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abstracts
Our first line of defense against viral pathogens is the innate immune system. Interferon-induced proteins with tetratricopeptide repeats (IFITs) are innate immune effector molecules that are thought to confer antiviral defense through the formation of the IFIT `Interactome', a multiprotein complex made up of IFIT1, IFIT2, IFIT3 and several other host factors1. Through IFIT1, this complex has the ability to distinguish self from non-self nucleic acids such as virus-derived RNA bearing 5-triphosphate or viral mRNA lacking 2-O methylation on the first two nucleotides1,2. We have limited information on the architecture of this complex, its role in innate immunity, and its activity downstream of RNA binding remain unclear. To better understand the mechanisms of Interactome formation, we are investigating the structure of its core, namely the IFIT1-IFIT2-IFIT3 complex. Since it is challenging to crystallize the complex as a whole, likely due to its size and heterogeneity, we are also targeting the structure of individual components and co-crystals of interacting domains. A crystal structure of human IFIT2 is available, and our lab has solved the structure of N-terminal human IFIT1 and, more recently, N-terminal IFIT3. In this study, we aim to characterize the interaction between IFIT1 and IFIT2, and between IFIT3 and IFIT2, through gel-filtration binding assays, in vitro pull-downs and deletion mutations. Preliminary results on the expression and purification of IFIT2-deletion mutants will be presented, as well as purification of IFIT subcomplexes. Understanding the molecular mechanisms behind IFIT-mediated virus elimination will help us unravel the complexities of these interactions and significantly advance our fundamental knowledge of innate immunity, paving the way for designing novel immunotherapeutics, which could potentially complement anti-cancer strategies that rely on oncolytic RNA viruses.
abstracts
The Immunity-Related GTPase Family M protein (IRGM) is involved in regulating cellular autophagy. Cellular knockdown of IRGM was shown to allow RNA viruses to hijack the autophagic immune response. Additionally, recent genetic studies have shown that underexpression of IRGM is associated with the incidence of Crohn's disease and infection by Mycobacterium tuberculosis. IRGM is an interferon-induced GTPase with an evolutionary conserved P-loop. It is an effector of the interferon-gamma pathway, but, unlike its protein family members, is not directly activated by the pathway. Its mechanism of action has been proposed to occur by translocation of IRGM to the mitochondria through recognition of cardiolipin, and affecting mitochondrial fission to induce autophagy. This potential interaction with cardiolipin might indicate the presence of a unique GTPase recognition and activation fold within IRGM. Our goal is to determine the X-ray crystal structure of IRGM in an effort to understand its molecular role in normal and diseased states. Additionally, we seek to test its interaction with and mechanism of recognition to mitochondrial cardiolipin as well as other autophagy-inducing binding partners. Currently, we have managed to express human IRGM in bacterial cells and have purified it to homogeneity using affinity and size-exclusion chromatography. These findings will serve to elucidate the mechanism of action of IRGM. Crucially, we hope to gain an understanding of its contributing role to Crohn's disease and tuberculosis infection at the molecular level, potentially paving the way to structure-based drug design and therapeutic opportunities.
abstracts
Mutations in the gene park2 that codes for a RING-In-Between-RING (RBR) E3 ubiquitin ligase are responsible for an autosomal recessive form of Parkinson's disease (PD). Compared to other ubiquitin ligases, the parkin protein exhibits low basal activity and requires activation both in vitro and in cells. Parkin is a 465-residue E3 ubiquitin ligase promoting mitophagy of damaged mitochondria. Parkin has two RING motifs RING1 and RING2 linked by a cysteine- rich in-between-RING (IBR) motif, a recently identified zinc-coordinating motif termed RING0, and an N-terminal ubiquitin-like domain (Ubl). It is believed that parkin may function as a RING/HECT hybrid, where ubiquitin is first transferred by the E2 enzyme onto parkin active cysteine and then to the substrate. Here, we report the crystal structure of full-length parkin at low resolution. This structure shows parkin in an auto-inhibited state and provides insight into how it is activated. In the structure RING0 occludes the ubiquitin acceptor site Cys431 in RING2 whereas a novel repressor element of parkin (REP) binds RING1 and blocks its E2-binding site. The ubiquitin-like domain (Ubl) binds adjacent to the REP through the hydrophobic surface centered around Ile44 and regulate parkin activity. Mutagenesis and NMR titrations verified interactions observed in the crystal. We also proposed the putative E2 binding site on RING1 and confirmed it by mutagenesis and NMR titrations. Importantly, mutations that disrupt these inhibitory interactions activate parkin both in vitro and in cells. The structure of the E3-ubiquitin ligase provides insights into how pathological mutations affect the protein integrity. Current work is directed towards obtaining high-resolution structure of full-length parkin in complex with E2 and substrates. The results will lead to new therapeutic strategies for treating and ultimately preventing PD.
abstracts
IFIT proteins are interferon-inducible, antiviral effectors that form a multiprotein complex with the ability to recognize markers of viral infection and subsequently restrict viruses. IFIT1, IFIT2 and IFIT3 are at the heart of the complex, interacting with each other and several host factors, forming what is known as the 'IFIT interactome'. Central to their ability to mediate complex formation is the tetratricopeptide repeat (TPR) motif, a general protein-protein interaction module comprising a pair of antiparallel alpha helices. Additionally The TPR motifs of IFIT proteins have the unique ability to recognize RNA. Whereas IFIT1 interacts with virus derived ssRNA, IFIT2 has been shown to interact with dsRNA; IFIT3 is not known to bind RNA. Importantly, structural information is available for the N-terminal domain of IFIT1 and full-length IFIT2, but not for IFIT3. To gain insight into the mechanisms regulating complex formation, we are targeting the structure of human IFIT3 before incorporation into the IFIT complex. To that end, we have determined a low resolution crystal structure of N-terminal IFIT3, which reveals a domain swapped dimer. Notably, IFIT3 dimerization is similar to IFIT2, but distinct from IFIT1, which dimerizes via its C-terminus. Sequence conservation and structural analysis suggest that IFIT2 and IFIT3 evolved a similar mechanism for domain swapping. We propose that IFIT2 and IFIT3 may interact by forming domain swapped heterodimers. Current work is aimed at investigating the mechanisms of domain swapping via mutational analysis, and determining the structure of C-terminal human IFIT3.
