Biology subjects

Dryden, K.

Publications and source records attributed to Dryden, K..

5 recordsLinked to original sources

bioRxiv2024

Interplay between Mg2+ and Ca2+ at multiple sites of the ryanodine receptor

RyR1 is an intracellular Ca2+ channel important in excitable cells. Ca2+ activates it at low concentrations and inhibits it at high concentrations. Mg2+ is the main physiological RyR1 inhibitor, an effect that is overridden upon activation. Despite the significance of Mg2+-mediated inhibition, the molecular-level mechanisms remain unclear. We determined two cryo-EM structures of RyR1 with Mg2+ up to 2.8 [A] resolution, identifying multiple Mg2+ binding sites. Mg2+ inhibits at the known Ca2+ activating site and we propose that the EF hand domain is an inhibitory divalent cation sensor. Both divalent cations bind to ATP within a crevice, contributing to the precise transmission of allosteric changes within the enormous channel protein. Notably, Mg2+ inhibits RyR1 by interacting with the gating helices as validated by molecular dynamics. This structural insight enhances our understanding of how Mg2+ inhibition is overcome during excitation.

biophysics↗

bioRxiv2023

Structural mechanism of HP1-dependent transcriptional repression and chromatin compaction

Heterochromatin protein 1 (HP1) plays a central role in establishing and maintaining constitutive heterochromatin. However, the mechanisms underlying HP1-nucleosome interactions and their contributions to heterochromatin functions remain elusive. In this study, we employed a multidisciplinary approach to unravel the interactions between human HP1 and nucleosomes. We have elucidated the cryo-EM structure of an HP1 dimer bound to an H2A.Z nucleosome, revealing that the HP1 dimer interfaces with nucleosomes at two distinct sites. The primary binding site is located at the N-terminus of histone H3, specifically at the trimethylated K9 (K9me3) region, while a novel secondary binding site is situated near histone H2B, close to nucleosome superhelical location 4 (SHL4). Our biochemical data further demonstrates that HP1 binding influences the dynamics of DNA on the nucleosome. It promotes DNA unwrapping near the nucleosome entry and exit sites while concurrently restricting DNA accessibility in the vicinity of SHL4. This study offers a model that explains how HP1 functions in heterochromatin maintenance and gene silencing, particularly in the context of H3K9me-dependent mechanisms. Additionally, it sheds light on the H3K9me-independent role of HP1 in responding to DNA damage.

biochemistry↗

bioRxiv2021

Minicells from Highly Genome Reduced Escherichia coli: Cytoplasmic and Surface Expression of Recombinant Proteins and Incorporation in the Minicells

Minicells, small cells lacking a chromosome, produced by bacteria with mutated min genes, which control cell division septum placement, have many potential uses. Minicells have contributed to basic bacterial physiology studies and can enable new biotechnological applications, including drug delivery and vaccines. Genome-reduced (GR) bacteria are another informative area of investigation. Investigators identified that with even almost 30% of the E. coli genome deleted, the bacteria still live. In biotechnology and synthetic biology, GR bacteria offer certain advantages. With GR bacteria, more recombinant genes can be placed into GR chromosomes and fewer cell resources are devoted to purposes apart from biotechnological goals. Here, we show that these two technologies can be combined: min mutants can be made in GR E. coli. The minCminD mutant GR E. coli produce minicells that concentrate engineered recombinant proteins within these spherical delivery systems. We expressed recombinant GFP protein in the cytoplasm of GR bacteria and showed that it is concentrated within the minicells. We also expressed proteins on the surfaces of minicells made from GR bacteria using a recombinant Gram-negative AIDA-I autotransporter expression cassette. As some autotransporters, like AIDA-I, are concentrated at the bacterial poles, where minicells bud, and because the surface-to-volume ratio of the small minicells is higher than bacteria, recombinant proteins expressed on surfaces of the GR bacteria are concentrated on the minicells. Minicells made from GR bacteria can enable useful biotechnological innovations, such as drug delivery vehicles and vaccine immunogens.

synthetic biology↗

bioRxiv2020

Structure and assembly of CAV1 8S complexes revealed by single particle electron microscopy

Highly stable oligomeric complexes of the monotopic membrane protein caveolin serve as fundamental building blocks of caveolae. Current evidence suggests these complexes are disc shaped, but the details of their structural organization and how they assemble are poorly understood. Here, we address these questions using single particle electron microscopy of negatively stained recombinant 8S complexes of human Caveolin-1. We show that 8S complexes are toroidal structures ~15 nm in diameter that consist of an outer ring, an inner ring, and central protruding stalk. Moreover, we map the position of the N- and C-termini and determine their role in complex assembly, and visualize the 8S complexes in heterologous caveolae. Our findings provide critical insights into the structural features of 8S complexes and allow us to propose a new model for how these highly stable membrane-embedded complexes are generated.

cell biology↗

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↗