The human pre-replication complex is an open complex.

In eukaryotes, DNA replication initiation requires assembly and activation of the minichromosome maintenance (MCM) 2-7 double hexamer (DH) to melt origin DNA strands. However, the mechanism for this initial melting is unknown. Here, we report a 2.59-Å cryo-electron microscopy structure of the human MCM-DH (hMCM-DH), also known as the pre-replication complex. In this structure, the hMCM-DH with a constricted central channel untwists and stretches the DNA strands such that almost a half turn of the bound duplex DNA is distorted with 1 base pair completely separated, generating an initial open structure (IOS) at the hexamer junction. Disturbing the IOS inhibits DH formation and replication initiation. Mapping of hMCM-DH footprints indicates that IOSs are distributed across the genome in large clusters aligning well with initiation zones designed for stochastic origin firing. This work unravels an intrinsic mechanism that couples DH formation with initial DNA melting to license replication initiation in human cells.

Parental histone transfer caught at the replication fork.

In eukaryotes, DNA compacts into chromatin through nucleosomes1,2. Replication of the eukaryotic genome must be coupled to the transmission of the epigenome encoded in the chromatin3,4. Here we report cryo-electron microscopy structures of yeast (Saccharomyces cerevisiae) replisomes associated with the FACT (facilitates chromatin transactions) complex (comprising Spt16 and Pob3) and an evicted histone hexamer. In these structures, FACT is positioned at the front end of the replisome by engaging with the parental DNA duplex to capture the histones through the middle domain and the acidic carboxyl-terminal domain of Spt16. The H2A-H2B dimer chaperoned by the carboxyl-terminal domain of Spt16 is stably tethered to the H3-H4 tetramer, while the vacant H2A-H2B site is occupied by the histone-binding domain of Mcm2. The Mcm2 histone-binding domain wraps around the DNA-binding surface of one H3-H4 dimer and extends across the tetramerization interface of the H3-H4 tetramer to the binding site of Spt16 middle domain before becoming disordered. This arrangement leaves the remaining DNA-binding surface of the other H3-H4 dimer exposed to additional interactions for further processing. The Mcm2 histone-binding domain and its downstream linker region are nested on top of Tof1, relocating the parental histones to the replisome front for transfer to the newly synthesized lagging-strand DNA. Our findings offer crucial structural insights into the mechanism of replication-coupled histone recycling for maintaining epigenetic inheritance.

ALS-linked C9orf72-SMCR8 complex is a negative regulator of primary ciliogenesis.

Massive GGGGCC (G4C2) repeat expansion in C9orf72 and the resulting loss of C9orf72 function are the key features of ~50% of inherited amyotrophic lateral sclerosis and frontotemporal dementia cases. However, the biological function of C9orf72 remains unclear. We previously found that C9orf72 can form a stable GTPase activating protein (GAP) complex with SMCR8 (Smith-Magenis chromosome region 8). Herein, we report that the C9orf72-SMCR8 complex is a major negative regulator of primary ciliogenesis, abnormalities in which lead to ciliopathies. Mechanistically, the C9orf72-SMCR8 complex suppresses the primary cilium as a RAB8A GAP. Moreover, based on biochemical analysis, we found that C9orf72 is the RAB8A binding subunit and that SMCR8 is the GAP subunit in the complex. We further found that the C9orf72-SMCR8 complex suppressed the primary cilium in multiple tissues from mice, including but not limited to the brain, kidney, and spleen. Importantly, cells with C9orf72 or SMCR8 knocked out were more sensitive to hedgehog signaling. These results reveal the unexpected impact of C9orf72 on primary ciliogenesis and elucidate the pathogenesis of diseases caused by the loss of C9orf72 function.

Cryo-EM structures of the TMEM16A calcium-activated chloride channel.

Calcium-activated chloride channels (CaCCs) encoded by TMEM16A control neuronal signalling, smooth muscle contraction, airway and exocrine gland secretion, and rhythmic movements of the gastrointestinal system. To understand how CaCCs mediate and control anion permeation to fulfil these physiological functions, knowledge of the mammalian TMEM16A structure and identification of its pore-lining residues are essential. TMEM16A forms a dimer with two pores. Previous CaCC structural analyses have relied on homology modelling of a homologue (nhTMEM16) from the fungus Nectria haematococca that functions primarily as a lipid scramblase, as well as subnanometre-resolution electron cryo-microscopy. Here we present de novo atomic structures of the transmembrane domains of mouse TMEM16A in nanodiscs and in lauryl maltose neopentyl glycol as determined by single-particle electron cryo-microscopy. These structures reveal the ion permeation pore and represent different functional states. The structure in lauryl maltose neopentyl glycol has one Ca2+ ion resolved within each monomer with a constricted pore; this is likely to correspond to a closed state, because a CaCC with a single Ca2+ occupancy requires membrane depolarization in order to open (C.J.P. et al., manuscript submitted). The structure in nanodiscs has two Ca2+ ions per monomer and its pore is in a closed conformation; this probably reflects channel rundown, which is the gradual loss of channel activity that follows prolonged CaCC activation in 1 mM Ca2+. Our mutagenesis and electrophysiological studies, prompted by analyses of the structures, identified ten residues distributed along the pore that interact with permeant anions and affect anion selectivity, as well as seven pore-lining residues that cluster near pore constrictions and regulate channel gating. Together, these results clarify the basis of CaCC anion conduction.

Biochemical and structural characterization of the cyanophage-encoded phosphate-binding protein: implications for enhanced phosphate uptake of infected cyanobacteria.

To acquire phosphorus, cyanobacteria use the typical bacterial ABC-type phosphate transporter, which is composed of a periplasmic high-affinity phosphate-binding protein PstS and a channel formed by two transmembrane proteins PstC and PstA. A putative pstS gene was identified in the genomes of cyanophages that infect the unicellular marine cyanobacteria Prochlorococcus and Synechococcus. However, it has not been determined whether the cyanophage PstS protein is functional during infection to enhance the phosphate uptake rate of host cells. Here we showed that the cyanophage P-SSM2 PstS protein was abundant in the infected Prochlorococcus NATL2A cells and the host phosphate uptake rate was enhanced after infection. This is consistent with our biochemical and structural analyses showing that the phage PstS protein is indeed a high-affinity phosphate-binding protein. We further modelled the complex structure of phage PstS with host PstCA and revealed three putative interfaces that may facilitate the formation of a chimeric ABC transporter. Our results provide insights into the molecular mechanism by which cyanophages enhance the phosphate uptake rate of cyanobacteria. Phosphate acquisition by infected bacteria can increase the phosphorus contents of released cellular debris and virus particles, which together constitute a significant proportion of the marine dissolved organic phosphorus pool.

Structural insight into TRPV5 channel function and modulation.

TRPV5 (transient receptor potential vanilloid 5) is a unique calcium-selective TRP channel essential for calcium homeostasis. Unlike other TRPV channels, TRPV5 and its close homolog, TRPV6, do not exhibit thermosensitivity or ligand-dependent activation but are constitutively open at physiological membrane potentials and modulated by calmodulin (CaM) in a calcium-dependent manner. Here we report high-resolution electron cryomicroscopy structures of truncated and full-length TRPV5 in lipid nanodiscs, as well as of a TRPV5 W583A mutant and TRPV5 in complex with CaM. These structures highlight the mechanism of calcium regulation and reveal a flexible stoichiometry of CaM binding to TRPV5.

Structure of a presenilin family intramembrane aspartate protease.

Presenilin and signal peptide peptidase (SPP) are intramembrane aspartyl proteases that regulate important biological functions in eukaryotes. Mechanistic understanding of presenilin and SPP has been hampered by lack of relevant structural information. Here we report the crystal structure of a presenilin/SPP homologue (PSH) from the archaeon Methanoculleus marisnigri JR1. The protease, comprising nine transmembrane segments (TMs), adopts a previously unreported protein fold. The amino-terminal domain, consisting of TM1-6, forms a horseshoe-shaped structure, surrounding TM7-9 of the carboxy-terminal domain. The two catalytic aspartate residues are located on the cytoplasmic side of TM6 and TM7, spatially close to each other and approximately 8 Å into the lipid membrane surface. Water molecules gain constant access to the catalytic aspartates through a large cavity between the amino- and carboxy-terminal domains. Structural analysis reveals insights into the presenilin/SPP family of intramembrane proteases.

Cleavage of amyloid precursor protein by an archaeal presenilin homologue PSH.

Aberrant cleavage of amyloid precursor protein (APP) by γ-secretase contributes to the development of Alzheimer's disease. More than 200 disease-derived mutations have been identified in presenilin (the catalytic subunit of γ-secretase), making modulation of γ-secretase activity a potentially attractive therapeutic opportunity. Unfortunately, the technical challenges in dealing with intact γ-secretase have hindered discovery of modulators and demand a convenient substitute approach. Here we report that, similar to γ-secretase, the archaeal presenilin homolog PSH faithfully processes the substrate APP C99 into Aß42, Aß40, and Aß38. The molar ratio of the cleavage products Aß42 over Aß40 by PSH is nearly identical to that by γ-secretase. The proteolytic activity of PSH is specifically suppressed by presenilin-specific inhibitors. Known modulators of γ-secretase also modulate PSH similarly in terms of the Aß42/Aß40 ratio. Structural analysis reveals association of a known γ-secretase inhibitor with PSH between its two catalytic aspartate residues. These findings identify PSH as a surrogate protease for the screening of agents that may regulate the protease activity and the cleavage preference of γ-secretase.

Structure and mechanism of the uracil transporter UraA.

The nucleobase/ascorbate transporter (NAT) proteins, also known as nucleobase/cation symporter 2 (NCS2) proteins, are responsible for the uptake of nucleobases in all kingdoms of life and for the transport of vitamin C in mammals. Despite functional characterization of the NAT family members in bacteria, fungi and mammals, detailed structural information remains unavailable. Here we report the crystal structure of a representative NAT protein, the Escherichia coli uracil/H(+) symporter UraA, in complex with uracil at a resolution of 2.8 Å. UraA has a novel structural fold, with 14 transmembrane segments (TMs) divided into two inverted repeats. A pair of antiparallel ß-strands is located between TM3 and TM10 and has an important role in structural organization and substrate recognition. The structure is spatially arranged into a core domain and a gate domain. Uracil, located at the interface between the two domains, is coordinated mainly by residues from the core domain. Structural analysis suggests that alternating access of the substrate may be achieved through conformational changes of the gate domain.

Structure of a fucose transporter in an outward-open conformation.

The major facilitator superfamily (MFS) transporters are an ancient and widespread family of secondary active transporters. In Escherichia coli, the uptake of l-fucose, a source of carbon for microorganisms, is mediated by an MFS proton symporter, FucP. Despite intensive study of the MFS transporters, atomic structure information is only available on three proteins and the outward-open conformation has yet to be captured. Here we report the crystal structure of FucP at 3.1 Å resolution, which shows that it contains an outward-open, amphipathic cavity. The similarly folded amino and carboxyl domains of FucP have contrasting surface features along the transport path, with negative electrostatic potential on the N domain and hydrophobic surface on the C domain. FucP only contains two acidic residues along the transport path, Asp 46 and Glu 135, which can undergo cycles of protonation and deprotonation. Their essential role in active transport is supported by both in vivo and in vitro experiments. Structure-based biochemical analyses provide insights into energy coupling, substrate recognition and the transport mechanism of FucP.

Evolutionary mix-and-match with MFS transporters.

Major facilitator superfamily (MFS) transport proteins are ubiquitous in the membranes of all living cells, and ∼25% of prokaryotic membrane transport proteins belong to this superfamily. The MFS represents the largest and most diverse group of transporters and includes members that are clinically important. A wide range of substrates is transported in many instances actively by transduction of the energy stored in an H(+) electrochemical gradient into a concentration gradient of substrate. MFS transporters are characterized by a deep central hydrophilic cavity surrounded by 12 mostly irregular transmembrane helices. An alternating inverted triple-helix structural symmetry within the N- and C-terminal six-helix bundles suggests that the proteins arose by intragenic multiplication. However, despite similar features, MFS transporters share only weak sequence homology. Here, we show that rearrangement of the structural symmetry motifs in the Escherichia coli fucose permease (FucP) results in remarkable homology to lactose permease (LacY). The finding is supported by comparing the location of 34 point mutations in FucP to the location of mutants in LacY. Furthermore, in contrast to the conventional, linear sequence alignment, homologies between sugar- and H(+)-binding sites in the two proteins are observed. Thus, LacY and FucP likely evolved from primordial helix-triplets that formed functional transporters; however, the functional segments assembled in a different consecutive order. The idea suggests a simple, parsimonious chain of events that may have led to the enormous sequence diversity within the MFS.

Metallo-supramolecular branched polymer protects particles from air-water interface in single-particle cryo-electron microscopy.

Recent technological breakthroughs in single-particle cryo-electron microscopy (cryo-EM) enable rapid atomic structure determination of biological macromolecules. A major bottleneck in the current single particle cryo-EM pipeline is the preparation of good quality frozen cryo-EM grids, which is mostly a trial-and-error process. Among many issues, preferred particle orientation and sample damage by air-water interface (AWI) are common practical problems. Here we report a method of applying metallo-supramolecular branched polymer (MSBP) in the cryo-sample preparation for high-resolution single-particle cryo-EM. Our data shows that MSBP keeps a majority of particles away from air-water interface and mitigates preferred orientation as verified by the analyses of apoferritin, hemagglutinin) trimer and various sample proteins. The use of MSBP is a simple method to improve particle distribution for high-resolution structure determination in single-particle cryo-EM.

Cryo-EM structure of cyanophage P-SCSP1u offers insights into DNA gating and evolution of T7-like viruses.

Cyanophages, together with their host cyanobacteria, play important roles in marine biogeochemical cycles and control of marine food webs. The recently identified MPP-C (Marine Picocyanobacteria Podovirus clade C) cyanophages, belonging to the T7-like podoviruses, contain the smallest genomes among cyanopodoviruses and exhibit distinct infection kinetics. However, understanding of the MPP-C cyanophage infection process is hindered by the lack of high-resolution structural information. Here, we report the cryo-EM structure of the cyanophage P-SCSP1u, a representative member of the MPP-C phages, in its native form at near-atomic resolution, which reveals the assembly mechanism of the capsid and molecular interaction of the portal-tail complex. Structural comparison of the capsid proteins of P-SCSP1u and other podoviruses with known structures provides insights into the evolution of T7-like viruses. Furthermore, our study provides the near-atomic resolution structure of portal-tail complex for T7-like viruses. On the basis of previously reported structures of phage T7, we identify an additional valve and gate to explain the DNA gating mechanism for the T7-like viruses.

Synergism between CMG helicase and leading strand DNA polymerase at replication fork.

The replisome that replicates the eukaryotic genome consists of at least three engines: the Cdc45-MCM-GINS (CMG) helicase that separates duplex DNA at the replication fork and two DNA polymerases, one on each strand, that replicate the unwound DNA. Here, we determined a series of cryo-electron microscopy structures of a yeast replisome comprising CMG, leading-strand polymerase Polε and three accessory factors on a forked DNA. In these structures, Polε engages or disengages with the motor domains of the CMG by occupying two alternative positions, which closely correlate with the rotational movement of the single-stranded DNA around the MCM pore. During this process, the polymerase remains stably coupled to the helicase using Psf1 as a hinge. This synergism is modulated by a concerted rearrangement of ATPase sites to drive DNA translocation. The Polε-MCM coupling is not only required for CMG formation to initiate DNA replication but also facilitates the leading-strand DNA synthesis mediated by Polε. Our study elucidates a mechanism intrinsic to the replisome that coordinates the activities of CMG and Polε to negotiate any roadblocks, DNA damage, and epigenetic marks encountered during translocation along replication forks.

Menin "reads" H3K79me2 mark in a nucleosomal context.

Methylation of histone H3 lysine-79 (H3K79) is an epigenetic mark for gene regulation in development, cellular differentiation, and disease progression. However, how this histone mark is translated into downstream effects remains poorly understood owing to a lack of knowledge about its readers. We developed a nucleosome-based photoaffinity probe to capture proteins that recognize H3K79 dimethylation (H3K79me2) in a nucleosomal context. In combination with a quantitative proteomics approach, this probe identified menin as a H3K79me2 reader. A cryo-electron microscopy structure of menin bound to an H3K79me2 nucleosome revealed that menin engages with the nucleosome using its fingers and palm domains and recognizes the methylation mark through a π-cation interaction. In cells, menin is selectively associated with H3K79me2 on chromatin, particularly in gene bodies.

Cryo-EM structure of TMEM63C suggests it functions as a monomer.

The TMEM63 family proteins (A, B, and C), calcium-permeable channels in animals that are preferentially activated by hypo-osmolality, have been implicated in various physiological functions. Deficiency of these channels would cause many diseases including hearing loss. However, their structures and physiological roles are not yet well understood. In this study, we determine the cryo-electron microscopy (cryo-EM) structure of the mouse TMEM63C at 3.56 Å, and revealed structural differences compared to TMEM63A, TMEM63B, and the plant orthologues OSCAs. Further structural guided mutagenesis and calcium imaging demonstrated the important roles of the coupling of TM0 and TM6 in channel activity. Additionally, we confirm that TMEM63C exists primarily as a monomer under physiological conditions, in contrast, TMEM63B is a mix of monomer and dimer in cells, suggesting that oligomerization is a regulatory mechanism for TMEM63 proteins.

Structural insights into broadly neutralizing antibodies elicited by hybrid immunity against SARS-CoV-2.

ABSTRACTIncreasing spread by SARS-CoV-2 Omicron variants challenges existing vaccines and broadly reactive neutralizing antibodies (bNAbs) against COVID-19. Here we determine the diversity, potency, breadth and structural insights of bNAbs derived from memory B cells of BNT162b2-vaccinee after homogeneous Omicron BA.1 breakthrough infection. The infection activates diverse memory B cell clonotypes for generating potent class I/II and III bNAbs with new epitopes mapped to the receptor-binding domain (RBD). The top eight bNAbs neutralize wildtype and BA.1 potently but display divergent IgH/IgL sequences and neuralization profiles against other variants of concern (VOCs). Two of them (P2D9 and P3E6) belonging to class III NAbs display comparable potency against BA.4/BA.5, although structural analysis reveals distinct modes of action. P3E6 neutralizes all variants tested through a unique bivalent interaction with two RBDs. Our findings provide new insights into hybrid immunity on BNT162b2-induced diverse memory B cells in response to Omicron breakthrough infection for generating diverse bNAbs with distinct structural basis.

Recent Technical Advances in Sample Preparation for Single-Particle Cryo-EM.

Cryo-sample preparation is a vital step in the process of obtaining high-resolution structures of macromolecules by using the single-particle cryo-electron microscopy (cryo-EM) method; however, cryo-sample preparation is commonly hampered by high uncertainty and low reproducibility. Specifically, the existence of air-water interfaces during the sample vitrification process could cause protein denaturation and aggregation, complex disassembly, adoption of preferred orientations, and other serious problems affecting the protein particles, thereby making it challenging to pursue high-resolution 3D reconstruction. Therefore, sample preparation has emerged as a critical research topic, and several new methods for application at various preparation stages have been proposed to overcome the aforementioned hurdles. Here, we summarize the methods developed for enhancing the quality of cryo-samples at distinct stages of sample preparation, and we offer insights for developing future strategies based on diverse viewpoints. We anticipate that cryo-sample preparation will no longer be a limiting step in the single-particle cryo-EM field as increasing numbers of methods are developed in the near future, which will ultimately benefit the entire research community.

Structural Insight into the MCM double hexamer activation by Dbf4-Cdc7 kinase.

The Dbf4-dependent kinase Cdc7 (DDK) regulates DNA replication initiation by phosphorylation of the MCM double hexamer (MCM-DH) to promote helicase activation. Here, we determine a series of cryo electron microscopy (cryo-EM) structures of yeast DDK bound to the MCM-DH. These structures, occupied by one or two DDKs, differ primarily in the conformations of the kinase core. The interactions of DDK with the MCM-DH are mediated exclusively by subunit Dbf4 straddling across the hexamer interface on the three N-terminal domains (NTDs) of subunits Mcm2, Mcm6, and Mcm4. This arrangement brings Cdc7 close to its only essential substrate, the N-terminal serine/threonine-rich domain (NSD) of Mcm4. Dbf4 further displaces the NSD from its binding site on Mcm4-NTD, facilitating an immediate targeting of this motif by Cdc7. Moreover, the active center of Cdc7 is occupied by a unique Dbf4 inhibitory loop, which is disengaged when the kinase core assumes wobbling conformations. This study elucidates the versatility of Dbf4 in regulating the ordered multisite phosphorylation of the MCM-DH by Cdc7 kinase during helicase activation.

A broadly neutralizing antibody protects Syrian hamsters against SARS-CoV-2 Omicron challenge.

The strikingly high transmissibility and antibody evasion of SARS-CoV-2 Omicron variants have posed great challenges to the efficacy of current vaccines and antibody immunotherapy. Here, we screen 34 BNT162b2-vaccinees and isolate a public broadly neutralizing antibody ZCB11 derived from the IGHV1-58 family. ZCB11 targets viral receptor-binding domain specifically and neutralizes all SARS-CoV-2 variants of concern, especially with great potency against authentic Omicron and Delta variants. Pseudovirus-based mapping of 57 naturally occurred spike mutations or deletions reveals that S371L results in 11-fold neutralization resistance, but it is rescued by compensating mutations in Omicron variants. Cryo-EM analysis demonstrates that ZCB11 heavy chain predominantly interacts with Omicron spike trimer with receptor-binding domain in up conformation blocking ACE2 binding. In addition, prophylactic or therapeutic ZCB11 administration protects lung infection against Omicron viral challenge in golden Syrian hamsters. These results suggest that vaccine-induced ZCB11 is a promising broadly neutralizing antibody for biomedical interventions against pandemic SARS-CoV-2.