptamers. Moreover, the binding to ATP{gamma}S 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 Reps structural flexibility required to fulfill its multifunctional role during the AAV life cycle.

biochemistry↗