The innate immune system functions as the first line of defense against invading bacteria and viruses. In this context, the cGAS STING cyclic guanosine monophosphate (GMP)-adenosine monophosphate ...(AMP) synthase STING signaling axis perceives the nonself DNA associated with bacterial and viral infections, as well as the leakage of self DNA by cellular dysfunction and stresses, to elicit the host's immune responses. In this pathway, the noncanonical cyclic dinucleotide 2′,3′-cyclic GMP-AMP (2′,3′-cGAMP) functions as a second messenger for signal transduction: 2′,3′-cGAMP is produced by the enzyme cGAS upon its recognition of double-stranded DNA, and then the 2′,3′-cGAMP is recognized by the receptor STING to induce the phosphorylation of downstream factors, including TBK1 (TANK binding kinase 1) and IRF3 (interferon regulatory factor 3). Numerous crystal structures of the components of this cGAS STING signaling axis have been reported and these clarify the structural basis for their signal transduction mechanisms. In this review, we summarize recent progress made in the structural dissection of this signaling pathway and indicate possible directions of forthcoming research.
In eukaryotic membranes, type IV P-type adenosine triphosphatases (P4-ATPases) mediate the translocation of phospholipids from the outer to the inner leaflet and maintain lipid asymmetry, which is ...critical for membrane trafficking and signaling pathways. Here, we report the cryo-electron microscopy structures of six distinct intermediates of the human ATP8A1-CDC50a heterocomplex at resolutions of 2.6 to 3.3 angstroms, elucidating the lipid translocation cycle of this P4-ATPase. ATP-dependent phosphorylation induces a large rotational movement of the actuator domain around the phosphorylation site in the phosphorylation domain, accompanied by lateral shifts of the first and second transmembrane helices, thereby allowing phosphatidylserine binding. The phospholipid head group passes through the hydrophilic cleft, while the acyl chain is exposed toward the lipid environment. These findings advance our understanding of the flippase mechanism and the disease-associated mutants of P4-ATPases.
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•Incredible structural and mechanistic diversity in the CRISPR-Cas effector nucleases.•Divergent evolution of the type I (Cascade-Cas3) and type III (Csm/Cmr) effector ...nucleases.•Functional convergence between the type II (Cas9) and type V (Cpf1) effector nucleases.
In the prokaryotic CRISPR-Cas adaptive immune systems, a CRISPR RNA (crRNA) assembles with multiple or single Cas proteins to form crRNA ribonucleoprotein (crRNP) effector complexes, responsible for the destruction of invading genetic elements. Although the mechanisms of target recognition and cleavage by the crRNP effectors are quite diverse among the different types of CRISPR-Cas systems, the basic action principles of these crRNA-guided effector nucleases are highly conserved. In all of the crRNP effectors, the repeat-derived invariant and spacer-derived variable segments of the crRNA are recognized by the Cas protein(s) in sequence-dependent and sequence-independent manners, respectively, with the spacer-derived guide segment available for base pairing with target nucleic acids. Over the past few years, intensive studies have provided an atomic view of the crRNA-guided target interference mechanisms in different types of CRISPR-Cas systems. Here, we review the recent progress toward structural and mechanistic understanding of the diverse crRNP effector nucleases.
Membrane and membrane-spanning proteins, and non-coding RNA are biomolecules to play central roles in beginning of life and distinguishing higher-order eukaryotes. We have determined the structures ...of membrane protein-lipids complexes and non-coding RNA-protein complexes by X-ray crystallography and Cryo-EM single particle analysis, and combined with complementary functional analyses, elucidate their molecular mechanisms at atomic resolutions, to promote creating drugs and medical technologies with two venture companies.
The variety of taste sensations, including sweet, umami, bitter, sour, and salty, arises from diverse taste cells, each of which expresses specific taste sensor molecules and associated components ...for downstream signal transduction cascades. Recent years have witnessed major advances in our understanding of the molecular mechanisms underlying transduction of basic tastes in taste buds, including the identification of the bona fide sour sensor H
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channel OTOP1, and elucidation of transduction of the amiloride-sensitive component of salty taste (the taste of sodium) and the TAS1R-independent component of sweet taste (the taste of sugar). Studies have also discovered an unconventional chemical synapse termed “channel synapse” which employs an action potential-activated CALHM1/3 ion channel instead of exocytosis of synaptic vesicles as the conduit for neurotransmitter release that links taste cells to afferent neurons. New images of the channel synapse and determinations of the structures of CALHM channels have provided structural and functional insights into this unique synapse. In this review, we discuss the current view of taste transduction and neurotransmission with emphasis on recent advances in the field.
Xenobiotic and metabolite extrusion is an important process for the proper functions of cells and their compartments, including acidic organelles. MATE (multidrug and toxic compound extrusion) is a ...large family of secondary active transporters involved in the transport of various compounds across cellular and organellar membranes, and is present in the three domains of life. The major substrates of the bacterial MATE transporters are cationic compounds, including clinically important antibiotics, and thereby MATE transporters confer multi-drug resistance to pathogenic bacteria. The plant MATE transporters are important for the accumulation of various metabolites in organelles, including vacuoles. The human MATE transporters are expressed in the brush-border membrane of the kidney, and are involved in the clearance of cationic drugs from the body. During the past decade, progress in structural biology has clarified the transport mechanism of these MATE transporters in atomic detail. The present review summarizes the reported structures of MATE family transporters, along with their structure-guided functional analyses. This integrated view of the structures of MATE transporters provides novel insights into their transport mechanism.
•MATE (multidrug and toxic compound extrusion) is a large family of secondary active transporters.•MATE is involved in the transport of various xenobiotics and metabolites across cellular and organellar membranes.•The present review summarizes the reported structures of MATE family transporters.•The integrated view of the structures of MATE transporters provides novel insights into their transport mechanism.
The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets bearing a PAM (protospacer adjacent motif) and complementarity to the guide RNA. A recent study showed that, whereas wild-type ...Streptococcus pyogenes Cas9 (SpCas9) recognizes the 5′-NGG-3′ PAM, the engineered VQR, EQR, and VRER SpCas9 variants recognize the 5′-NGA-3′, 5′-NGAG-3′, and 5′-NGCG-3′ PAMs, respectively, thus expanding the targetable sequences in Cas9-mediated genome editing applications. Here, we present the high-resolution crystal structures of the three SpCas9 variants in complexes with a single-guide RNA and its altered PAM-containing, partially double-stranded DNA targets. A structural comparison of the three SpCas9 variants with wild-type SpCas9 revealed that the multiple mutations synergistically induce an unexpected displacement in the phosphodiester backbone of the PAM duplex, thereby allowing the SpCas9 variants to directly recognize the altered PAM nucleotides. Our findings explain the altered PAM specificities of the SpCas9 variants and establish a framework for further rational engineering of CRISPR-Cas9.
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•Crystal structures of SpCas9 variants in complexes with sgRNA and target DNA•Multiple mutations synergistically induce PAM duplex displacement•Structural basis for the altered PAM specificities of the SpCas9 variants
Hirano and Nishimasu et al. solved the high-resolution crystal structures of the VQR (D1135V/R1335Q/T1337R), EQR (D1135E/R1335Q/T1337R), and VRER (D1135V/G1218R/R1335E/T1337R) SpCas9 variants in complexes with a guide RNA and its target DNA, thereby explaining their altered PAM specificities.
The RNA-guided Cpf1 (also known as Cas12a) nuclease associates with a CRISPR RNA (crRNA) and cleaves the double-stranded DNA target complementary to the crRNA guide. The two Cpf1 orthologs from ...Acidaminococcus sp. (AsCpf1) and Lachnospiraceae bacterium (LbCpf1) have been harnessed for eukaryotic genome editing. Cpf1 requires a specific nucleotide sequence, called a protospacer adjacent motif (PAM), for target recognition. Besides the canonical TTTV PAM, Cpf1 recognizes suboptimal C-containing PAMs. Here, we report four crystal structures of LbCpf1 in complex with the crRNA and its target DNA containing either TTTA, TCTA, TCCA, or CCCA as the PAM. These structures revealed that, depending on the PAM sequences, LbCpf1 undergoes conformational changes to form altered interactions with the PAM-containing DNA duplexes, thereby achieving the relaxed PAM recognition. Collectively, the present structures advance our mechanistic understanding of the PAM-dependent, crRNA-guided DNA cleavage by the Cpf1 family nucleases.
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•Cpf1 recognizes canonical TTTV and non-canonical C-containing PAMs•Crystal structures of L. bacterium Cpf1 in complex with the crRNA and its target DNA•PAM recognition mechanism is highly conserved among the Cpf1 enzymes•PAM-interacting domain displacement plays roles in the relaxed PAM recognition
Yamano et al. report the crystal structures of L. bacterium Cpf1 bound to the crRNA and its target DNA with distinct PAMs, providing insights into the recognition mechanism of the optimal TTTV and suboptimal C-containing PAMs.
Tetraspanins play critical roles in various physiological processes, ranging from cell adhesion to virus infection. The members of the tetraspanin family have four membrane-spanning domains and short ...and large extracellular loops, and associate with a broad range of other functional proteins to exert cellular functions. Here we report the crystal structure of CD9 and the cryo-electron microscopic structure of CD9 in complex with its single membrane-spanning partner protein, EWI-2. The reversed cone-like molecular shape of CD9 generates membrane curvature in the crystalline lipid layers, which explains the CD9 localization in regions with high membrane curvature and its implications in membrane remodeling. The molecular interaction between CD9 and EWI-2 is mainly mediated through the small residues in the transmembrane region and protein/lipid interactions, whereas the fertilization assay revealed the critical involvement of the LEL region in the sperm-egg fusion, indicating the different dependency of each binding domain for other partner proteins.
The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used
Cas9 enzyme (SpCas9) requires an NGG protospacer adjacent motif (PAM) for ...target recognition, thereby restricting the targetable genomic loci. Here, we report a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize relaxed NG PAMs. The crystal structure revealed that the loss of the base-specific interaction with the third nucleobase is compensated by newly introduced non-base-specific interactions, thereby enabling the NG PAM recognition. We showed that SpCas9-NG induces indels at endogenous target sites bearing NG PAMs in human cells. Furthermore, we found that the fusion of SpCas9-NG and the activation-induced cytidine deaminase (AID) mediates the C-to-T conversion at target sites with NG PAMs in human cells.
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