Title: The role of conformational dynamics in regulating the translation of TOP mRNAs by La-related protein 1 (LARP1)
Abstract:
Intrinsically disordered regions (IDRs) are segments of proteins that lack predicted secondary or tertiary structures, allowing for highly dynamic and flexible folding. Dogma suggests that protein structure determines function in a cell, so, it is important to elucidate how these disordered regions become functional, either as disordered entities or by assuming relative order. The mechanisms underlying IDR functionality remain poorly understood due to difficulties in capturing a stable conformation, both computationally and experimentally. The flexibility of IDRs facilitates interactions with various partners to fulfill diverse cellular roles. One IDR-containing protein, La-related protein 1 (LARP1), regulates the translation of terminal oligopyrimidine (TOP) mRNAs, which encode the components of the translational machinery. LARP1 consists of at least two functional domains: the well-characterized DM15 domain, and the less understood La-module, containing both a La-motif and an IDR. The IDR in the La-module binds multiple targets, including TOP mRNAs, poly(A) RNA, Poly(A) Binding Protein (PABP), various kinases, and eukaryotic translation initiation factor 4A (eIF)4A to influence downstream processes. This putative functional diversity may explain how LARP1 stabilizes, promotes, or represses the translation of TOP mRNAs. I hypothesize that the La-module’s IDR undergoes conformational changes that enable it to interact with different targets, and in some cases, adopt more structured forms. Initial biochemical experiments indicate potential structural changes in the La-module upon addition of a binding partner, and that the DM15 may stabilize the La-module—highlighting the importance of this inter-domain interaction. To further investigate, we will employ biochemical, structural, and computational tools.
Title: The intrinsically disordered region of the Listeria monocytogenes secretion chaperone PrsA2 is required for bacterial virulence
Abstract:
Listeria monocytogenes (Lm) is a Gram-positive bacterium that can survive in the environment as a saprophyte yet can cause life-threatening infections once ingested by a susceptible mammalian host. The switch from life in the environment to inside a host requires the transcriptional activator PrfA, a master regulator of bacterial virulence. Within the host, PrfA exists in a high activity state and regulates the expression of secreted virulence factors such as the pore forming toxin, listeriolysin O (LLO), necessary for host cell phagosome escape. The maturation of LLO and other factors are dependent on PrsA2, a secreted peptidyl-prolyl isomerase (PPIase) chaperone. The PrsA2 PPIase and foldase domains have been characterized for ΔprsA2-associated phenotypes, including reduced virulence and motility. However, an uncharacterized region of PrsA2 is the intrinsically disordered C-terminal serine-rich tail (C-tail). Intrinsically disordered regions of chaperones are highly flexible, allowing them to carry out multiple biologically relevant functions such as protein recognition and interaction. Therefore, we hypothesize that the PrsA2 C-tail is critical for client protein interactions. We first generated Lm prsA2 C-tail deletion and serine-to-alanine substitution strains and characterized them for ΔprsA2-dependent phenotypes. We determined that the PrsA2 C-tail is critical for bacterial virulence and required for secreted LLO activity. To further probe the interaction of PrsA2 with LLO, we used isothermal titration calorimetry which suggests that the PrsA2 C-tail deletion mutant interacts with LLO at lower affinity than wild-type PrsA2. Additionally, using a chaperone-assisted folding assay, we demonstrate that the PrsA2 C-tail is critical for folding LLO. Taken together, we show that the PrsA2 C-tail is required for full functional activity of LLO, and we anticipate that it is important for interaction with additional key virulence factors.
Berman/Durrant and Cahoon Lab
Friday, October 18th, 2024
12:00PM
Langley A219B