Structure

Trafficking receptors control protein localization through the recognition of specific signal sequences that specify unique cellular locations. Differences in luminal pH are important for the vectorial trafficking of cargo receptors. The KDEL receptor is responsible for maintaining the integrity of the ER by retrieving luminally localized folding chaperones in a pH-dependent mechanism. Structural studies have revealed the end states of KDEL receptor activation and the mechanism of selective cargo binding. However, precisely how the KDEL receptor responds to changes in luminal pH remains unclear...

Nectin and nectin-like (Necl) co-receptor axis, comprised of receptors DNAM-1, TIGIT, CD96, PVRIG, and nectin/Necl ligands, is gaining prominence in immuno-oncology. Within this axis, the inhibitory receptor PVRIG recognizes Nectin-2 with high affinity, but the underlying molecular basis remains unknown. By determining the crystal structure of PVRIG in complex with Nectin-2, we identified a unique CC' loop in PVRIG, which complements the double-lock-and-key binding mode and contributes to its high affinity for Nectin-2...

3-Methylcrotonyl-CoA carboxylase (MCC) catalyzes the two-step, biotin-dependent production of 3-methylglutaconyl-CoA, an essential intermediate in leucine catabolism. Given the critical metabolic role of MCC, deficiencies in this enzyme lead to organic aciduria, while its overexpression is linked to tumor development. MCC is a dodecameric enzyme composed of six copies of each α- and β-subunit. We present the cryo-EM structure of the endogenous MCC holoenzyme from Trypanosoma brucei in a non-filamentous state at 2...

The flagellotropic bacteriophage χ (Chi) infects bacteria via the flagellar filament. Despite years of study, its structural architecture remains partly characterized. Through cryo-EM, we unveil χ's nearly complete structure, encompassing capsid, neck, tail, and tail tip. While the capsid and tail resemble phage YSD1, the neck and tail tip reveal new proteins and their arrangement. The neck shows a unique conformation of the tail tube protein, forming a socket-like structure for attachment to the neck...

Proteins that contain a highly conserved TLDc domain (Tre2/Bub2/Cdc16 LysM domain catalytic) offer protection against oxidative stress and are widely implicated in neurological health and disease. How this family of proteins exerts their function, however, is poorly understood. We have recently found that the yeast TLDc protein, Oxr1p, inhibits the proton pumping vacuolar ATPase (V-ATPase) by inducing disassembly of the pump. While loss of TLDc protein function in mammals shares disease phenotypes with V-ATPase defects, whether TLDc proteins impact human V-ATPase activity directly is unclear...

The translation factor IF5A is highly conserved in Eukarya and Archaea and undergoes a unique post-translational hypusine modification by the deoxyhypusine synthase (DHS) enzyme. DHS transfers the butylamine moiety from spermidine to IF5A using NAD as a cofactor, forming a deoxyhypusine intermediate. IF5A is a key player in protein synthesis, preventing ribosome stalling in proline-rich sequences during translation elongation and facilitating translation elongation and termination. Additionally, human eIF5A participates in various essential cellular processes and contributes to cancer metastasis, with inhibiting hypusination showing anti-proliferative effects...

Human mixed lineage leukemia 4 (MLL4), also known as KMT2D, regulates cell type specific transcriptional programs through enhancer activation. Along with the catalytic methyltransferase domain, MLL4 contains seven less characterized plant homeodomain (PHD) fingers. Here, we report that the sixth PHD finger of MLL4 (MLL4PHD6 ) binds to the hydrophobic motif of ten-eleven translocation 3 (TET3), a dioxygenase that converts methylated cytosine into oxidized derivatives. The solution NMR structure of the TET3-MLL4PHD6 complex and binding assays show that, like histone H4 tail, TET3 occupies the hydrophobic site of MLL4PHD6 , and that this interaction is conserved in the seventh PHD finger of homologous MLL3 (MLL3PHD7 )...

Integrin αIIbβ3 is the key receptor regulating platelet retraction and accumulation and a proven drug-target for antithrombotic therapies. Here we resolve the cryo-EM structures of the full-length αIIbβ3, which covers three distinct states along the activation pathway. Firstly, we obtain the αIIbβ3 structure at 3 Å resolution in the inactive state, revealing the overall topology of the heterodimer with the transmembrane (TM) helices and the ligand-binding domain tucked in a specific angle proximity to the TM region...

PI3Kα is a lipid kinase that phosphorylates PIP2 and generates PIP3. The hyperactive PI3Kα mutation, H1047R, accounts for about 14% of breast cancer, making it a highly attractive target for drug discovery. Here, we report the cryo-EM structures of PI3KαH1047R bound to two different allosteric inhibitors QR-7909 and QR-8557 at a global resolution of 2.7 Å and 3.0 Å, respectively. The structures reveal two distinct binding pockets on the opposite sides of the activation loop...

There are three key components at the core of the mpox virus (MPXV) DNA polymerase holoenzyme: DNA polymerase F8, processivity factors A22, and the Uracil-DNA glycosylase E4. The holoenzyme is recognized as a vital antiviral target because MPXV replicates in the cytoplasm of host cells. Nucleotide analogs such as cidofovir and cytarabine (Ara-C) have shown potential in curbing MPXV replication and they also display promise against other poxviruses. However, the mechanism behind their inhibitory effects remains unclear...

The human CDK-activating kinase (CAK) is a multifunctional protein complex and key regulator of cell growth and division. Because of its critical functions in regulating the cell cycle and transcription initiation, it is a key target for multiple cancer drug discovery programs. However, the structure of the active human CAK, insights into its regulation, and its interactions with cellular substrates and inhibitors remained elusive until recently due to the lack of high-resolution structures of the intact complex...

Endolysosomal exonucleases PLD3 and PLD4 (phospholipases D3 and D4) are associated with autoinflammatory and autoimmune diseases. We report structures of these enzymes, and the molecular basis of their catalysis. The structures reveal an intra-chain dimer topology forming a basic active site at the interface. Like other PLD superfamily members, PLD3 and PLD4 carry HxKxxxxD/E motifs and participate in phosphodiester-bond cleavage. The enzymes digest ssDNA and ssRNA in a 5'-to-3' manner and are blocked by 5'-phosphorylation...

GCN2 is a stress response kinase that phosphorylates the translation initiation factor eIF2α to inhibit general protein synthesis when activated by uncharged tRNA and stalled ribosomes. The presence of a HisRS-like domain in GCN2, normally associated with tRNA aminoacylation, led to the hypothesis that eIF2α kinase activity is regulated by the direct binding of this domain to uncharged tRNA. Here we solved the structure of the HisRS-like domain in the context of full-length GCN2 by cryoEM. Structure and function analysis shows the HisRS-like domain of GCN2 has lost histidine and ATP binding but retains tRNA binding abilities...

The centromere is epigenetically marked by a histone H3 variant-CENP-A. The budding yeast CENP-A called Cse4, consists of an unusually long N-terminus that is known to be involved in kinetochore assembly. Its disordered chaperone, Scm3 is responsible for the centromeric deposition of Cse4 as well as in the maintenance of a segregation-competent kinetochore. In this study, we show that the Cse4 N-terminus is intrinsically disordered and interacts with Scm3 at multiple sites, and the complex does not gain any substantial structure...

Membrane forces shift the equilibria of mechanosensitive channels enabling them to convert mechanical cues into electrical signals. Molecular tools to stabilize and methods to capture their highly dynamic states are lacking. Cyclodextrins can mimic tension through the sequestering of lipids from membranes. Here we probe the conformational ensemble of MscS by EPR spectroscopy, the lipid environment with NMR, and function with electrophysiology under cyclodextrin-induced tension. We show the extent of MscS activation depends on the cyclodextrin-to-lipid ratio, and that lipids are depleted slower when MscS is present...

Entry of Salmonella into host enterocytes relies on its pathogenicity island 1 effector SipA. We found that SipA binds to F-actin in a 1:2 stoichiometry with sub-nanomolar affinity. A cryo-EM reconstruction revealed that SipA's globular core binds at the groove between actin strands, whereas the extended C-terminal arm penetrates deeply into the inter-strand space, stabilizing F-actin from within. The unusually strong binding of SipA is achieved by a combination of fast association via the core and very slow dissociation dictated by the arm...

Designed protein cages and related materials provide unique opportunities for applications in biotechnology and medicine, but their creation remains challenging. Here, we apply computational approaches to design a suite of tetrahedrally symmetric, self-assembling protein cages. For the generation of docked conformations, we emphasize a protein fragment-based approach, while for sequence design of the de novo interface, a comparison of knowledge-based and machine learning protocols highlights the power and increased experimental success achieved using ProteinMPNN...

Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, is a major enzyme of energy metabolism that couples NADH oxidation and ubiquinone reduction with proton translocation. The NADH oxidation site features different enzymatic activities with various nucleotides. While the kinetics of these reactions are well described, only binding of NAD+ and NADH have been structurally characterized. Here, we report the structures of the electron input module of Aquifex aeolicus complex I with bound ADP-ribose and 3-acetylpyridine adenine dinucleotides at resolutions better than 2...

Mollusk hemocyanins, among the largest known proteins, are used as immunostimulants in biomedical and clinical applications. The hemocyanin of the Chilean gastropod Concholepas concholepas (CCH) exhibits unique properties, which makes it safe and effective for human immunotherapy, as observed in animal models of bladder cancer and melanoma, and dendritical cell vaccine trials. Despite its potential, the structure and amino acid sequence of CCH remain unknown. This study reports two sequence fragments of CCH, representing three complete functional units (FUs)...

Protein missense mutations and resulting protein stability changes are important causes for many human genetic diseases. However, the accurate prediction of stability changes due to mutations remains a challenging problem. To address this problem, we have developed an unbiased effective model: PMSPcnn that is based on a convolutional neural network. We have included an anti-symmetry property to build a balanced training dataset, which improves the prediction, in particular for stabilizing mutations. Persistent homology, which is an effective approach for characterizing protein structures, is used to obtain topological features...