Journal of Molecular Biology最新文献

{"title":"Assembly of the human multi-tRNA synthetase complex through leucine zipper motifs.","authors":"Dong Kyu Kim, Kayoung Lee, Beom Sik Kang","doi":"10.1016/j.jmb.2024.168865","DOIUrl":"https://doi.org/10.1016/j.jmb.2024.168865","url":null,"abstract":"<p><p>Aminoacyl-tRNA synthetases (ARSs) are responsible for the ligation of amino acids to their cognate tRNAs. In human, nine ARSs form a multi-tRNA synthetase complex (MSC) with three ARS-interacting multifunctional proteins (AIMPs). Among the components of MSC, arginyl-tRNA synthetase 1 (RARS1) and two AIMPs (AIMP1 and AIMP2) have leucine zipper (LZ) motifs, which they utilize for their assembly in an MSC. RARS1 and AIMP1 have two LZ motifs (LZ1 and LZ2) in their N-terminus, respectively, while AIMP2 has one LZ motif between its lysyl-tRNA synthetase 1 (KARS1)-binding motif and glutathione transferase-homology domain, which links aspartyl-tRNA synthetase 1 (DARS1). Although the interaction mode between AIMP1 and RARS1, which also binds glutaminyl-tRNA synthetase 1 (QARS1), has been revealed, the mode in the presence of AIMP2 is still ambiguous since AIMP2 is known to not only bind to AIMP1 but also form a homodimer through its LZ. Here, we determined a crystal structure of the LZ complex of AIMP1 and AIMP2 and revealed the interaction mode of a heterotrimeric complex of RARS1, AIMP1, and AIMP2. The complex is established by a three-stranded coiled-coil structure with RARS1 LZ1, AIMP1 LZ1, and AIMP2 LZ and is completed with a two-stranded coiled-coil structure of RARS1 LZ2 and AIMP1 LZ2. In the human MSC, this heterotrimeric complex of RARS1, AIMP1, and AIMP2 allows for a subcomplex of fourteen protein molecules, in which two QARS1-RARS1-AIMP1-AIMP2-2×KARS1 complexes are linked separately to a dimeric DARS1.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"168865"},"PeriodicalIF":4.7,"publicationDate":"2024-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142611380","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Corrigendum to “The Role of ATG9 Vesicles in Autophagosome Biogenesis” [J. Mol. Biol. 436(15) (2024) 168489]","authors":"Elisabeth Holzer ,&nbsp;Sascha Martens ,&nbsp;Susanna Tulli","doi":"10.1016/j.jmb.2024.168849","DOIUrl":"10.1016/j.jmb.2024.168849","url":null,"abstract":"","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 23","pages":"Article 168849"},"PeriodicalIF":4.7,"publicationDate":"2024-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142611395","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structural studies on Mycobacterial NudC reveal a class of zinc independent NADH pyrophosphatase.","authors":"Lingyu Meng, Zhaojian Sun, Yulong Zhang, Yan Dong, Xiaoan Du, Yujian Wu, Yuan Yuan, Yirong Sun, Yong Xu, Huaiwei Ding, Jinsong Liu, Jinxin Xu","doi":"10.1016/j.jmb.2024.168864","DOIUrl":"https://doi.org/10.1016/j.jmb.2024.168864","url":null,"abstract":"<p><p>Non-tuberculous mycobacteria (NTM) have emerged as an increasing threat to public health, due to the extreme antibiotic resistance. NADH pyrophosphatase (NudC) was proposed involving in mycobacterial resistance to the first line anti-tubercular drug isoniazid (INH) or its analog ethionamide (ETH), by hydrolyzing their NAD modified active forms (NAD-INH and NAD-ETH). In this study, we performed enzymatic and structural studies on NudC from M. abscessus (NudC_Mab), which is highly resistant to isoniazid and emerging as the most worrisome NTM. We determined the crystal structures of NudC_Mab in apo form, substrate NAD-bound form and product AMP-bound form. We observed the mode for the Nudix motif of NudC_Mab capturing the pyrophosphate group of NAD mediated by three divalent cation ions, which provides details for understanding the mechanism on NudC hydrolyzing NAD(H) or NAD-capped substrate. Interestingly, our structures revealed a novel subclass NudC from mycobacteria characterized by a unique arginine residue on the conserved QPWPFPxS motif, as well as a unique tower domain that replaces a well-defined zinc-binding motif in E.coli NudC and catalytic domain of mammalian Nudt12. Thus, our structural studies on NudC_Mab not only present a class of zinc independent NADH pyrophosphatase in mycobacteria, but also may facilitate the design of NudC inhibitors for the treatment of mycobacteria infections in combination with INH or ETH.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"168864"},"PeriodicalIF":4.7,"publicationDate":"2024-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142611386","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Revealing Missing Protein–Ligand Interactions Using AlphaFold Predictions","authors":"Nahuel Escobedo ,&nbsp;Tadeo Saldaño ,&nbsp;Juan Mac Donagh ,&nbsp;Luciana Rodriguez Sawicki ,&nbsp;Nicolas Palopoli ,&nbsp;Sebastian Fernandez Alberti ,&nbsp;Maria Silvina Fornasari ,&nbsp;Gustavo Parisi","doi":"10.1016/j.jmb.2024.168852","DOIUrl":"10.1016/j.jmb.2024.168852","url":null,"abstract":"<div><div>Protein–ligand interactions represent an essential step to understand the bases of molecular recognition, an intense field of research in many scientific areas. Structural biology has played a central role in unveiling protein–ligand interactions, but current techniques are still not able to reliably describe the interactions of ligands with highly flexible regions. In this work, we explored the capacity of AlphaFold2 (AF2) to estimate the presence of interactions between ligands and residues belonging to disordered regions. As these interactions are missing in the crystallographic-derived structures, we called them “ghost interactions”. Using a set of protein structures experimentally obtained after AF2 was trained, we found that the obtained models are good predictors of regions associated with order–disorder transitions. Additionally, we found that AF2 predicts residues making ghost interactions with ligands, which are mostly buried and show differential evolutionary conservation with the rest of the residues located in the flexible region. Our findings could fuel current areas of research that consider, given their biological relevance and their involvement in diseases, intrinsically disordered proteins as potentially valuable targets for drug development.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 23","pages":"Article 168852"},"PeriodicalIF":4.7,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142602664","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Pim1 is Critical in T-cell-independent B-cell Response and MAPK Activation in B Cells","authors":"Dongya Cui ,&nbsp;Yongguang Zhang ,&nbsp;Baijiao Zheng ,&nbsp;Liling Chen ,&nbsp;Jianhui Wei ,&nbsp;Danfeng Lin ,&nbsp;Miaohui Huang ,&nbsp;Hekang Du ,&nbsp;Qi Chen","doi":"10.1016/j.jmb.2024.168824","DOIUrl":"10.1016/j.jmb.2024.168824","url":null,"abstract":"<div><div>The Pim family consists of three members that encode a distinct class of highly conserved serine/threonine kinases. In this study, we generated and examined mice with hematopoiesis-specific deletion of Pim1 and bone marrow (BM) chimeric mice with B-cell-specific targeted deletion of Pim1. Pim1 was expressed at all stages of B-cell development and hematopoietic-specific deletion of Pim1 altered B-cell development in BM, spleen and peritoneal. However, Pim1 deficiency did not affect T-cell development. Studies of BM chimeric mice showed that Pim1 is required in a cell-intrinsic manner to maintain normal B-cell development. Pim1 deficiency led to significant changes in B cell antibody responses. Additionally, Pim1 deficiency resulted in reduced B cell receptor (BCR)-induced cell proliferation and cell cycle progression. Examination of the various BCR-activated signaling pathways in Pim1-deficient B cells reveals defective activation of mitogen-activated protein kinases (MAPKs), which are known to regulate genes involved in cell proliferation and survival. qRT-PCR analysis of BCR-engaged B cells from Pim1-deficient B cells revealed reduced expression of cyclin-dependent kinase (CDK) and cyclin genes, including CDK2, CCNB1 and CCNE1. In conclusion, Pim1 plays a crucial role in B-cell development and B cell activation.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 23","pages":"Article 168824"},"PeriodicalIF":4.7,"publicationDate":"2024-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142589676","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Nanobody Toolbox for Recognizing Distinct Epitopes on Cas9","authors":"Jack Boylan ,&nbsp;Rebecca A Shrem ,&nbsp;Isabel C. Vallecillo-Viejo ,&nbsp;Craig L. Duvall ,&nbsp;Brian E. Wadzinski ,&nbsp;Benjamin W. Spiller","doi":"10.1016/j.jmb.2024.168836","DOIUrl":"10.1016/j.jmb.2024.168836","url":null,"abstract":"<div><div>Cas9s and fusions of Cas9s have emerged as powerful tools for genetic manipulations. Fusions of Cas9 with other DNA editing enzymes have led to variants capable of single base editing and catalytically dead Cas9s have emerged as tools to specifically target desired regions of a genome. Here we describe the generation of a panel of nanobodies directed against three unique epitopes on <em>Streptococcus pyogenes</em> Cas9. The nanobodies were identified from a nanobody library derived from an alpaca that had been immunized with Cas9. The most potent binders recognize Cas9 and RNA bound Cas9 equally well and do not inhibit Cas9 cleavage of target DNA. These nanobodies bind non-overlapping epitopes as determined by ELISA based epitope binning experiments and mass photometry. We present the sequences of these clones and supporting biochemical data so the broader scientific community can access these reagents.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 23","pages":"Article 168836"},"PeriodicalIF":4.7,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142556804","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Translation Complex Profile Sequencing Allows Discrimination of Leaky Scanning and Reinitiation in Upstream Open Reading Frame-controlled Translation","authors":"Dmitri E. Andreev ,&nbsp;Jack A.S. Tierney ,&nbsp;Pavel V. Baranov","doi":"10.1016/j.jmb.2024.168850","DOIUrl":"10.1016/j.jmb.2024.168850","url":null,"abstract":"<div><div>Upstream open reading frames (uORFs) are a class of translated regions (translons) in mRNA 5′ leaders. uORFs are believed to be pervasive regulators of the translation of mammalian mRNAs. Some uORFs are highly repressive but others have little or no impact on downstream mRNA translation either due to inefficient recognition of their start codon(s) or/and due to efficient reinitiation after uORF translation. While experiments with uORF reporter constructs proved to be instrumental in the investigation of uORF-mediated mechanisms of translation control, they can have serious limitations as manipulations with uORF sequences can yield various artefacts. Here we propose a general approach for using translation complex profiling (TCP-seq) data for exploring uORF regulatory characteristics. Using several examples, we show how TCP-seq could be used to estimate both repressiveness and modes of action of individual uORFs. We demonstrate how this approach could be used to assess the mechanisms of uORF-mediated translation control in the mRNA of several human genes, including <em>EIF5</em>, <em>IFRD1</em>, <em>MDM2</em>, <em>MIEF1</em>, <em>PPP1R15B</em>, <em>TAF7,</em> and <em>UCP2</em>.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 23","pages":"Article 168850"},"PeriodicalIF":4.7,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142563952","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"HulaCCR1, a pump-like cation channelrhodopsin discovered in a lake microbiome","authors":"Shunki Takaramoto ,&nbsp;Shai Fainsod ,&nbsp;Takashi Nagata ,&nbsp;Andrey Rozenberg ,&nbsp;Oded Béjà ,&nbsp;Keiichi Inoue","doi":"10.1016/j.jmb.2024.168844","DOIUrl":"10.1016/j.jmb.2024.168844","url":null,"abstract":"<div><div>Channelrhodopsins are light-gated ion channels consisting of seven transmembrane helices and a retinal chromophore, which are used as popular optogenetic tools for modulating neuronal activity. Cation channelrhodopsins (CCRs), first recognized as the photoreceptors in the chlorophyte <em>Chlamydomonas reinhardtii</em>, have since been identified in diverse species of green algae, as well in other unicellular eukaryotes. The CCRs from non-chlorophyte species are commonly referred to as bacteriorhodopsin-like cation channelrhodopsins, or BCCRs, as most of them feature the three characteristic amino acid residues of the “DTD motif” in the third transmembrane helix (TM3 or helix C) matching the canonical DTD motif of the well-studied archaeal light-driven proton pump bacteriorhodopsin. Here, we report characterization of HulaCCR1, a novel BCCR identified through metatranscriptomic analysis of a unicellular eukaryotic community in Lake Hula, Israel. Interestingly, HulaCCR1 has an ETD motif in which the first residue of the canonical motif is substituted for glutamate. Electrophysiological measurements of the wild-type and a mutant with a DTD motif of HulaCCR1 suggest the critical role of the first glutamate in spectral tuning and channel gating. Additionally, HulaCCR1 exhibits long extensions at the N- and C-termini. Photocurrents recorded from a truncated variant without the signal peptide predicted at the N-terminus were diminished, and membrane localization of the truncated variant significantly decreased, indicating that the signal peptide is important for membrane trafficking of HulaCCR1. These characteristics of HulaCCR1 would be related to a new biological significance in the original unidentified species, distinct from those known for other BCCRs.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 23","pages":"Article 168844"},"PeriodicalIF":4.7,"publicationDate":"2024-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142542654","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The Hsp90 Molecular Chaperone as a Global Modifier of the Genotype-Phenotype-Fitness Map: An Evolutionary Perspective","authors":"José Aguilar-Rodríguez ,&nbsp;Christopher M. Jakobson ,&nbsp;Daniel F. Jarosz","doi":"10.1016/j.jmb.2024.168846","DOIUrl":"10.1016/j.jmb.2024.168846","url":null,"abstract":"<div><div>Global modifier genes influence the mapping of genotypes onto phenotypes and fitness through their epistatic interactions with genetic variants on a massive scale. The first such factor to be identified, Hsp90, is a highly conserved molecular chaperone that plays a central role in protein homeostasis. Hsp90 is a “hub of hubs” that chaperones proteins engaged in many key cellular and developmental regulatory networks. These clients, which are enriched in kinases, transcription factors, and E3 ubiquitin ligases, drive diverse cellular functions and are themselves highly connected. By contrast to many other hub proteins, the abundance and activity of Hsp90 changes substantially in response to shifting environmental conditions. As a result, Hsp90 modifies the functional impact of many genetic variants simultaneously in a manner that depends on environmental stress. Studies in diverse organisms suggest that this coupling between Hsp90 function and challenging environments exerts a substantial impact on what parts of the genome are visible to natural selection, expanding adaptive opportunities when most needed. In this Perspective, we explore the multifaceted role of Hsp90 as global modifier of the genotype-phenotype-fitness map as well as its implications for evolution in nature and the clinic.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 23","pages":"Article 168846"},"PeriodicalIF":4.7,"publicationDate":"2024-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142556806","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Conformational Differences in the Light Chain Constant Domain of Immunoglobulin G and Free Light Chain May Influence Proteolysis in AL Amyloidosis","authors":"Elena S. Klimtchuk ,&nbsp;Tatiana Prokaeva ,&nbsp;Brian H. Spencer ,&nbsp;Sherry Wong ,&nbsp;Shreya Ghosh ,&nbsp;Angela Urdaneta ,&nbsp;Gareth Morgan ,&nbsp;Thomas E. Wales ,&nbsp;Olga Gursky","doi":"10.1016/j.jmb.2024.168837","DOIUrl":"10.1016/j.jmb.2024.168837","url":null,"abstract":"<div><div>Immunoglobulin light chain amyloidosis (AL) is a life-threatening disease caused by the deposition of light chain (LC) and its fragments containing variable (V_L) and portions of constant (C_L) domains. AL patients feature either monoclonal free LCs (FLCs) circulating as covalent and noncovalent homodimers, or monoclonal immunoglobulin (Ig) wherein the LC and heavy chain (HC) form disulfide-linked heterodimers, or both. The role of full-length Ig in AL amyloidosis is unclear as prior studies focused on FLC or V_L domain. We used a mammalian cell-based expression system to generate four AL patient-derived full-length IgGs, two non-AL IgG controls, and six corresponding FLC proteins derived from an <em>IGLV6-57</em> germline precursor. Comparison of proteins’ secondary structure, thermal stability, proteolytic susceptibility, and disulfide link reduction suggested the importance of local <em>vs.</em> global conformational stability. Analysis of IgGs <em>vs.</em> corresponding FLCs using hydrogen–deuterium exchange mass spectrometry revealed major differences in the local conformation/dynamics of the C_L domain. In all IgGs <em>vs.</em> FLCs, segments containing β-strand and α-helix βA_C-αA_CB_C were more dynamic/exposed while segment βD_C-βE_C was less dynamic/exposed. Notably, these segments overlapped proteolysis-prone regions whose <em>in vivo</em> cleavage generates LC fragments found in AL deposits. Altogether, the results suggest that preferential cleavage in segments βA_C-αA_CB_C of FLC or βD_C-βE_C of LC in IgG helps generate amyloid protein precursors. We propose that protecting these segments using small-molecule stabilizers, which bind to the interfacial cavities C_L-C_L in FLC and/or C_L-C_H1 in IgG, is a potential therapeutic strategy to complement current approaches targeting V_L-V_L or V_L-C_L stabilization of LC dimer.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"436 23","pages":"Article 168837"},"PeriodicalIF":4.7,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142566743","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}