The receptor binding domain of SARS-CoV-2 Omicron subvariants targets Siglec-9 to decrease its immunogenicity by preventing macrophage phagocytosis.

The development of a vaccine specific to severe acute respiratory syndrome coronavirus 2 Omicron has been hampered due to its low immunogenicity. Here, using reverse mutagenesis, we found that a phenylalanine-to-serine mutation at position 375 (F375S) in the spike protein of Omicron to revert it to the sequence found in Delta and other ancestral strains significantly enhanced the immunogenicity of Omicron vaccines. Sequence FAPFFAF at position 371-377 in Omicron spike had a potent inhibitory effect on macrophage uptake of receptor-binding domain (RBD) nanoparticles or spike-pseudovirus particles containing this sequence. Omicron RBD enhanced binding to Siglec-9 on macrophages to impair phagocytosis and antigen presentation and promote immune evasion, which could be abrogated by the F375S mutation. A bivalent F375S Omicron RBD and Delta-RBD nanoparticle vaccine elicited potent and broad nAbs in mice, rabbits and rhesus macaques. Our research suggested that manipulation of the Siglec-9 pathway could be a promising approach to enhance vaccine response.

Allosteric role of a structural NADP+ molecule in glucose-6-phosphate dehydrogenase activity.

Human glucose-6-phosphate dehydrogenase (G6PD) is the main cellular source of NADPH, and thus plays a key role in maintaining reduced glutathione to protect cells from oxidative stress disorders such as hemolytic anemia. G6PD is a multimeric enzyme that uses the cofactors ß-D-glucose 6-phosphate (G6P) and "catalytic" NADP+ (NADP+c), as well as a "structural" NADP+ (NADP+s) located ∼25 Å from the active site, to generate NADPH. While X-ray crystallographic and biochemical studies have revealed a role for NADP+s in maintaining the catalytic activity by stabilizing the multimeric G6PD conformation, other potential roles for NADP+s have not been evaluated. Here, we determined the high resolution cryo-electron microscopy structures of human wild-type G6PD in the absence of bound ligands and a catalytic G6PD-D200N mutant bound to NADP+c and NADP+s in the absence or presence of G6P. A comparison of these structures, together with previously reported structures, reveals that the unliganded human G6PD forms a mixture of dimers and tetramers with similar overall folds, and binding of NADP+s induces a structural ordering of a C-terminal extension region and allosterically regulates G6P binding and catalysis. These studies have implications for understanding G6PD deficiencies and for therapy of G6PD-mediated disorders.

Structure of spinach photosystem II-LHCII supercomplex at 3.2 Å resolution.

During photosynthesis, the plant photosystem II core complex receives excitation energy from the peripheral light-harvesting complex II (LHCII). The pathways along which excitation energy is transferred between them, and their assembly mechanisms, remain to be deciphered through high-resolution structural studies. Here we report the structure of a 1.1-megadalton spinach photosystem II-LHCII supercomplex solved at 3.2 Å resolution through single-particle cryo-electron microscopy. The structure reveals a homodimeric supramolecular system in which each monomer contains 25 protein subunits, 105 chlorophylls, 28 carotenoids and other cofactors. Three extrinsic subunits (PsbO, PsbP and PsbQ), which are essential for optimal oxygen-evolving activity of photosystem II, form a triangular crown that shields the Mn4CaO5-binding domains of CP43 and D1. One major trimeric and two minor monomeric LHCIIs associate with each core-complex monomer, and the antenna-core interactions are reinforced by three small intrinsic subunits (PsbW, PsbH and PsbZ). By analysing the closely connected interfacial chlorophylls, we have obtained detailed insights into the energy-transfer pathways between the antenna and core complexes.

ATP-citrate lyase multimerization is required for coenzyme-A substrate binding and catalysis.

ATP-citrate lyase (ACLY) is a major source of nucleocytosolic acetyl-CoA, a fundamental building block of carbon metabolism in eukaryotes. ACLY is aberrantly regulated in many cancers, cardiovascular disease, and metabolic disorders. However, the molecular mechanisms determining ACLY activity and function are unclear. To this end, we investigated the role of the uncharacterized ACLY C-terminal citrate synthase homology domain in the mechanism of acetyl-CoA formation. Using recombinant, purified ACLY and a suite of biochemical and biophysical approaches, including analytical ultracentrifugation, dynamic light scattering, and thermal stability assays, we demonstrated that the C terminus maintains ACLY tetramerization, a conserved and essential quaternary structure in vitro and likely also in vivo Furthermore, we show that the C terminus, only in the context of the full-length enzyme, is necessary for full ACLY binding to CoA. Together, we demonstrate that ACLY forms a homotetramer through the C terminus to facilitate CoA binding and acetyl-CoA production. Our findings highlight a novel and unique role of the C-terminal citrate synthase homology domain in ACLY function and catalysis, adding to the understanding of the molecular basis for acetyl-CoA synthesis by ACLY. This newly discovered means of ACLY regulation has implications for the development of novel ACLY modulators to target acetyl-CoA-dependent cellular processes for potential therapeutic use.

Structural Mechanism Underlying the Specific Recognition between the Arabidopsis State-Transition Phosphatase TAP38/PPH1 and Phosphorylated Light-Harvesting Complex Protein Lhcb1.

During state transitions, plants regulate energy distribution between photosystems I and II through reversible phosphorylation and lateral migration of the major light-harvesting complex LHCII. Dephosphorylation of LHCII and the transition from state 2 to state 1 requires a thylakoid membrane-associated phosphatase named TAP38 or PPH1. TAP38/PPH1 specifically targets LHCII but not the core subunits of photosystem II, whereas the underlying molecular mechanism of their mutual recognition is currently unclear. Here, we present the structures of Arabidopsis thaliana TAP38/PPH1 in the substrate-free and substrate-bound states. The protein contains a type 2C serine/threonine protein phosphatase (PP2C) core domain, a Mn(2+) (or Mg(2+)) binuclear center and two additional motifs contributing to substrate recognition. A 15-mer phosphorylated N-terminal peptide of Lhcb1 binds to TAP38/PPH1 on two surface clefts enclosed by the additional motifs. The first segment of the phosphopeptide is clamped by a pair of tooth-like arginine residues at Cleft 1 site. The binding adopts the lock-and-key mechanism with slight rearrangement of the substrate binding residues on TAP38/PPH1. Meanwhile, a more evident substrate-induced fitting occurs on Cleft 2 harboring the extended part of the phosphopeptide. The results unravel the bases for the specific recognition between TAP38/PPH1 and phosphorylated Lhcb1, a crucial step in state transitions.

Molecular Basis for RNA Cytidine Acetylation by NAT10.

Human NAT10 acetylates the N4 position of cytidine in RNA, predominantly on rRNA and tRNA, to facilitate ribosome biogenesis and protein translation. NAT10 has been proposed as a therapeutic target in cancers as well as aging-associated pathologies such as Hutchinson-Gilford Progeria Syndrome (HGPS). The ∼120 kDa NAT10 protein uses its acetyl-CoA-dependent acetyltransferase, ATP-dependent helicase, and RNA binding domains in concert to mediate RNA-specific N4-cytidine acetylation. While the biochemical activity of NAT10 is well known, the molecular basis for catalysis of eukaryotic RNA acetylation remains relatively undefined. To provide molecular insights into the RNA-specific acetylation by NAT10, we determined the single particle cryo-EM structures of Chaetomium thermophilum NAT10 ( Ct NAT10) bound to a bisubstrate cytidine-CoA probe with and without ADP. The structures reveal that NAT10 forms a symmetrical heart-shaped dimer with conserved functional domains surrounding the acetyltransferase active sites harboring the cytidine-CoA probe. Structure-based mutagenesis with analysis of mutants in vitro supports the catalytic role of two conserved active site residues (His548 and Tyr549 in Ct NAT10), and two basic patches, both proximal and distal to the active site for RNA-specific acetylation. Yeast complementation analyses and senescence assays in human cells also implicates NAT10 catalytic activity in yeast thermoadaptation and cellular senescence. Comparison of the NAT10 structure to protein lysine and N-terminal acetyltransferase enzymes reveals an unusually open active site suggesting that these enzymes have been evolutionarily tailored for RNA recognition and cytidine-specific acetylation.

The histamine receptor H1 acts as an alternative receptor for SARS-CoV-2.

Numerous host factors, in addition to human angiotensin-converting enzyme 2 (hACE2), have been identified as coreceptors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), demonstrating broad viral tropism and diversified druggable potential. We and others have found that antihistamine drugs, particularly histamine receptor H1 (HRH1) antagonists, potently inhibit SARS-CoV-2 infection. In this study, we provided compelling evidence that HRH1 acts as an alternative receptor for SARS-CoV-2 by directly binding to the viral spike protein. HRH1 also synergistically enhanced hACE2-dependent viral entry by interacting with hACE2. Antihistamine drugs effectively prevent viral infection by competitively binding to HRH1, thereby disrupting the interaction between the spike protein and its receptor. Multiple inhibition assays revealed that antihistamine drugs broadly inhibited the infection of various SARS-CoV-2 mutants with an average IC50 of 2.4 µM. The prophylactic function of these drugs was further confirmed by authentic SARS-CoV-2 infection assays and humanized mouse challenge experiments, demonstrating the therapeutic potential of antihistamine drugs for combating coronavirus disease 19.IMPORTANCEIn addition to human angiotensin-converting enzyme 2, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can utilize alternative cofactors to facilitate viral entry. In this study, we discovered that histamine receptor H1 (HRH1) not only functions as an independent receptor for SARS-CoV-2 but also synergistically enhances ACE2-dependent viral entry by directly interacting with ACE2. Further studies have demonstrated that HRH1 facilitates the entry of SARS-CoV-2 by directly binding to the N-terminal domain of the spike protein. Conversely, antihistamine drugs, primarily HRH1 antagonists, can competitively bind to HRH1 and thereby prevent viral entry. These findings revealed that the administration of repurposable antihistamine drugs could be a therapeutic intervention to combat coronavirus disease 19.

Allosteric role of the citrate synthase homology domain of ATP citrate lyase.

ATP citrate lyase (ACLY) is the predominant nucleocytosolic source of acetyl-CoA and is aberrantly regulated in many diseases making it an attractive therapeutic target. Structural studies of ACLY reveal a central homotetrameric core citrate synthase homology (CSH) module flanked by acyl-CoA synthetase homology (ASH) domains, with ATP and citrate binding the ASH domain and CoA binding the ASH-CSH interface to produce acetyl-CoA and oxaloacetate products. The specific catalytic role of the CSH module and an essential D1026A residue contained within it has been a matter of debate. Here, we report biochemical and structural analysis of an ACLY-D1026A mutant demonstrating that this mutant traps a (3S)-citryl-CoA intermediate in the ASH domain in a configuration that is incompatible with the formation of acetyl-CoA, is able to convert acetyl-CoA and OAA to (3S)-citryl-CoA in the ASH domain, and can load CoA and unload acetyl-CoA in the CSH module. Together, this data support an allosteric role for the CSH module in ACLY catalysis.

Molecular mechanism of N-terminal acetylation by the ternary NatC complex.

Protein N-terminal acetylation is predominantly a ribosome-associated modification, with NatA-E serving as the major enzymes. NatC is the most unusual of these enzymes, containing one Naa30 catalytic subunit and two auxiliary subunits, Naa35 and Naa38; and substrate selectivity profile that overlaps with NatE. Here, we report the cryoelectron microscopy structure of S. pombe NatC with a NatE/C-type bisubstrate analog and inositol hexaphosphate (IP6), and associated biochemistry studies. We find that the presence of three subunits is a prerequisite for normal NatC acetylation activity in yeast and that IP6 binds tightly to NatC to stabilize the complex. We also describe the molecular basis for IP6-mediated NatC complex stabilization and the overlapping yet distinct substrate profiles of NatC and NatE.

Molecular basis for N-terminal acetylation by human NatE and its modulation by HYPK.

The human N-terminal acetyltransferase E (NatE) contains NAA10 and NAA50 catalytic, and NAA15 auxiliary subunits and associates with HYPK, a protein with intrinsic NAA10 inhibitory activity. NatE co-translationally acetylates the N-terminus of half the proteome to mediate diverse biological processes, including protein half-life, localization, and interaction. The molecular basis for how NatE and HYPK cooperate is unknown. Here, we report the cryo-EM structures of human NatE and NatE/HYPK complexes and associated biochemistry. We reveal that NAA50 and HYPK exhibit negative cooperative binding to NAA15 in vitro and in human cells by inducing NAA15 shifts in opposing directions. NAA50 and HYPK each contribute to NAA10 activity inhibition through structural alteration of the NAA10 substrate-binding site. NAA50 activity is increased through NAA15 tethering, but is inhibited by HYPK through structural alteration of the NatE substrate-binding site. These studies reveal the molecular basis for coordinated N-terminal acetylation by NatE and HYPK.

The importance of preoperative T1 slope for determining proper postoperative C2-7 Cobb's angle in patients undergoing cervical reconstruction.

BACKGROUND: This study aimed to explore the relationship among different cervical sagittal parameters in asymptomatic volunteers and the correlation between surgical efficacy and difference of presumed and actual postoperative C2-7 Cobbs's angle (C2-7COBB), which was calculated based on preoperative T1 slope (T1S) in patients undergoing cervical reconstruction. METHODS: In total, 158 inpatients with cervical spondylosis and 274 asymptomatic volunteers were retrospectively reviewed. Cervical sagittal parameters, such as C2-7COBB, T1S, thoracic inlet angle (TIA), and neck tilt (NT), were compared. Then, the correlation among these parameters was analyzed in asymptomatic volunteers, and a regression equation between T1S and C2-7COBB was established and used to analyze the correlation among the Japanese Orthopaedic Association (JOA) score improvement, the sagittal parameters, and the difference between presumed and actual postoperative C2-7COBB in patients after cervical reconstruction. RESULTS: The mean T1S, C2-7COBB, and TIA were significantly decreased in patients (P < 0.01). T1S and NT had a strong correlation with TIA (P < 0.01). T1S demonstrated a moderate correlation with C2-7COBB in asymptomatic volunteers (r = 0.569, P < 0.01). A regression equation had been established as C2-7COBB = 0.742 × T1S - 0.866. The mean C2-7COBB and JOA score improved significantly (P < 0.05) postoperatively. Moreover, the JOA improvement rate showed a significant negative correlation with the difference in the presumed and actual postoperative C2-7COBB (r = - 0.696, P < 0.01). CONCLUSION: Our study successfully established a regression equation for calculating postsurgical C2-7COBB based on the correlation between T1S and C2-7COBB in asymptomatic volunteers. The regression equation could be used for guiding surgeons to accomplish an ideal postsurgical C2-7COBB for patients with cervical spondylosis.

Author Correction: Molecular basis for acetyl-CoA production by ATP-citrate lyase.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Molecular basis for acetyl-CoA production by ATP-citrate lyase.

ATP-citrate lyase (ACLY) synthesizes cytosolic acetyl coenzyme A (acetyl-CoA), a fundamental cellular building block. Accordingly, aberrant ACLY activity is observed in many diseases. Here we report cryo-EM structures of human ACLY, alone or bound to substrates or products. ACLY forms a homotetramer with a rigid citrate synthase homology (CSH) module, flanked by four flexible acetyl-CoA synthetase homology (ASH) domains; CoA is bound at the CSH-ASH interface in mutually exclusive productive or unproductive conformations. The structure of a catalytic mutant of ACLY in the presence of ATP, citrate and CoA substrates reveals a phospho-citryl-CoA intermediate in the ASH domain. ACLY with acetyl-CoA and oxaloacetate products shows the products bound in the ASH domain, with an additional oxaloacetate in the CSH domain, which could function in ACLY autoinhibition. These structures, which are supported by biochemical and biophysical data, challenge previous proposals of the ACLY catalytic mechanism and suggest additional therapeutic possibilities for ACLY-associated metabolic disorders.

Structure and Mechanism of Acetylation by the N-Terminal Dual Enzyme NatA/Naa50 Complex.

NatA co-translationally acetylates the N termini of over 40% of eukaryotic proteins and can associate with another catalytic subunit, Naa50, to form a ternary NatA/Naa50 dual enzyme complex (also called NatE). The molecular basis of association between Naa50 and NatA and the mechanism for how their association affects their catalytic activities in yeast and human are poorly understood. Here, we determined the X-ray crystal structure of yeast NatA/Naa50 as a scaffold to understand coregulation of NatA/Naa50 activity in both yeast and human. We find that Naa50 makes evolutionarily conserved contacts to both the Naa10 and Naa15 subunits of NatA. These interactions promote catalytic crosstalk within the human complex, but do so to a lesser extent in the yeast complex, where Naa50 activity is compromised. These studies have implications for understanding the role of the NatA/Naa50 complex in modulating the majority of the N-terminal acetylome in diverse species.

Genomic characterization of genes encoding histone acetylation modulator proteins identifies therapeutic targets for cancer treatment.

A growing emphasis in anticancer drug discovery efforts has been on targeting histone acetylation modulators. Here we comprehensively analyze the genomic alterations of the genes encoding histone acetylation modulator proteins (HAMPs) in the Cancer Genome Atlas cohort and observe that HAMPs have a high frequency of focal copy number alterations and recurrent mutations, whereas transcript fusions of HAMPs are relatively rare genomic events in common adult cancers. Collectively, 86.3% (63/73) of HAMPs have recurrent alterations in at least 1 cancer type and 16 HAMPs, including 9 understudied HAMPs, are identified as putative therapeutic targets across multiple cancer types. For example, the recurrent focal amplification of BRD9 is observed in 9 cancer types and genetic depletion of BRD9 inhibits tumor growth. Our systematic genomic analysis of HAMPs across a large-scale cancer specimen cohort may facilitate the identification and prioritization of potential drug targets and selection of suitable patients for precision treatment.

Structure and assembly mechanism of plant C2S2M2-type PSII-LHCII supercomplex.

In plants, the photosynthetic machinery photosystem II (PSII) consists of a core complex associated with variable numbers of light-harvesting complexes II (LHCIIs). The supercomplex, comprising a dimeric core and two strongly bound and two moderately bound LHCIIs (C2S2M2), is the dominant form in plants acclimated to limited light. Here we report cryo-electron microscopy structures of two forms of C2S2M2 (termed stacked and unstacked) from Pisum sativum at 2.7- and 3.2-angstrom resolution, respectively. In each C2S2M2, the moderately bound LHCII assembles specifically with a peripheral antenna complex CP24-CP29 heterodimer and the strongly bound LHCII, to establish a pigment network that facilitates light harvesting at the periphery and energy transfer into the core. The high mobility of peripheral antennae, including the moderately bound LHCII and CP24, provides insights into functional regulation of plant PSII.

Structure of the catalytic domain of a state transition kinase homolog from Micromonas algae.

Under natural environments, plants and algae have evolved various photosynthetic acclimation mechanisms in response to the constantly changing light conditions. The state transition and long-term response processes in photosynthetic acclimation involve remodeling and composition alteration of thylakoid membrane. A chloroplast protein kinase named Stt7/STN7 has been found to have pivotal roles in both state transition and long-term response. Here we report the crystal structures of the kinase domain of a putative Stt7/STN7 homolog from Micromonas sp. RCC299 (MsStt7d) in the apo form and in complex with various nucleotide substrates. MsStt7d adopts a canonical protein kinase fold and contains all the essential residues at the active site. A novel hairpin motif, found to be a conserved feature of the Stt7/STN7 family and indispensable for the kinase stability, interacts with the activation loop and fixes it in an active conformation. We have also demonstrated that MsStt7d is a dualspecifi city kinase that phosphorylates both Thr and Tyr residues. Moreover, preliminary in vitro data suggest that it might be capable of phosphorylating a consensus N-terminal pentapeptide of light-harvesting proteins Micromonas Lhcp4 and Arabidopsis Lhcb1 directly. The potential peptide/protein substrate binding site is predicted based on the location of a pseudo-substrate contributed by the adjacent molecule within the crystallographic dimer. The structural and biochemical data presented here provide a framework for an improved understanding on the role of Stt7/STN7 in photosynthetic acclimation.