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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 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 [...]

Protein translocation across the bacterial membrane, mediated by the secretory translocon SecYEG and the SecA ATPase (1-4),is enhanced by proton motive force5,6 and membrane-integrated SecDF7-9, which associates with SecYEG. The role of SecDF has remained unclear, although it is proposed to function in later stages of translocation as well as in membrane protein biogenesis (4,10-13). Here, we determined the crystal structure of Thermus thermophilus SecDF at 3.3 Å resolution, revealing a pseudo-symmetrical, 12-helix transmembrane domain belonging to the RND superfamily and two major periplasmic domains, P1 and P4. Higher-resolution analysis of the periplasmic domains suggested that P1, which binds an unfolded protein, undergoes functionally important conformational changes. In vitro analyses identified an ATP-independent step of protein translocation that requires both SecDF and proton motive force. Electrophysiological analyses revealed that SecDF conducts protons in a manner dependent on pH and the presence of an unfolded protein, with conserved Asp and Arg residues at the transmembrane interface between SecD and SecF playing essential roles in the movements of protons and preproteins. Therefore, we propose that SecDF functions as a membrane-integrated chaperone, powered by proton motive force, to achieve ATP-independent protein translocation., The functional importance of SecDF in protein translocation was previously shown in vivo (7,9,14): the SecD- and SecF-deficient Escherichia coli mutant secDl (producing negligible levels of SecD and SecF and [...]

Maturation of precursor transfer RNA (pre-tRNA) includes excision of the 5' leader and 3' trailer sequences, removal of introns and addition of the CCA terminus (1-3). Nucleotide modifications are incorporated [...]

tRNAs from all 3 phylogenetic domains have a 5-methyluridine at position 54 (T54) in the T-loop. The methyl group is transferred from S-adenosylmethionine by TrmA methyltransferase in most Gram-negative bacteria and some archaea and eukaryotes, whereas it is transferred from 5,10-methylenetetrahydrofolate (MTHF) by TrmFO, a folate/FAD-dependent methyltransferase, in most Gram-positive bacteria and some Gram-negative bacteria. However, the catalytic mechanism remains unclear, because the crystal structure of TrmFO has not been solved. Here, we report the crystal structures of Thermus thermophilus TrmFO in its free form, tetrahydrofolate (THF)-bound form, and glutathione-bound form at 2.1-, 1.6-, and 1.05-[Angstrom] resolutions, respectively. TrmFO consists of an FAD-binding domain and an insertion domain, which both share structural similarity with those of GidA, an enzyme involved in the 5-carboxymethylaminomethylation of U34 of some tRNAs. However, the overall structures of TrmFO and GidA are basically different because of their distinct domain orientations, which are consistent with their respective functional specificities. In the THF complex, the pteridin ring of THF is sandwiched between the flavin ring of FAD and the imidazole ring of a His residue. This structure provides a snapshot of the folate/FAD-dependent methyl transfer, suggesting that the transferring methylene group of MTHF is located close to the redox-active N5 atom of FAD. Furthermore, we established an in vitro system to measure the methylation activity. Our TrmFO-tRNA docking model, in combination with mutational analyses, suggests a catalytic mechanism, in which the methylene of MTHF is directly transferred onto U54, and then the exocyclic methylene of U54 is reduced by [FADH.sub.2]. modification | X-ray crystallography | TrmFO

Pyrrolysine (pyl), the 22nd natural amino acid, is genetically encoded by UAG and inserted into proteins by the unique suppressor [tRNA.sup.Pyl] (ref. 1). The Methanosarcinaceae produce Pyl and express Pyl-containing methyltransferases that allow growth on methylamines (2). Homologous methyltransferases and the Pyl biosynthetic and coding machinery are also found in two bacterial species (1,3). Pyl coding is maintained by pyrrolysyl-tRNA synthetase (PylRS), which catalyses the formation of Pyl-[tRNA.sup.Pyl] (refs 4, 5). Pyl is not a recent addition to the genetic code. PylRS was already present in the last universal common ancestorb; it then persisted in organisms that utilize methylamines as energy sources. Recent protein engineering efforts added non-canonical amino acids to the genetic code (7,8). This technology relies on the directed evolution of an 'orthogonal' tRNA synthetase-tRNA pair in which an engineered aminoaryl-tRNA synthetase (aaRS) specifically and exclusively arylates the orthogonal tRNA with a non-canonical amino acid. For Pyl the natural evolutionary process developed such a system some 3 billion years ago. When transformed into Escherichia coli, Methanosarcina barkeri PylRS and [tRNA.sup.Pyl] function as an orthogonal pair in vivos (5,9). Here we show that Desulfitobacterium hafniense PylRS-[tRNA.sup.Pyl] is an orthogonal pair in vitro and in vivo, and present the crystal structure of this orthogonal pair. The ancient emergence of PylRS-[tRNA.sup.Pyl] allowed the evolution of unique structural features in both the protein and the tRNA. These structural elements manifest an intricate, specialized aaRS-tRNA interaction surface that is highly distinct from those observed in any other known aaRS-tRNA complex; it is this general property that underlies the molecular basis of orthogonality., Unlike the archaeal PylRS sequences, the bacterial versions are encoded in two separate genes. The pylS gene encodes the D. hafniense PylRS (DhPylRS) presented here, which includes a tRNA recognition [...]

Over 30% of proteins are secreted across or integrated into membranes. Their newly synthesized forms contain either cleavable signal sequences or non-cleavable membrane anchor sequences, which direct them to the [...]

Author(s): Reiya Taniguchi [1, 2]; Asuka Inoue [3, 4]; Misa Sayama [5]; Akiharu Uwamizu [3]; Keitaro Yamashita [6]; Kunio Hirata [4, 6]; Masahito Yoshida [7]; Yoshiki Tanaka [8]; Hideaki E. [...]

Author(s): Kozo Tomita [1]; Ryuichiro Ishitani [2]; Shuya Fukai [2]; Osamu Nureki (corresponding author) [2, 3] Genetic information is replicated and transcribed by polynucleotide polymerases. Conventional template-dependent DNA/RNA polymerases undergo [...]

Author(s): Kozo Tomita [1, 2, 7]; Shuya Fukai [3, 7]; Ryuichiro Ishitani [4]; Takuya Ueda [2]; Nono Takeuchi [2]; Dmitry G. Vassylyev [5]; Osamu Nureki (corresponding author) [3, 6] The [...]