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Ligand binding site prediction is a crucial initial step in structure-based drug discovery. Although several methods have been proposed previously, including those using geometry based and machine learning techniques, their accuracy is considered to be still insufficient. In this study, we introduce an approach that leverages a graph transformer neural network to rank the results of a geometry-based pocket detection method. We also created a larger training dataset compared to the conventionally used sc-PDB and investigated the correlation between the dataset size and prediction performance. Our findings indicate that utilizing a graph transformer-based method alongside a larger training dataset could enhance the performance of ligand binding site prediction. [ABSTRACT FROM AUTHOR]

The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novicida (FnCas9), one of the largest Cas9 orthologs, in complex with a guide RNA and its PAM-containing DNA targets. A structural comparison of FnCas9 with other Cas9 orthologs revealed striking conserved and divergent features among distantly related CRISPR-Cas9 systems. We found that FnCas9 recognizes the 5'-NGG-3' PAM, and used the structural information to create a variant that can recognize the more relaxed 5'-YG-3' PAM. Furthermore, we demonstrated that the FnCas9-ribonucleoprotein complex can be microinjected into mouse zygotes to edit endogenous sites with the 5'-YG-3' PAM, thus expanding the target space of the CRISPR-Cas9 toolbox.

The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs). Here, we report the crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 Å resolution. The structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA:DNA heteroduplex in a positively charged groove at their interface. Whereas the recognition lobe is essential for binding sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease domains, which are properly positioned for cleavage of the complementary and noncomplementary strands of the target DNA, respectively. The nuclease lobe also contains a carboxyl-terminal domain responsible for the interaction with the protospacer adjacent motif (PAM). This high-resolution structure and accompanying functional analyses have revealed the molecular mechanism of RNA-guided DNA targeting by Cas9, thus paving the way for the rational design of new, versatile genome-editing technologies.

LUBAC (linear ubiquitin chain assembly complex) activates the canonical NF-κB pathway through linear polyubiquitination of NEMO (NF-κB essential modulator, also known as IKKγ) and RIP1. However, the regulatory mechanism of LUBAC-mediated NF-κB activation remains elusive. Here, we show that A20 suppresses LUBAC-mediated NF-κB activation by binding linear polyubiquitin via the C-terminal seventh zinc finger (ZF7), whereas CYLD suppresses it through deubiquitinase (DUB) activity. We determined the crystal structures of A20 ZF7 in complex with linear diubiquitin at 1.70-1.98 Å resolutions. The crystal structures revealed that A20 ZF7 simultaneously recognizes the Met1-linked proximal and distal ubiquitins, and that genetic mutations associated with B cell lymphomas map to the ubiquitin-binding sites. Our functional analysis indicated that the binding of A20 ZF7 to linear polyubiquitin contributes to the recruitment of A20 into a TNF receptor (TNFR) signalling complex containing LUBAC and IκB kinase (IKK), which results in NF-κB suppression. These findings provide new insight into the regulation of immune and inflammatory responses.

The drug/metabolite transporter (DMT) superfamily is a large group of membrane transporters ubiquitously found in eukaryotes, bacteria and archaea, and includes exporters for a remarkably wide range of substrates, such as toxic compounds and metabolites (1). YddG is a bacterial DMT protein that expels aromatic amino acids and exogenous toxic compounds, thereby contributing to cellular homeostasis (2,3). Here we present structural and functional analyses of YddG. Using liposome-based analyses, we show that Escherichia coli and Starkeya novella YddG export various amino acids. The crystal structure of S. novella YddG at 2.4 Å resolution reveals a new membrane transporter topology, with ten transmembrane segments in an outward-facing state. The overall structure is basket-shaped, with a large substrate-binding cavity at the centre of the molecule, and is composed of inverted structural repeats related by two-fold pseudosymmetry. On the basis of this intramolecular symmetry, we propose a structural model for the inward-facing state and a mechanism of the conformational change for substrate transport, which we confirmed by biochemical analyses. These findings provide a structural basis for the mechanism of transport of DMT superfamily proteins., An important physiological function is the expulsion of various compounds from cells to the extracellular space, which is essential for cellular homeostasis. This process involves specific membrane transporters that export [...]

Krokinobacter eikastus rhodopsin 2 (KR2) is the first light-driven [Na.sup.+] pump discovered, and is viewed as a potential next-generation optogenetics tool. Since the positively charged Schiff base proton, located within the ion-conducting pathway of all light-driven ion pumps, was thought to prohibit the transport of a non-proton cation, the discovery of KR2 raised the question of how it achieves [Na.sup.+] transport. Here we present crystal structures of KR2 under neutral and acidic conditions, which represent the resting and M-like intermediate states, respectively. Structural and spectroscopic analyses revealed the gating mechanism, whereby the flipping of Asp116 sequesters the Schiff base proton from the conducting pathway to facilitate [Na.sup.+] transport. Together with the structure-based engineering of the first light-driven [K.sup.+] pumps, electrophysiological assays in mammalian neurons and behavioural assays in a nematode, our studies reveal the molecular basis for light-driven non-proton cation pumps and thus provide a framework that may advance the development of next-generation optogenetics., Many organisms capture light energy and information using the rhodopsin family of proteins, which comprise the heptahelical transmembrane (7-TM) proteins called opsins covalently linked to retinal. Based on their primary [...]

The crystal structure of the bacterial protein YidC is reported, together with a structure-based functional analysis, providing insight into the role of YidC in inserting single-spanning membrane proteins into the membrane. Structure of YidC from Bacillus halodurans The bacterial protein YidC, a homologue of mitochondrial Oxa1 and chloroplast Alb3, is not just a chaperone that facilitates proper folding and membrane topology of its membrane-protein substrates, in cooperation with the Sec machinery. It also inserts several single- or double-spanning membrane proteins into the membrane independently of Sec. Osamu Nureki and colleagues present the long-awaited structure of YidC, which provides insights into the second role of this membrane protein. The structure suggests that YidC does not adopt a polypeptide-conducting channel-like architecture. Instead, a novel fold within the protein forms a positively charged hydrophilic groove. The authors, using structure-based functional analysis, reveal that electrostatic interactions between a conserved Arg residue in the groove and the acidic residues at the N-terminal region of a substrate protein are essential for the YidC-mediated insertion of the substrate into the membrane. Newly synthesized membrane proteins must be accurately inserted into the membrane, folded and assembled for proper functioning. The protein YidC inserts its substrates into the membrane, thereby facilitating membrane protein assembly in bacteria; the homologous proteins Oxa1 and Alb3 have the same function in mitochondria and chloroplasts, respectively.sup.1,2. In the bacterial cytoplasmic membrane, YidC functions as an independent insertase and a membrane chaperone in cooperation with the translocon SecYEG.sup.3,4,5. Here we present the crystal structure of YidC from Bacillus halodurans, at 2.4 Å resolution. The structure reveals a novel fold, in which five conserved transmembrane helices form a positively charged hydrophilic groove that is open towards both the lipid bilayer and the cytoplasm but closed on the extracellular side. Structure-based in vivo analyses reveal that a conserved arginine residue in the groove is important for the insertion of membrane proteins by YidC. We propose an insertion mechanism for single-spanning membrane proteins, in which the hydrophilic environment generated by the groove recruits the extracellular regions of substrates into the low-dielectric environment of the membrane., Author(s): Kaoru Kumazaki [sup.1] [sup.2] , Shinobu Chiba [sup.3] , Mizuki Takemoto [sup.1] [sup.2] , Arata Furukawa [sup.4] , Ken-ichi Nishiyama [sup.5] , Yasunori Sugano [sup.4] , Takaharu Mori [sup.6] [...]

Multidrug and toxic compound extrusion (MATE) family transporters are conserved in the three primary domains of life (Archaea, Bacteria and Eukarya), and export xenobiotics using an electrochemical gradient of [H.sup.+] [...]

PIWI-interacting RNAs (piRNAs) silence transposons to maintain genome integrity in animal germ lines (1-4). piRNAs are classified as primary and secondary piRNAs, depending on their biogenesis machinery5-10. Primary piRNAs are [...]

Channelrhodopsins (ChRs) are light-gated cation channels derived from algae that have shown experimental utility in optogenetics; for example, neurons expressing ChRs can be optically controlled with high temporal precision within systems as complex as freely moving mammals. Although ChRs have been broadly applied to neuroscience research, little is known about the molecular mechanisms by which these unusual and powerful proteins operate. Here we present the crystal structure of a ChR (a C1C2 chimaera between ChR1 and ChR2 from Chlamydomonas reinhardtii) at 2.3 Å resolution. The structure reveals the essential molecular architecture of ChRs, including the retinal-binding pocket and cation conduction pathway. This integration of structural and electrophysiological analyses provides insight into the molecular basis for the remarkable function of ChRs, and paves the way for the precise and principled design of ChR variants with novel properties., Organisms ranging from archaebacteria to human beings capture energy and/or information contained within environmental sources of light by using photoreceptors called rhodopsins, which consist of seven-transmembrane-helix proteins, called opsins, covalently [...]