MFN1 structures reveal nucleotide-triggered dimerization critical for mitochondrial fusion.

Mitochondria are double-membraned organelles with variable shapes influenced by metabolic conditions, developmental stage, and environmental stimuli. Their dynamic morphology is a result of regulated and balanced fusion and fission processes. Fusion is crucial for the health and physiological functions of mitochondria, including complementation of damaged mitochondrial DNAs and the maintenance of membrane potential. Mitofusins are dynamin-related GTPases that are essential for mitochondrial fusion. They are embedded in the mitochondrial outer membrane and thought to fuse adjacent mitochondria via combined oligomerization and GTP hydrolysis. However, the molecular mechanisms of this process remain unknown. Here we present crystal structures of engineered human MFN1 containing the GTPase domain and a helical domain during different stages of GTP hydrolysis. The helical domain is composed of elements from widely dispersed sequence regions of MFN1 and resembles the 'neck' of the bacterial dynamin-like protein. The structures reveal unique features of its catalytic machinery and explain how GTP binding induces conformational changes to promote GTPase domain dimerization in the transition state. Disruption of GTPase domain dimerization abolishes the fusogenic activity of MFN1. Moreover, a conserved aspartate residue trigger was found to affect mitochondrial elongation in MFN1, probably through a GTP-loading-dependent domain rearrangement. Thus, we propose a mechanistic model for MFN1-mediated mitochondrial tethering, and our results shed light on the molecular basis of mitochondrial fusion and mitofusin-related human neuromuscular disorders.

Structures of mammalian GLD-2 proteins reveal molecular basis of their functional diversity in mRNA and microRNA processing.

The stability and processing of cellular RNA transcripts are efficiently controlled via non-templated addition of single or multiple nucleotides, which is catalyzed by various nucleotidyltransferases including poly(A) polymerases (PAPs). Germline development defective 2 (GLD-2) is among the first reported cytoplasmic non-canonical PAPs that promotes the translation of germline-specific mRNAs by extending their short poly(A) tails in metazoan, such as Caenorhabditis elegans and Xenopus. On the other hand, the function of mammalian GLD-2 seems more diverse, which includes monoadenylation of certain microRNAs. To understand the structural basis that underlies the difference between mammalian and non-mammalian GLD-2 proteins, we determine crystal structures of two rodent GLD-2s. Different from C. elegans GLD-2, mammalian GLD-2 is an intrinsically robust PAP with an extensively positively charged surface. Rodent and C. elegans GLD-2s have a topological difference in the ß-sheet region of the central domain. Whereas C. elegans GLD-2 prefers adenosine-rich RNA substrates, mammalian GLD-2 can work on RNA oligos with various sequences. Coincident with its activity on microRNAs, mammalian GLD-2 structurally resembles the mRNA and miRNA processor terminal uridylyltransferase 7 (TUT7). Our study reveals how GLD-2 structurally evolves to a more versatile nucleotidyltransferase, and provides important clues in understanding its biological function in mammals.

Aurora kinase A-mediated phosphorylation triggers structural alteration of Rab1A to enhance ER complexity during mitosis.

Morphological rearrangement of the endoplasmic reticulum (ER) is critical for metazoan mitosis. Yet, how the ER is remodeled by the mitotic signaling remains unclear. Here, we report that mitotic Aurora kinase A (AURKA) employs a small GTPase, Rab1A, to direct ER remodeling. During mitosis, AURKA phosphorylates Rab1A at Thr75. Structural analysis demonstrates that Thr75 phosphorylation renders Rab1A in a constantly active state by preventing interaction with GDP-dissociation inhibitor (GDI). Activated Rab1A is retained on the ER and induces the oligomerization of ER-shaping protein RTNs and REEPs, eventually triggering an increase of ER complexity. In various models, from Caenorhabditis elegans and Drosophila to mammals, inhibition of Rab1AThr75 phosphorylation by genetic modifications disrupts ER remodeling. Thus, our study reveals an evolutionarily conserved mechanism explaining how mitotic kinase controls ER remodeling and uncovers a critical function of Rab GTPases in metaphase.

Mitochondria-ER contact mediated by MFN2-SERCA2 interaction supports CD8+ T cell metabolic fitness and function in tumors.

Metabolic fitness of T cells is essential for their vitality, which is largely dependent on the behavior of the mitochondria. The nature of mitochondrial behavior in tumor-infiltrating T cells remains poorly understood. In this study, we show that mitofusin-2 (MFN2) expression is positively correlated with the prognosis of multiple cancers. Genetic ablation of Mfn2 in CD8+ T cells dampens mitochondrial metabolism and function and promotes tumor progression. In tumor-infiltrating CD8+ T cells, MFN2 enhances mitochondria-endoplasmic reticulum (ER) contact by interacting with ER-embedded Ca2+-ATPase SERCA2, facilitating the mitochondrial Ca2+ influx required for efficient mitochondrial metabolism. MFN2 stimulates the ER Ca2+ retrieval activity of SERCA2, thereby preventing excessive mitochondrial Ca2+ accumulation and apoptosis. Elevating mitochondria-ER contact by increasing MFN2 in CD8+ T cells improves the efficacy of cancer immunotherapy. Thus, we reveal a tethering-and-buffering mechanism of organelle cross-talk that regulates the metabolic fitness of tumor-infiltrating CD8+ T cells and highlights the therapeutic potential of enhancing MFN2 expression to optimize T cell function.

Structure of Epstein-Barr virus tegument protein complex BBRF2-BSRF1 reveals its potential role in viral envelopment.

Epstein-Barr virus (EBV) is a γ-herpesvirus associated with the occurrence of several human malignancies. BBRF2 and BSRF1 are two EBV tegument proteins that have been suggested to form a hetero-complex and mediate viral envelopment, but the molecular basis of their interaction and the functional mechanism of this complex remains unknown. Here, we present crystal structures of BBRF2 alone and in complex with BSRF1. BBRF2 has a compact globular architecture featuring a central ß-sheet that is surrounded by 10 helices, it represents a novel fold distinct from other known protein structures. The central portion of BSRF1 folds into two tightly associated antiparallel α-helices, forming a composite four-helix bundle with two α-helices from BBRF2 via a massive hydrophobic network. In vitro, a BSRF1-derived peptide binds to BBRF2 and reduces the number of viral genome copies in EBV-positive cells. Exogenous BBRF2 and BSRF1 co-localize at the Golgi apparatus. Furthermore, BBRF2 binds capsid and capsid-associated proteins, whereas BSRF1 associates with glycoproteins. These findings indicate that the BBRF2-BSRF1 complex tethers EBV nucleocapsids to the glycoprotein-enriched Golgi membrane, facilitating secondary envelopment.

Structural insights of human mitofusin-2 into mitochondrial fusion and CMT2A onset.

Mitofusin-2 (MFN2) is a dynamin-like GTPase that plays a central role in regulating mitochondrial fusion and cell metabolism. Mutations in MFN2 cause the neurodegenerative disease Charcot-Marie-Tooth type 2A (CMT2A). The molecular basis underlying the physiological and pathological relevance of MFN2 is unclear. Here, we present crystal structures of truncated human MFN2 in different nucleotide-loading states. Unlike other dynamin superfamily members including MFN1, MFN2 forms sustained dimers even after GTP hydrolysis via the GTPase domain (G) interface, which accounts for its high membrane-tethering efficiency. The biochemical discrepancy between human MFN2 and MFN1 largely derives from a primate-only single amino acid variance. MFN2 and MFN1 can form heterodimers via the G interface in a nucleotide-dependent manner. CMT2A-related mutations, mapping to different functional zones of MFN2, lead to changes in GTP hydrolysis and homo/hetero-association ability. Our study provides fundamental insight into how mitofusins mediate mitochondrial fusion and the ways their disruptions cause disease.