Loop Region Research Articles

Molecular dynamics simulations reveal concentration-dependent blockage of graphene quantum dots to water channel protein openings

Graphene quantum dots (GQDs) have attracted significant attention across various scientific research areas due to their exceptional properties. However, studies on the potential toxicity of GQDs have yielded conflicting results. Therefore, a comprehensive evaluation of the toxicity profile of GQDs is essential for a thorough understanding of their biosafety. In this work, employing a molecular dynamics (MD) simulation approach, we investigate the interactions between GQDs and graphene oxide quantum dots (GOQDs) with the AQP1 water channel protein, aiming to explore the potential biological influence of GQDs/GOQDs. Our MD simulation results reveal that GQDs can adsorb to the loop region around the openings of AQP1 water channels, resulting in the blockage of these channels and potential toxicity. Interestingly, this blockage is concentration-dependent, with higher GQD concentrations leading to a greater likelihood of blockage. Additionally, GOQDs show a lower probability of blocking the openings of AQP1 water channels compared to GQDs, due to the hydrophobicity of the loop regions around the openings, which ultimately leads to lower interaction energy. Therefore, these findings provide new insights into the potential adverse impact of GQDs on AQP1 water channels through the blockage of their openings, offering valuable molecular insights into the toxicity profile of GQD nanomaterials.

Impact of missense mutations on the structure–function relationship of human succinyl-CoA synthetase using in silico analysis

The encephalomyopathic mtDNA depletion syndrome with methylmalonic aciduria is associated with succinyl-CoA synthetase (SCS) deficiency caused by pathogenic variants in genes encoding its two subunits. SCS is a mitochondrial enzyme involved in several metabolic pathways and acts as a heterodimer composed of α and β subunits encoded by SUCLG1 and SUCLA2 genes, respectively. The purpose of this study was to analyze the effects of the most pathogenic non-synonymous single nucleotide polymorphisms (nsSNPs) by applying, using different prediction tools, a filtering strategy, on the 343 and 365 nsSNPs found in SUCLG1 and SUCLA2 genes, respectively, retrieved from the databases, then to evaluate their structural and functional effects using homology modeling and molecular docking. Results showed that most deleterious mutations selected for structural analysis were located in loop regions critical for protein stability and function, especially, variants altering glycine and proline residues in these regions supporting their importance. We also showed that variants leading to hydrophobic and hydrophilic residues can destabilize the folding and binding of the protein. Molecular docking has also been used to identify the most important regions of ligand binding site (CoA binding site, ADP-Mg2+ binding site and phosphate ion binding site) and between the two subunits themselves, which mainly involving the ligase CoA domain. Our structural analysis, performed on selected nsSNP, are in accordance with experimental studies reported in the literature and predicted that they would responsible to either nonfunctional protein, subunit instability resulting in reduced amounts of misassembled protein, or in a protein unable to phosphorylate ADP.

Preferential cleavage of the coronavirus defective viral genome by cellular endoribonuclease with characteristics of RNase L

In testing whether coronavirus defective viral genome 12.7 (DVG12.7) with transcription regulating sequence (TRS) can synthesize subgenomic mRNA (sgmRNA) in coronavirus-infected cells, it was unexpectedly found by Northern blot assay that not only sgmRNA (designated sgmDVG 12.7) but also an RNA fragment with a size less than sgmDVG 12.7 was identified. A subsequent study demonstrated that the identified RNA fragment (designated clvDVG) was a cleaved RNA product originating from DVG12.7, and the cleaved sites were located in the loop region of stem‒loop structure and after UU and UA dinucleotides. clvDVG was also identified in mock-infected HRT-18 cells transfected with DVG12.7 transcript, indicating that cellular endoribonuclease is responsible for the cleavage. In addition, the sequence and structure surrounding the cleavage sites can affect the cleavage efficiency of DVG12.7. The cleavage features are therefore consistent with the general criteria for RNA cleavage by cellular RNase L. Furthermore, both the cleavage of rRNA and the synthesis of clvDVG were also identified in A549 cells. Because (i) the cleavage sites occurred predominantly after single-stranded UA and UU dinucleotides, (ii) the sequence and structure surrounding the cleavage sites affected the cleavage efficiency, (iii) the cleavage of rRNA is an index of the activation of RNase L, and (iv) the cleavage of both rRNA and DVG12.7 was identified in A549 cells, the results together indicated that the preferential cleavage of DVG12.7 is correlated with cellular endoribonuclease with the characteristics of RNase L and such cleavage features have not been previously characterized in coronaviruses.

An internal loop region is responsible for inherent target specificity of bacterial Cold-shock proteins.

Cold shock proteins (Csps), of around 70 amino acids, share a protein fold for the cold shock domain (CSD) that contains RNA binding motifs, RNP1 and RNP2, and constitute one family of bacterial RNA-binding proteins. Despite similar amino acid composition, Csps have been shown to individually possess inherent specific functions. Here we identify the molecular differences in Csps that allow selective recognition of RNA targets. Using chimeras and mutants of Escherichia coli CspD and CspA, we demonstrate that Lys43-Ala44 in an internal loop of CspD and the N-terminal portion with Lys4 of CspA are important for determining their target specificities. Pull-down assays suggest these distinct specificities reflect differences in the ability to act on the target RNAs rather than differences in binding to the RNA targets. A phylogenetic tree constructed from 1,573 Csps reveals that the Csps containing Lys-Ala in the loop form a monophyletic clade, and the members in this clade are shown to have target specificities similar to E. coli CspD. The phylogenetic tree also finds a small cluster of Csps containing Lys-Glu in the loop, and these exhibit different specificity than E. coli CspD. Examination of this difference suggests a role of the loop of CspD type proteins in recognition of specific targets. Additionally, each identified type of Csp shows a different distribution pattern among bacteria. Our findings provide a basis for subclassification of Csps based on target RNA specificity, which will be useful for understanding of the functional specialization of Csps.

Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1) lncRNA Conformational Dynamics in Complex with RNA-Binding Protein with Serine-Rich Domain 1 (RNPS1) in the Pan-cancer Splicing and Gene Expression.

Alternative splicing events increase the transcriptomic and proteomic complexity in cancers. Overexpression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a highly conserved lncRNA, is widely known to promote cancer development, one mechanism for which may be through the regulation of alternative splicing and, thereby, gene expression. Its regulatory interactions with proteins have been a subject of much interest, yet little research has been carried out on the mechanisms adopted. It has been observed that MALAT1 binds to RNA-binding protein with serine-rich domain 1 (RNPS1), being colocalized in the nuclear speckles, and together, these two binding partners may regulate alternative splicing. Upregulated RNPS1 is predicted to play a key role in the pan-cancer development. Experimental tertiary structure of full-length MALAT1 is currently lacking despite the availability of the 3D structure of 3' expression and nuclear retention element. We hypothesize that the computationally modeled tertiary structures of the specific binding motifs in the M-region, E-region, and full-length structures of MALAT1 may adopt a modular structure and bind to the RNPS1 loop region of RS/P domain involved in exon skipping, interacting in a manner fully consistent with the biochemical experiments. Extensive observations using the powerful molecular dynamics (MD) simulations of MALAT1 regions bound to RNPS1 suggested that all three regions form interactive, yet stable complexes. The ranking of the MM-GBSA- and MM-PBSA-derived binding free energies between these complexes corroborated well in the MD simulations and experiments. Energy decomposition analyses suggested that arginines in the RNPS1 protein are among the major contributors toward the binding free energies as calculated by MM-GBSA present in the Amber package; while among the nucleotides, the major contributors were nucleotides with G and A nucleobases, with more contributory effect in comparison to arginines, across the bound M-region, E-region, and full-length MALAT1. This suggests that specific purines play a greater role in the complex formation, in a loop-specific manner, and the more proactive approach in complexation tilts toward MALAT1. To the best of our knowledge, our studies are the first studies taking a unique approach, utilizing the binding motifs to deduce a tertiary structure of MALAT1, toward our understanding of the lncRNA-protein interactions, stability, and binding on a structural basis. The therapeutic implications of targeting this complex formation to regulate splicing and hence, oncogenesis, is further envisaged.

In situ crystalline structure of the human eosinophil major basic protein-1.

Eosinophils are white blood cells that participate in innate immune responses and have an essential role in the pathogenesis of inflammatory and neoplastic disorders. Upon activation, eosinophils release cytotoxic proteins such as major basic protein-1 (MBP-1) from cytoplasmic secretory granules (SGr) wherein MBP-1 is stored as nanocrystals. How the MBP-1 nanocrystalline core is formed, stabilized, and subsequently mobilized remains unknown. Here, we report the in-situ structure of crystalline MBP-1 within SGrs of human eosinophils. The structure reveals a mechanism for intragranular crystal packing and stabilization of MBP-1 via a structurally conserved loop region that is associated with calcium-dependent carbohydrate binding in other C-type lectin (CTL) proteins. Single-cell and single-SGr profiling correlating real-space three-dimensional information from cellular montage cryo-electron tomography (cryo-ET) and microcrystal electron diffraction (MicroED) data obtained from non-activated and IL33-activated eosinophils revealed activation-dependent crystal expansion and extrusion of expanded crystals from SGr. These results suggest that MBP-1 crystals play a dynamic role in the release of SGr contents. Collectively, this research demonstrates the importance of in-situ macromolecular structure determination.

Natural selection on apical membrane antigen 1 (AMA1) of an emerging zoonotic malaria parasite Plasmodium inui

Apical membrane antigen 1 (AMA1) of malaria parasites plays an important role in host cell invasion. Antibodies to AMA1 can inhibit malaria merozoite invasion of erythrocytes while vaccine-induced specific cytotoxic T cell responses to this protein are associated with clinical protection. Polymorphisms in AMA1 of Plasmodium falciparum (PfAMA1) and P. vivax (PvAMA1) are of concern for vaccine development. To date, little is known about sequence diversity in ama1 of P. inui (Piama1), an emerging zoonotic malaria parasite. In this study, 80 complete Piama1 coding sequences were obtained from 57 macaques in Thailand that defined 60 haplotypes clustering in two phylogenetic lineages. In total, 74 nucleotide substitutions were identified and distributed unevenly across the gene. Blockwise analysis of the rates of synonymous (dS) and nonsynonymous (dN) nucleotide substitutions did not show a significant deviation from neutrality among Thai isolates. However, significantly negative Tajima’s D values were detected in domain I and the loop region of domain II, implying purifying selection. Codon-based analysis of dN/dS has identified 12 and 14 codons under positive and negative selections, respectively. Meanwhile, 85 amino acid substitutions were identified among 80 Thai and 11 non-Thai PiAMA1 sequences. Of these, 48 substituted residues had a significant alteration in physicochemical properties, suggesting positive selection. More than half of these positively selected amino acids (32 of 48) corresponded to the predicted B-cell or T-cell epitopes, suggesting that selective pressure could be mediated by host immunity. Importantly, 14 amino acid substitutions were singletons and predicted to be deleterious that could be subject to ongoing purifying selection or elimination. Besides genetic drift and natural selection, intragenic recombination identified in domain II could generate sequence variation in Piama1. It is likely that malarial ama1 exhibits interspecies differences in evolutionary histories. Knowledge of the sequence diversity of the Piama1 locus further provides an evolutionary perspective of this important malaria vaccine candidate.

Zebrafish Polymerase Theta and human Polymerase Theta: orthologues with homologous function.

DNA Polymerase Theta (Pol θ) is a conserved an A-family polymerase that plays an essential role in repairing double strand breaks, through micro-homology end joining, and bypassing DNA lesions, through translesion synthesis, to protect genome integrity. Despite its essential role in DNA repair, Pol θ is inherently error-prone. Recently, key loop regions were identified to play an important role in key functions of Pol θ. Here we present a comparative structure-function study of the polymerase domain of zebrafish and human Pol θ. We show that these two proteins share a large amount of sequence and structural homology. However, we identify differences in the amino acid composition within the key loop areas shown to drive characteristic Pol θ functions. Despite these differences zebrafish Pol θ still displays characteristics identify in human Pol θ, including DNA template extension in the presence of different divalent metals, microhomology-mediated end joining, and translesion synthesis. These results will support future studies looking to gain insight into Pol θ function on the basis of evolutionarily conserved features.

Single-Stranded Hairpin Loop RNAs (loopmeRNAs) Potently Induce Gene Silencing through the RNA Interference Pathway.

Synthetic small interfering RNAs conjugated to trivalent N-acetylgalactosamine (GalNAc) are clinically validated drugs for treatment of liver diseases. Incorporation of phosphorothioate linkages and ribose modifications are necessary for stability, potency, and duration of pharmacology. Although multiple alternative siRNA designs such as Dicer-substrate RNA, shRNA, and circular RNA have been evaluated in vitro and in preclinical studies with some success, clinical applications of these designs are limited as it is difficult to incorporate chemical modifications in these designs. An alternative siRNA design that can incorporate chemical modifications through straightforward synthesis without compromising potency will significantly advance the field. Here, we report a facile synthesis of GalNAc ligand-containing single-stranded loop hairpin RNAs (loopmeRNAs) with clinically relevant chemical modifications. We evaluated the efficiency of novel loopmeRNA designs in vivo and correlated their structure-activity relationship with the support of in vitro metabolism data. Sequences and chemical modifications in the loop region of the loopmeRNA design were optimized for maximal potency. Our studies demonstrate that loopmeRNAs can efficiently silence expression of target genes with comparable efficacy to conventional double-stranded siRNAs but reduced environmental and regulatory burdens.

A Versatile Method to Create Antibody/Split‐Enzyme Complexes and Its Application to a Rapid, Homogeneous, and Universal Electrochemical Immunosensing System

AbstractIn this study, a versatile method is developed to create an antibody/split‐enzyme complex for use in electrochemical immunosensors. SpyCatchers are genetically fused at each terminus of flavin adenine dinucleotide‐dependent glucose dehydrogenase (FAD‐GDH) and a protease recognition sequence is inserted into a loop region to prepare a soluble protein and the split GDH. SpyTag‐fused single‐chain variable fragments (scFvs) are employed, which leads to the simple preparation of antibody‐split enzyme complexes (split AECs). The addition of pentameric C‐reactive protein (CRP), as a representative multimeric protein, restored the GDH activity of the split AECs, as the two split AEC fragments formed active GDH when in close proximity. CRP in human serum is electrochemically detected within 5 min without any washing steps with a clinically relevant CRP detection range (0.03–1 mg dL−1). The detection system is expanded to detect hemoglobin, SARS‐CoV‐2 spike protein, and inactivated SARS‐CoV‐2 by exchanging the scFvs. These results show that the convenient preparation method of the split GDH and the split AEC by the insertion of the protease recognition sequence and the use of SpyCatcher/SpyTag can realize a rapid, homogeneous, and universal electrochemical detection system that can be integrated into a commercially available electrochemical glucose sensor.

Regulate catalytic performance by engineering non-regular structure of extradiol dioxygenase: An entrance to bottom strategy

Extradiol dioxygenase Tcu3516 is a home-sourced enzyme demonstrating potent aromatic phenol degradation capacity. To add to the advantageous modifications inside active cavity, this work reported a novel strategy to engineer rarely concerned non-regular structures around the entrance towards the active site at the bottom of cavity. Three structures, Loop region 1 (Loop1: Met173-Arg185), Loop region 2 (Loop2: Ala201-Val212) and C-terminal (C-tail: His290-Lys306) were therefore identified through structural flexibility analysis. Highly rigid prolines within the structures were mutated into smaller alanine, glycine, or serine to improve structural flexibilities; while only P183S on Loop1 showed 3-fold activity enhancement vs the WT when subjected to cleavage of mono-cyclic catechol analogues. The analysis of Root Mean Square Fluctuation showed that P183S presents certain enhancement on Loop1 flexibility without dramatic changes of other domains. Furthermore, the synergetic effects from mutation P183S and cavity-based mutations V186L, V212N and D285A were evaluated by characterizing combinatorial mutants. Temperature dependence and thermostability of the combined mutants showed a more flexible catalytic domain without sacrificing structural integrity and stability. kcat value of P183S/V186L (SL) towards monocyclic catechols significantly surpasses any other combinatorial mutants around Tcu3516 active sites. Moreover, the synergetic effects on conformational plasticity were analyzed by molecular dynamic simulations to shed light into the interplay between structural changes and catalytic performance.

Crystal Structure and Molecular Mechanism of Isocitrate Lyase from Chloroflexus aurantiacus.

Chloroflexus aurantiacus is a green, nonsulfur bacterium that employs the 3-hydroxypropionate cycle to grow, using carbon dioxide/bicarbonate as its primary carbon source. Like most bacteria, it possesses the glyoxylate cycle, facilitated by malate synthase and isocitrate lyase (ICL), allowing a "tricarboxylic acid cycle" bypass. C. aurantiacus also harbors ICL, an enzyme that catalyzes reversible isocitrate cleavage into glyoxylate and succinate. This study presents the crystal structures of C. aurantiacus-derived ICL (CaICL), in its Mg2+-bound and Mn2+ and isocitrate-bound forms, elucidating its substrate-binding mechanism and catalytic loop dynamics. CaICL forms a homotetramer and interacts with isocitrate via critical active-site residues, revealing its catalytic mechanism. The stabilization of the catalytic loop and adjacent terminal regions upon isocitrate binding underscores its functional significance. These findings advance our understanding regarding ICL enzymes, offering a basis for future investigations into their biological roles and potential applications.

Structural insights into molecular-targeting helix–loop–helix peptide against vascular endothelial growth factor-A

Mid-sized binding peptides have recently emerged as a new therapeutic modality. A helix-loop-helix (HLH) peptide was designed as a scaffold for combinatorial peptide libraries. We screened the HLH peptide libraries against human vascular endothelial growth factor-A (VEGF) to generate a peptide, VS42-LR3, which inhibited VEGF/receptor interaction and suppressed tumor growth in a murine xenograft model of human colorectal cancer. Here, we report the first crystal structure of the HLH peptide in a complex with VEGF at high resolution using space-grown protein crystals. The X-ray structural analysis revealed that the monomeric VS42-LR3 adopted an HLH structure and bound to VEGF at the VEGF receptor-binding site. Interestingly, from the site-directed mutagenesis, thermodynamic analysis, and molecular dynamic simulations, it turned out that the loop region in the non-interacting surface to VEGF affected the structural rigidity of the whole HLH to increase the binding affinity. These findings provide valuable insights for the design of more structurally stable and higher affinity mid-sized binding peptides as well as HLH peptides, that could play a crucial role in advancing molecular-targeting therapies.

Rapid affinity optimization of an anti-TREM2 clinical lead antibody by cross-lineage immune repertoire mining

We describe a process for rapid antibody affinity optimization by repertoire mining to identify clones across B cell clonal lineages based on convergent immune responses where antigen-specific clones with the same heavy (VH) and light chain germline segment pairs, or parallel lineages, bind a single epitope on the antigen. We use this convergence framework to mine unique and distinct VH lineages from rat anti-triggering receptor on myeloid cells 2 (TREM2) antibody repertoire datasets with high diversity in the third complementarity-determining loop region (CDR H3) to further affinity-optimize a high-affinity agonistic anti-TREM2 antibody while retaining critical functional properties. Structural analyses confirm a nearly identical binding mode of anti-TREM2 variants with subtle but significant structural differences in the binding interface. Parallel lineage repertoire mining is uniquely tailored to rationally explore the large CDR H3 sequence space in antibody repertoires and can be easily and generally applied to antibodies discovered in vivo.

Diversity, Distribution and Structural Prediction of the Pathogenic Bacterial Effectors EspN and EspS.

Many Gram-negative enterobacteria translocate virulence proteins (effectors) into intestinal epithelial cells using a type III secretion system (T3SS) to subvert the activity of various cell functions possess. Many T3SS effectors have been extensively characterized, but there are still some effector proteins whose functional information is completely unknown. In this study, two predicted effectors of unknown function, EspN and EspS (Escherichia coli secreted protein N and S), were selected for analysis of translocation, distribution and structure prediction. The TEM1 (β-lactamase) translocation assay was performed, which showed that EspN and EspS are translocated into host cells in a T3SS-dependent manner during bacterial infection. A phylogenetic tree analysis revealed that homologs of EspN and EspS are widely distributed in pathogenic bacteria. Multiple sequence alignment revealed that EspN and its homologs share a conserved C-terminal region (673-1133 a.a.). Furthermore, the structure of EspN (673-1133 a.a.) was also predicted and well-defined, which showed that it has three subdomains connected by a loop region. EspS and its homologs share a sequence-conserved C-terminal (146-291 a.a.). The predicted structure of EspS (146-291 a.a.) is composed of a β-sheet consisting of four β-strands and several short helices, which has a TM score of 0.5014 with the structure of the Vibrio cholerae RTX cysteine protease domain (PDBID: 3eeb). These results suggest that EspN and EspS may represent two important classes of T3SS effectors associated with pathogen virulence, and our findings provide important clues to understanding the potential functions of EspN and EspS.

Ebola Virus Matrix Protein VP40 Single Mutations G198R and G201R Significantly Enhance Plasma Membrane Localization.

Viral proteins frequently undergo single or multiple amino acid mutations during replication, which can significantly alter their functionality. The Ebola virus matrix protein VP40 is multifunctional but primarily responsible for creating the viral envelope by binding to the inner leaflet of the host cell plasma membrane (PM). Changes to the VP40 surface cationic charge via mutations can influence PM interactions, resulting in altered viral assembly and budding. A recent mutagenesis study evaluated the effects of several mutations and found that mutations G198R and G201R enhanced VP40 assembly at the PM and virus-like particle budding. These two mutations lie in the loop region of the C-terminal domain (CTD), which directly interacts with the PM. To understand the role of these mutations in PM localization at the molecular level, we performed both all-atom and coarse-grained molecular dynamics simulations using a dimer-dimer configuration of VP40, which contains the CTD-CTD interface. Our studies indicate that the location of mutations on the outer surface of the CTD regions can lead to changes in membrane binding orientation and degree of membrane penetration. Direct PI(4,5)P2 interactions with the mutated residues seem to further stabilize and pull VP40 into the PM, thereby enhancing interactions with numerous amino acids that were otherwise infrequently or completely inaccessible. These multiscale computational studies provide new insights at the atomic and molecular level as to how VP40-PM interactions are altered through single amino acid mutations. Given the high case fatality rates associated with Ebola virus disease in humans, it is essential to explore the mechanisms of viral assembly in the presence of mutations to mitigate the severity of the disease and understand the potential of future outbreaks.

The influence of residue interaction on thermal stability of lipase based on dynamic graph embedding

Lipases with high thermal stability have greater application in industrial production. Focusing on wild-type lipase and their thermotolerant mutants, this study employs dynamic graph embedding to explore the change of spatiotemporal characteristics in residue-residue interactions, analyzing the relationship between residue mutations and the thermal stability of lipase. K-means clustering is used to analyze the synergistic relationships between residues, revealing that thermotolerant mutants achieve higher thermal stability by enhancing the synergistic interactions between secondary structures, resulting in a more balanced and tightly-knit internal structure. Critical residues are identified using anomaly node detection methods, showing that residues with spatiotemporal instability are mainly distributed on highly flexible loop regions. Specifically, mutations at Tyr89 and Asp132 restrict the motion range of the loops, making the lipase structure more stable. The study demonstrates that mutating highly flexible regions to increase structural rigidity can effectively enhance the thermal stability of lipases.

Insights into VDAC Gating: Room-Temperature X-ray Crystal Structure of mVDAC-1.

The voltage-dependent anion channel (VDAC) is a crucial mitochondrial protein that facilitates ion and metabolite exchange between mitochondria and the cytosol. Initially characterized over three decades ago, the structure of VDAC-1 was resolved in 2008, revealing a novel β-barrel protein architecture. This study presents the first room-temperature crystal structure of mouse VDAC-1 (mVDAC-1), which is a significant step toward understanding the channel's gating mechanism. The new structure, obtained at a 3.3 Å resolution, demonstrates notable differences from the previously determined cryogenic structure, particularly in the loop regions, which may be critical for the transition between the 'open' and 'closed' states of VDAC-1. Comparative analysis of the root-mean-square deviation (R.M.S.D.) and B-factors between the cryogenic and room-temperature structures suggests that these conformational differences, although subtle, are important for VDAC's functional transitions. The application of electric field-stimulated X-ray crystallography (EF-X) is proposed as a future direction to resolve the 'closed' state of VDAC-1 by inducing voltage-driven conformational changes in order to elucidate the dynamic gating mechanism of VDAC-1. Our findings have profound implications for understanding the molecular basis of VDAC's role in mitochondrial function and its regulation under physiological conditions.

Mettl1-dependent m7G tRNA modification is essential for maintaining spermatogenesis and fertility in Drosophila melanogaster

Modification of guanosine to N7-methylguanosine (m7G) in the variable loop region of tRNA is catalyzed by the METTL1/WDR4 heterodimer and stabilizes target tRNA. Here, we reveal essential functions of Mettl1 in Drosophila fertility. Knockout of Mettl1 (Mettl1-KO) causes no major effect on the development of non-gonadal tissues, but abolishes the production of elongated spermatids and mature sperm, which is fully rescued by expression of a Mettl1-transgene, but not a catalytic-dead Mettl1 transgene. This demonstrates that Mettl1-dependent m7G is required for spermatogenesis. Mettl1-KO results in a loss of m7G modification on a subset of tRNAs and decreased tRNA abundance. Ribosome profiling shows that Mettl1-KO led to ribosomes stalling at codons decoded by tRNAs that were reduced in abundance. Mettl1-KO also significantly reduces the translation efficiency of genes involved in elongated spermatid formation and sperm stability. Germ cell-specific expression of Mettl1 rescues disrupted m7G tRNA modification and tRNA abundance in Mettl1-KO testes but not in non-gonadal tissues. Ribosome stalling is much less detectable in non-gonadal tissues than in Mettl1-KO testes. These findings reveal a developmental role for m7G tRNA modification and indicate that m7G modification-dependent tRNA abundance differs among tissues.

Residue-Specific Epitope Mapping of the PD-1/Nivolumab Interaction Using X-ray Footprinting Mass Spectrometry.

X-ray footprinting coupled with mass spectrometry (XFMS) presents a novel approach in structural biology, offering insights into protein conformation and dynamics in the solution state. The interaction of the cancer-immunotherapy monoclonal antibody nivolumab with its antigen target PD-1 was used to showcase the utility of XFMS against the previously published crystal structure of the complex. Changes in side-chain solvent accessibility, as determined by the oxidative footprint of free PD-1 versus PD-1 bound to nivolumab, agree with the binding interface side-chain interactions reported from the crystal structure of the complex. The N-linked glycosylation sites of PD-1 were confirmed through an LC-MS/MS-based deglycosylation analysis of asparagine deamidation. In addition, subtle changes in side-chain solvent accessibility were observed in the C'D loop region of PD-1 upon complex formation with nivolumab.