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Spinal cord injury (SCI) disrupts the structural and functional connectivity between the higher center and the spinal cord, resulting in severe motor, sensory, and autonomic dysfunction with a variety of complications. The pathophysiology of SCI is complicated and multifaceted, and thus individual treatments acting on a specific aspect or process are inadequate to elicit neuronal regeneration and functional recovery after SCI. Combinatory strategies targeting multiple aspects of SCI pathology have achieved greater beneficial effects than individual therapy alone. Although many problems and challenges remain, the encouraging outcomes that have been achieved in preclinical models offer a promising foothold for the development of novel clinical strategies to treat SCI. In this review, we characterize the mechanisms underlying axon regeneration of adult neurons and summarize recent advances in facilitating functional recovery following SCI at both the acute and chronic stages. In addition, we analyze the current status, remaining problems, and realistic challenges towards clinical translation. Finally, we consider the future of SCI treatment and provide insights into how to narrow the translational gap that currently exists between preclinical studies and clinical practice. Going forward, clinical trials should emphasize multidisciplinary conversation and cooperation to identify optimal combinatorial approaches to maximize therapeutic benefit in humans with SCI., (©The Author(s) 2023. Published by Oxford University Press on behalf of Higher Education Press.)

CRISPR-Cas systems are bacterial adaptive immune systems, and phages counteract these systems using many approaches such as producing anti-CRISPR (Acr) proteins. Here, we report the structures of both AcrIF14 and its complex with the crRNA-guided surveillance (Csy) complex. Our study demonstrates that apart from interacting with the Csy complex to block the hybridization of target DNA to the crRNA, AcrIF14 also endows the Csy complex with the ability to interact with non-sequence-specific dsDNA as AcrIF9 does. Further structural studies of the Csy-AcrIF14-dsDNA complex and biochemical studies uncover that the PAM recognition loop of the Cas8f subunit of the Csy complex and electropositive patches within the N-terminal domain of AcrIF14 are essential for the non-sequence-specific dsDNA binding to the Csy-AcrIF14 complex, which is different from the mechanism of AcrIF9. Our findings highlight the prevalence of Acr-induced non-specific DNA binding and shed light on future studies into the mechanisms of such Acr proteins., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Respirasome, as a vital part of the oxidative phosphorylation system, undertakes the task of transferring electrons from the electron donors to oxygen and produces a proton concentration gradient across the inner mitochondrial membrane through the coupled translocation of protons. Copious research has been carried out on this lynchpin of respiration. From the discovery of individual respiratory complexes to the report of the high-resolution structure of mammalian respiratory supercomplex I 1 III 2 IV 1 , scientists have gradually uncovered the mysterious veil of the electron transport chain (ETC). With the discovery of the mammalian respiratory mega complex I 2 III 2 IV 2 , a new perspective emerges in the research field of the ETC. Behind these advances glitters the light of the revolution in both theory and technology. Here, we give a short review about how scientists 'see' the structure and the mechanism of respirasome from the macroscopic scale to the atomic scale during the past decades.

Mechanosensitive (MS) channels are extensively studied membrane protein for maintaining intracellular homeostasis through translocating solutes and ions across the membrane, but its mechanisms of channel gating and ion selectivity are largely unknown. Here, we identified the YnaI channel as the Na + /K + cation-selective MS channel and solved its structure at 3.8 Å by cryo-EM single-particle method. YnaI exhibits low conductance among the family of MS channels in E. coli, and shares a similar overall heptamer structure fold with previously studied MscS channels. By combining structural based mutagenesis, quantum mechanical and electrophysiological characterizations, we revealed that ion selective filter formed by seven hydrophobic methionine (YnaI Met158 ) in the transmembrane pore determined ion selectivity, and both ion selectivity and gating of YnaI channel were affected by accompanying anions in solution. Further quantum simulation and functional validation support that the distinct binding energies with various anions to YnaI Met158 facilitate Na + /K + pass through, which was defined as binding-block mechanism. Our structural and functional studies provided a new perspective for understanding the mechanism of how MS channels select ions driven by mechanical force.

TRPML1 channel is a non-selective group-2 transient receptor potential (TRP) channel with Ca 2+ permeability. Located mainly in late endosome and lysosome of all mammalian cell types, TRPML1 is indispensable in the processes of endocytosis, membrane trafficking, and lysosome biogenesis. Mutations of TRPML1 cause a severe lysosomal storage disorder called mucolipidosis type IV (MLIV). In the present study, we determined the cryo-electron microscopy (cryo-EM) structures of Mus musculus TRPML1 (mTRPML1) in lipid nanodiscs and Amphipols. Two distinct states of mTRPML1 in Amphipols are added to the closed state, on which could represent two different confirmations upon activation and regulation. The polycystin-mucolipin domain (PMD) may sense the luminal/extracellular stimuli and undergo a "move upward" motion during endocytosis, thus triggering the overall conformational change in TRPML1. Based on the structural comparisons, we propose TRPML1 is regulated by pH, Ca 2+ , and phosphoinositides in a combined manner so as to accommodate the dynamic endocytosis process.

Respirasome, a huge molecular machine that carries out cellular respiration, has gained growing attention since its discovery, because respiration is the most indispensable biological process in almost all living creatures. The concept of respirasome has renewed our understanding of the respiratory chain organization, and most recently, the structure of respirasome solved by Yang's group from Tsinghua University (Gu et al. Nature 237(7622):639-643, 2016) firstly presented the detailed interactions within this huge molecular machine, and provided important information for drug design and screening. However, the study of cellular respiration went through a long history. Here, we briefly showed the detoured history of respiratory chain investigation, and then described the amazing structure of respirasome.

Bone sialoprotein-binding protein (Bbp), a MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules) family protein expressed on the surface of Staphylococcus aureus (S. aureus), mediates adherence to fibrinogen α (Fg α), a component in the extracellular matrix of the host cell and is important for infection and pathogenesis. In this study, we solved the crystal structures of apo-Bbp(273-598) and Bbp(273-598)-Fg α(561-575) complex at a resolution of 2.03 Å and 1.45 Å, respectively. Apo-Bbp(273-598) contained the ligand binding region N2 and N3 domains, both of which followed a DE variant IgG fold characterized by an additional D1 strand in N2 domain and D1' and D2' strands in N3 domain. The peptide mapped to the Fg α(561-575) bond to Bbp(273-598) on the open groove between the N2 and N3 domains. Strikingly, the disordered C-terminus in the apo-form reorganized into a highly-ordered loop and a β-strand G'' covering the ligand upon ligand binding. Bbp(Ala298-Gly301) in the N2 domain of the Bbp(273-598)-Fg α(561-575) complex, which is a loop in the apo-form, formed a short α-helix to interact tightly with the peptide. In addition, Bbp(Ser547-Gln561) in the N3 domain moved toward the binding groove to make contact directly with the peptide, while Bbp(Asp338-Gly355) and Bbp(Thr365-Tyr387) in N2 domain shifted their configurations to stabilize the reorganized C-terminus mainly through strong hydrogen bonds. Altogether, our results revealed the molecular basis for Bbp-ligand interaction and advanced our understanding of S. aureus infection process.

DraIII is a type IIP restriction endonucleases (REases) that recognizes and creates a double strand break within the gapped palindromic sequence CAC↑NNN↓GTG of double-stranded DNA (↑ indicates nicking on the bottom strand; ↓ indicates nicking on the top strand). However, wild type DraIII shows significant star activity. In this study, it was found that the prominent star site is CAT↑GTT↓GTG, consisting of a star 5' half (CAT) and a canonical 3' half (GTG). DraIII nicks the 3' canonical half site at a faster rate than the 5' star half site, in contrast to the similar rate with the canonical full site. The crystal structure of the DraIII protein was solved. It indicated, as supported by mutagenesis, that DraIII possesses a ββα-metal HNH active site. The structure revealed extensive intra-molecular interactions between the N-terminal domain and the C-terminal domain containing the HNH active site. Disruptions of these interactions through site-directed mutagenesis drastically increased cleavage fidelity. The understanding of fidelity mechanisms will enable generation of high fidelity REases.

The transition metal cobalt, an essential cofactor for many enzymes in prokaryotes, is taken up by several specific transport systems. The CbiMNQO protein complex belongs to type-1 energy-coupling factor (ECF) transporters and is a widespread group of microbial cobalt transporters. CbiO is the ATPase subunit (A-component) of the cobalt transporting system in the gram-negative thermophilic bacterium Thermoanaerobacter tengcongensis. Here we report the crystal structure of a nucleotide-free CbiO at a resolution of 2.3 Å. CbiO contains an N-terminal canonical nucleotide-binding domain (NBD) and C-terminal helical domain. Structural and biochemical data show that CbiO forms a homodimer mediated by the NBD and the C-terminal domain. Interactions mainly via conserved hydrophobic amino acids between the two C-terminal domains result in formation of a four-helix bundle. Structural comparison with other ECF transporters suggests that non-conserved residues outside the T-component binding groove in the A component likely act as a specificity determinant for T components. Together, our data provide information on understanding of the structural organization and interaction of the CbiMNQO system.

Staphylococcus aureus is the most important Gram-positive colonizer of human skin and nasal passage, causing high morbidity and mortality. SD-repeat containing protein D (SdrD), an MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules) family surface protein, plays an important role in S. aureus adhesion and pathogenesis, while its binding target and molecular mechanism remain largely unknown. Here we solved the crystal structures of SdrD N2-N3 domain and N2-N3-B1 domain. Through structural analysis and comparisons, we characterized the ligand binding site of SdrD, and proposed a featured sequence motif of its potential ligands. In addition, the structures revealed for the first time the interactions between B1 domain and N2-N3 domain among B domain-containing MSCRAMMs. Our results may help in understanding the roles SdrD plays in S. aureus adhesion and shed light on the development of novel antibiotics.

The melanoma antigen (MAGE) family proteins are well known as tumor-specific antigens and comprise more than 60 genes, which share a conserved MAGE homology domain (MHD). Type I MAGEs are highly expressed cancer antigens, and they play an important role in tumorigenesis and cancer cell survival. Recently, several MAGE proteins were identified to interact with RING domain proteins, including a sub-family of E3 ubiquitin ligases. The binding mode between MAGEs and RING proteins was investigated and one important structure of these MAGE-RING complexes was solved: the MAGE-G1-NSE1 complex. Structural and biochemical studies indicated that MAGE proteins could adjust the E3 ubiquitin ligase activity of its cognate RING partner both in vitro and in vivo. However, the underlying mechanism was not fully understood. Here, we review these exciting advances in the studies on MAGE family, suggest potential mechanisms by which MAGEs activate the E3 activity of their binding RING proteins and highlight the anticancer potential of this family proteins.