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iLight2: A near‐infrared optogenetic tool for gene transcription with low background activation Protein Sci. (IF 8.0) Pub Date : 2024-04-22 Mikhail Baloban, Ludmila A. Kasatkina, Vladislav V. Verkhusha
Optogenetic tools (OTs) operating in the far‐red and near‐infrared (NIR) region offer advantages for light‐controlling biological processes in deep tissues and spectral multiplexing with fluorescent probes and OTs acting in the visible range. However, many NIR OTs suffer from background activation in darkness. Through shortening linkers, we engineered a novel NIR OT, iLight2, which exhibits a significantly
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Biophysical insights into the dimer formation of human Sirtuin 2 Protein Sci. (IF 8.0) Pub Date : 2024-04-22 Noa Suzuki, Tsuyoshi Konuma, Takahisa Ikegami, Satoko Akashi
Sirtuin 2 (SIRT2) is a class III histone deacetylase that is highly conserved from bacteria to mammals. We prepared and characterized the wild‐type (WT) and mutant forms of the histone deacetylase (HDAC) domain of human SIRT2 (hSIRT2) using various biophysical methods and evaluated their deacetylation activity. We found that WT hSIRT2 HDAC (residues 52–357) forms a homodimer in a concentration‐dependent
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The Phenix‐AlphaFold webservice: Enabling AlphaFold predictions for use in Phenix Protein Sci. (IF 8.0) Pub Date : 2024-04-22 Billy K. Poon, Thomas C. Terwilliger, Paul D. Adams
Advances in machine learning have enabled sufficiently accurate predictions of protein structure to be used in macromolecular structure determination with crystallography and cryo‐electron microscopy data. The Phenix software suite has AlphaFold predictions integrated into an automated pipeline that can start with an amino acid sequence and data, and automatically perform model‐building and refinement
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How hydrophobicity, side chains, and salt affect the dimensions of disordered proteins Protein Sci. (IF 8.0) Pub Date : 2024-04-12 Michael C. Baxa, Xiaoxuan Lin, Cedrick D. Mukinay, Srinivas Chakravarthy, Joseph R. Sachleben, Sarah Antilla, Nina Hartrampf, Joshua A. Riback, Isabelle A. Gagnon, Bradley L. Pentelute, Patricia L. Clark, Tobin R. Sosnick
Despite the generally accepted role of the hydrophobic effect as the driving force for folding, many intrinsically disordered proteins (IDPs), including those with hydrophobic content typical of foldable proteins, behave nearly as self‐avoiding random walks (SARWs) under physiological conditions. Here, we tested how temperature and ionic conditions influence the dimensions of the N‐terminal domain
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Lipid exchange of apolipoprotein A‐I amyloidogenic variants in reconstituted high‐density lipoprotein with artificial membranes Protein Sci. (IF 8.0) Pub Date : 2024-04-12 Yubexi Correa, Mathilde Ravel, Marie Imbert, Sarah Waldie, Luke Clifton, Ann Terry, Felix Roosen‐Runge, Jens O. Lagerstedt, Michael Moir, Tamim Darwish, Marité Cárdenas, Rita Del Giudice
High‐density lipoproteins (HDLs) are responsible for removing cholesterol from arterial walls, through a process known as reverse cholesterol transport. The main protein in HDL, apolipoprotein A‐I (ApoA‐I), is essential to this process, and changes in its sequence significantly alter HDL structure and functions. ApoA‐I amyloidogenic variants, associated with a particular hereditary degenerative disease
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Specific features of a scaffolding antibody light chain Protein Sci. (IF 8.0) Pub Date : 2024-04-12 Johanna Trommer, Florian Lesniowski, Johannes Buchner, Hristo L. Svilenov
The antigen‐binding sites in conventional antibodies are formed by hypervariable complementarity‐determining regions (CDRs) from both heavy chains (HCs) and light chains (LCs). A deviation from this paradigm is found in a subset of bovine antibodies that bind antigens via an ultra‐long CDR. The HCs bearing ultra‐long CDRs pair with a restricted set of highly conserved LCs that convey stability to the
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Engineering bio‐brick protein scaffolds for organizing enzyme assemblies Protein Sci. (IF 8.0) Pub Date : 2024-04-12 Alba Ledesma‐Fernandez, Susana Velasco‐Lozano, Pedro Campos‐Muelas, Ricardo Madrid, Fernando López‐Gallego, Aitziber L. Cortajarena
Enzyme scaffolding is an emerging approach for enhancing the catalytic efficiency of multi‐enzymatic cascades by controlling their spatial organization and stoichiometry. This study introduces a novel family of engineered SCAffolding Bricks, named SCABs, utilizing the consensus tetratricopeptide repeat (CTPR) domain for organized multi‐enzyme systems. Two SCAB systems are developed, one employing head‐to‐tail
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Retromer‐mediated recruitment of the WASH complex involves discrete interactions between VPS35, VPS29, and FAM21 Protein Sci. (IF 8.0) Pub Date : 2024-04-12 Miguel Romano‐Moreno, Elsa N. Astorga‐Simón, Adriana L. Rojas, Aitor Hierro
Endosomal trafficking ensures the proper distribution of lipids and proteins to various cellular compartments, facilitating intracellular communication, nutrient transport, waste disposal, and the maintenance of cell structure. Retromer, a peripheral membrane protein complex, plays an important role in this process by recruiting the associated actin‐polymerizing WASH complex to establish distinct sorting
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Proteasome‐dependent degradation of histone H1 subtypes is mediated by its C‐terminal domain Protein Sci. (IF 8.0) Pub Date : 2024-04-09 D. García‐Gomis, J. López, A. Calderón, M. Andrés, I. Ponte, A. Roque
Histone H1 is involved in chromatin compaction and dynamics. In human cells, the H1 complement is formed by different amounts of somatic H1 subtypes, H1.0‐H1.5 and H1X. The amount of each variant depends on the cell type, the cell cycle phase, and the time of development and can be altered in disease. However, the mechanisms regulating H1 protein levels have not been described. We have analyzed the
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Diverse crystalline protein scaffolds through metal‐dependent polymorphism Protein Sci. (IF 8.0) Pub Date : 2024-04-09 Mantas Liutkus, Ivan R. Sasselli, Adriana L. Rojas, Aitziber L. Cortajarena
As protein crystals are increasingly finding diverse applications as scaffolds, controlled crystal polymorphism presents a facile strategy to form crystalline assemblies with controllable porosity with minimal to no protein engineering. Polymorphs of consensus tetratricopeptide repeat proteins with varying porosity were obtained through co‐crystallization with metal salts, exploiting the innate metal
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Disentangling the formation, mechanism, and evolvement of the covalent methanesulfonyl fluoride acetylcholinesterase adduct: Insights into an aged‐like inactive complex susceptible to reactivation by a combination of nucleophiles Protein Sci. (IF 8.0) Pub Date : 2024-04-09 Jure Stojan, Alessandro Pesaresi, Anže Meden, Doriano Lamba
Chemical warfare nerve agents and pesticides, known as organophosphorus compounds inactivate cholinesterases (ChEs) by phosphorylating the serine hydroxyl group located at the active site of ChEs. Over the course of time, phosphorylation is followed by loss of an organophosphate‐leaving group and the bond with ChEs becomes irreversible, a process known as aging. Differently, structurally related irreversible
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Computational and experimental identification of keystone interactions in Ebola virus matrix protein VP40 dimer formation Protein Sci. (IF 8.0) Pub Date : 2024-04-09 Yogesh Narkhede, Roopashi Saxena, Tej Sharma, Jacob P. Conarty, Valentina Toro Ramirez, Balindile B. Motsa, Souad Amiar, Sheng Li, Prem P. Chapagain, Olaf Wiest, Robert V. Stahelin
The Ebola virus (EBOV) is a lipid‐enveloped virus with a negative sense RNA genome that can cause severe and often fatal viral hemorrhagic fever. The assembly and budding of EBOV is regulated by the matrix protein, VP40, which is a peripheral protein that associates with anionic lipids at the inner leaflet of the plasma membrane. VP40 is sufficient to form virus‐like particles (VLPs) from cells, which
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Myxococcus xanthus translesion DNA synthesis protein ImuA is an ATPase enhanced by DNA Protein Sci. (IF 8.0) Pub Date : 2024-04-09 Kristi Lichimo, Dana J. Sowa, Andriana Tetenych, Monica M. Warner, Caitlin Doubleday, Harman S. Dev, Catie Luck, Sara N. Andres
Translesion DNA synthesis pathways are necessary to ensure bacterial replication in the presence of DNA damage. Translesion DNA synthesis carried out by the PolV mutasome is well‐studied in Escherichia coli, but ~one third of bacteria use a functionally homologous protein complex, consisting of ImuA, ImuB, and ImuC (also called DnaE2). Numerous in vivo studies have shown that all three proteins are
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Disruption of Ca2+/calmodulin:KSR1 interaction lowers ERK activation Protein Sci. (IF 8.0) Pub Date : 2024-04-09 Louise Thines, Hyunbum Jang, Zhigang Li, Samar Sayedyahossein, Ryan Maloney, Ruth Nussinov, David B. Sacks
KSR1, a key scaffold protein for the MAPK pathway, facilitates ERK activation upon growth factor stimulation. We recently demonstrated that KSR1 binds the Ca2+‐binding protein calmodulin (CaM), thereby providing an intersection between KSR1‐mediated and Ca2+ signaling. In this study, we set out to generate a KSR1 point mutant with reduced Ca2+/CaM binding in order to unravel the functional implications
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Transient interdomain interactions in free USP14 shape its conformational ensemble Protein Sci. (IF 8.0) Pub Date : 2024-04-08 Johannes Salomonsson, Björn Wallner, Linda Sjöstrand, Pádraig D'Arcy, Maria Sunnerhagen, Alexandra Ahlner
The deubiquitinase (DUB) ubiquitin‐specific protease 14 (USP14) is a dual domain protein that plays a regulatory role in proteasomal degradation and has been identified as a promising therapeutic target. USP14 comprises a conserved USP domain and a ubiquitin‐like (Ubl) domain separated by a 25‐residue linker. The enzyme activity of USP14 is autoinhibited in solution, but is enhanced when bound to the
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Inhibitory protein–protein interactions of the SIRT1 deacetylase are choreographed by post‐translational modification Protein Sci. (IF 8.0) Pub Date : 2024-03-27 Troy C. Krzysiak, You‐Jin Choi, Yong Joon Kim, Yunhan Yang, Christopher DeHaven, Lariah Thompson, Ryan Ponticelli, Mara M. Mermigos, Laurel Thomas, Andrea Marquez, Ian Sipula, Jongsook Kim Kemper, Michael Jurczak, Gary Thomas, Angela M. Gronenborn
Regulation of SIRT1 activity is vital to energy homeostasis and plays important roles in many diseases. We previously showed that insulin triggers the epigenetic regulator DBC1 to prime SIRT1 for repression by the multifunctional trafficking protein PACS‐2. Here, we show that liver DBC1/PACS‐2 regulates the diurnal inhibition of SIRT1, which is critically important for insulin‐dependent switch in fuel
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MA‐PEP: A novel anticancer peptide prediction framework with multimodal feature fusion based on attention mechanism Protein Sci. (IF 8.0) Pub Date : 2024-03-27 Xiao Liang, Haochen Zhao, Jianxin Wang
AntiCancer Peptides (ACPs) have emerged as promising therapeutic agents for cancer treatment. The time‐consuming and costly nature of wet‐lab discriminatory methods has spurred the development of various machine learning and deep learning‐based ACP classification methods. Nonetheless, current methods encountered challenges in efficiently integrating features from various peptide modalities, thereby
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KCD: A prediction web server of knowledge‐based circular dichroism Protein Sci. (IF 8.0) Pub Date : 2024-03-27 Damián Jacinto‐Méndez, Carmen Giovana Granados‐Ramírez, Mauricio D. Carbajal‐Tinoco
We present a web server that predicts the far‐UV circular dichroism (CD) spectra of proteins by utilizing their three‐dimensional (3D) structures from the Protein Data Bank (PDB). The main algorithm is based on the classical theory of optical activity together with a set of atomic complex polarizabilities, which are obtained from the analysis of a series of synchrotron radiation CD spectra and their
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Protein structure–function continuum model: Emerging nexuses between specificity, evolution, and structure Protein Sci. (IF 8.0) Pub Date : 2024-03-27 Munishwar Nath Gupta, Vladimir N. Uversky
The rationale for replacing the old binary of structure–function with the trinity of structure, disorder, and function has gained considerable ground in recent years. A continuum model based on the expanded form of the existing paradigm can now subsume importance of both conformational flexibility and intrinsic disorder in protein function. The disorder is actually critical for understanding the protein–protein
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Role of UDP‐N‐acetylmuramic acid in the regulation of MurA activity revealed by molecular dynamics simulations Protein Sci. (IF 8.0) Pub Date : 2024-03-27 Maycon Vinicius Damasceno de Oliveira, Kauê S. da Costa, José Rogério A. Silva, Jerônimo Lameira, Anderson H. Lima
The peptidoglycan biosynthesis pathway plays a vital role in bacterial cells, and facilitates peptidoglycan layer formation, a fundamental structural component of the bacterial cell wall. The enzymes in this pathway are candidates for antibiotic development, as most do not have mammalian homologues. The UDP‐N‐acetylglucosamine (UNAG) enolpyruvyl transferase enzyme (MurA) in the peptidoglycan pathway
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Conserved proline residues prevent dimerization and aggregation in the β‐lactamase BlaC Protein Sci. (IF 8.0) Pub Date : 2024-03-27 A. Chikunova, M. P. Manley, C. N. Heijjer, C. S. Drenth, A. J. Cramer‐Blok, M. Ud Din Ahmad, A. Perrakis, M. Ubbink
Evolution leads to conservation of amino acid residues in protein families. Conserved proline residues are usually considered to ensure the correct folding and to stabilize the three‐dimensional structure. Surprisingly, proline residues that are highly conserved in class A β‐lactamases were found to tolerate various substitutions without large losses in enzyme activity. We investigated the roles of
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Design of a symmetry‐broken tetrahedral protein cage by a method of internal steric occlusion Protein Sci. (IF 8.0) Pub Date : 2024-03-27 Nika Gladkov, Elena A. Scott, Kyle Meador, Eric J. Lee, Arthur D. Laganowsky, Todd O. Yeates, Roger Castells‐Graells
Methods in protein design have made it possible to create large and complex, self‐assembling protein cages with diverse applications. These have largely been based on highly symmetric forms exemplified by the Platonic solids. Prospective applications of protein cages would be expanded by strategies for breaking the designed symmetry, for example, so that only one or a few (instead of many) copies of
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Viral envelope proteins fused to multiple distinct fluorescent reporters to probe receptor binding Protein Sci. (IF 8.0) Pub Date : 2024-03-27 Ilhan Tomris, Roosmarijn van der Woude, Rebeca de Paiva Froes Rocha, Alba Torrents de la Peña, Andrew B. Ward, Robert P. de Vries
Enveloped viruses carry one or multiple proteins with receptor‐binding functionalities. Functional receptors can be glycans, proteinaceous, or both; therefore, recombinant protein approaches are instrumental in attaining new insights regarding viral envelope protein receptor‐binding properties. Visualizing and measuring receptor binding typically entails antibody detection or direct labeling, whereas
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Integrating the analysis of human biopsies using post‐translational modifications proteomics Protein Sci. (IF 8.0) Pub Date : 2024-03-27 Sonali Bhardwaj, Mitchell Bulluss, Ana D'Aubeterre, Afshin Derakhshani, Regan Penner, MaryAnn Mahajan, Vinit B. Mahajan, Antoine Dufour
Proteome diversities and their biological functions are significantly amplified by post‐translational modifications (PTMs) of proteins. Shotgun proteomics, which does not typically survey PTMs, provides an incomplete picture of the complexity of human biopsies in health and disease. Recent advances in mass spectrometry‐based proteomic techniques that enrich and study PTMs are helping to uncover molecular
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Identification of a novel DNA repair inhibitor using an in silico driven approach shows effective combinatorial activity with genotoxic agents against multidrug‐resistant Escherichia coli Protein Sci. (IF 8.0) Pub Date : 2024-03-27 Lorenzo Bernacchia, Antoine Paris, Arya Gupta, Robert J. Charman, Jake McGreig, Mark N. Wass, Neil M. Kad
Increasing antimicrobial drug resistance represents a global existential threat. Infection is a particular problem in immunocompromised individuals, such as patients undergoing cancer chemotherapy, due to the targeting of rapidly dividing cells by antineoplastic agents. We recently developed a strategy that targets bacterial nucleotide excision DNA repair (NER) to identify compounds that act as antimicrobial
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Unraveling the underlying mechanisms of reduced amyloidogenic properties in human calcitonin via double mutations Protein Sci. (IF 8.0) Pub Date : 2024-03-24 Yu‐Pei Chang, Pei‐Chun Pan, Ling‐Hsien Tu
The therapeutic efficacy of peptide‐based drugs is commonly hampered by the intrinsic propensity to aggregation. A notable example is human calcitonin (hCT), a peptide hormone comprising 32 amino acids, which is synthesized and secreted by thyroid gland parafollicular cells (C cells). This hormone plays a vital role in regulating blood calcium levels and upholding bone integrity. Despite its physiological
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Engineering quorum quenching acylases with improved kinetic and biochemical properties Protein Sci. (IF 8.0) Pub Date : 2024-03-23 Kitty Sompiyachoke, Mikael H. Elias
Many Gram‐negative bacteria use N‐acyl‐L‐homoserine lactone (AHL) signals to coordinate phenotypes such as biofilm formation and virulence factor production. Quorum‐quenching enzymes, such as AHL acylases, chemically degrade these molecules which prevents signal reception by bacteria and inhibits undesirable biofilm‐related traits. These capabilities make acylases appealing candidates for controlling
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Adaptable SEC‐SAXS data collection for higher quality structure analysis in solution Protein Sci. (IF 8.0) Pub Date : 2024-03-23 Tsutomu Matsui, Ivan Rajkovic, Blaine H. M. Mooers, Ping Liu, Thomas M. Weiss
The two major challenges in synchrotron size‐exclusion chromatography coupled in‐line with small‐angle x‐ray scattering (SEC‐SAXS) experiments are the overlapping peaks in the elution profile and the fouling of radiation‐damaged materials on the walls of the sample cell. In recent years, many post‐experimental analyses techniques have been developed and applied to extract scattering profiles from these
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Proinsulin folding and trafficking defects trigger a common pathological disturbance of endoplasmic reticulum homeostasis Protein Sci. (IF 8.0) Pub Date : 2024-03-21 Anoop Arunagiri, Maroof Alam, Leena Haataja, Hassan Draz, Bashiyer Alasad, Praveen Samy, Nadeed Sadique, Yue Tong, Ying Cai, Hadis Shakeri, Federica Fantuzzi, Hazem Ibrahim, Insook Jang, Vaibhav Sidarala, Scott A. Soleimanpour, Leslie S. Satin, Timo Otonkoski, Miriam Cnop, Pamela Itkin‐Ansari, Randal J. Kaufman, Ming Liu, Peter Arvan
Primary defects in folding of mutant proinsulin can cause dominant‐negative proinsulin accumulation in the endoplasmic reticulum (ER), impaired anterograde proinsulin trafficking, perturbed ER homeostasis, diminished insulin production, and β‐cell dysfunction. Conversely, if primary impairment of ER‐to‐Golgi trafficking (which also perturbs ER homeostasis) drives misfolding of nonmutant proinsulin—this
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Rational evolution for altering the ligand preference of estrogen receptor alpha Protein Sci. (IF 8.0) Pub Date : 2024-03-21 Roy Eerlings, Purvi Gupta, Xiao Yin Lee, Tien Nguyen, Sarah El Kharraz, Florian Handle, Elien Smeets, Lisa Moris, Wout Devlies, Bram Vandewinkel, Irina Thiry, Duy Tien Ta, Anton Gorkovskiy, Karin Voordeckers, Els Henckaerts, Vitor B. Pinheiro, Frank Claessens, Kevin J. Verstrepen, Arnout Voet, Christine Helsen
Estrogen receptor α is commonly used in synthetic biology to control the activity of genome editing tools. The activating ligands, estrogens, however, interfere with various cellular processes, thereby limiting the applicability of this receptor. Altering its ligand preference to chemicals of choice solves this hurdle but requires adaptation of unspecified ligand‐interacting residues. Here, we provide
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Disrupted protein interaction dynamics in a genetic neurodevelopmental disorder revealed by structural bioinformatics and genetic code expansion Protein Sci. (IF 8.0) Pub Date : 2024-03-21 Valerio Marino, Wanchana Phromkrasae, Michele Bertacchi, Paul Cassini, Krittalak Chakrabandhu, Daniele Dell'Orco, Michèle Studer
Deciphering the structural effects of gene variants is essential for understanding the pathophysiological mechanisms of genetic diseases. Using a neurodevelopmental disorder called Bosch‐Boonstra‐Schaaf Optic Atrophy Syndrome (BBSOAS) as a genetic disease model, we applied structural bioinformatics and Genetic Code Expansion (GCE) strategies to assess the pathogenic impact of human NR2F1 variants and
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Copper ion incorporation in α‐synuclein amyloids Protein Sci. (IF 8.0) Pub Date : 2024-03-21 Gulshan Walke, Ranjeet Kumar, Pernilla Wittung‐Stafshede
Copper ion dys‐homeostasis is linked to neurodegenerative diseases involving amyloid formation. Even if many amyloidogenic proteins can bind copper ions as monomers, little is known about copper interactions with the resulting amyloid fibers. Here, we investigate copper interactions with α‐synuclein, the amyloid‐forming protein in Parkinson's disease. Copper (Cu(II)) binds tightly to monomeric α‐synuclein
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DNA‐dependent phase separation by human SSB2 (NABP1/OBFC2A) protein points to adaptations to eukaryotic genome repair processes Protein Sci. (IF 8.0) Pub Date : 2024-03-21 Zoltán J. Kovács, Gábor M. Harami, János Pálinkás, Natalie Kuljanishvili, József Hegedüs, Hajnalka Harami‐Papp, Lamiya Mahmudova, Lana Khamisi, Gergely Szakács, Mihály Kovács
Single‐stranded DNA binding proteins (SSBs) are ubiquitous across all domains of life and play essential roles via stabilizing and protecting single‐stranded (ss) DNA as well as organizing multiprotein complexes during DNA replication, recombination, and repair. Two mammalian SSB paralogs (hSSB1 and hSSB2 in humans) were recently identified and shown to be involved in various genome maintenance processes
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Oligomerization mediated by the D2 domain of DTX3L is critical for DTX3L‐PARP9 reading function of mono‐ADP‐ribosylated androgen receptor Protein Sci. (IF 8.0) Pub Date : 2024-03-21 Carlos Vela‐Rodríguez, Chunsong Yang, Heli I. Alanen, Rebeka Eki, Tarek A. Abbas, Mirko M. Maksimainen, Tuomo Glumoff, Ramona Duman, Armin Wagner, Bryce M. Paschal, Lari Lehtiö
Deltex proteins are a family of E3 ubiquitin ligases that encode C‐terminal RING and DTC domains that mediate interactions with E2 ubiquitin‐conjugating enzymes and recognize ubiquitination substrates. DTX3L is unique among the Deltex proteins based on its N‐terminal domain architecture. The N‐terminal D1 and D2 domains of DTX3L mediate homo‐oligomerization, and the D3 domain interacts with PARP9,
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Efficient and economic protein labeling for NMR in mammalian expression systems: Application to a preT‐cell and T‐cell receptor protein Protein Sci. (IF 8.0) Pub Date : 2024-03-21 Robert J. Mallis, Jonathan J. Lee, Arjen Van den Berg, Kristine N. Brazin, Thibault Viennet, Jonathan Zmuda, Melissa Cross, Denitsa Radeva, Ricard Rodriguez‐Mias, Judit Villén, Vladimir Gelev, Ellis L. Reinherz, Haribabu Arthanari
Protein nuclear magnetic resonance (NMR) spectroscopy relies on the ability to isotopically label polypeptides, which is achieved through heterologous expression in various host organisms. Most commonly, Escherichia coli is employed by leveraging isotopically substituted ammonium and glucose to uniformly label proteins with 15N and 13C, respectively. Moreover, E. coli can grow and express proteins
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Engineering a monobody specific to monomeric Cu/Zn‐superoxide dismutase associated with amyotrophic lateral sclerosis Protein Sci. (IF 8.0) Pub Date : 2024-03-21 Hiroshi Amesaka, Mizuho Hara, Yuki Sakai, Atsuko Shintani, Kaori Sue, Tomoyuki Yamanaka, Shun‐ichi Tanaka, Yoshiaki Furukawa
Misfolding of mutant Cu/Zn‐superoxide dismutase (SOD1) has been implicated in familial form of amyotrophic lateral sclerosis (ALS). A natively folded SOD1 forms a tight homodimer, and the dimer dissociation has been proposed to trigger the oligomerization/aggregation of SOD1. Besides increasing demand for probes allowing the detection of monomerized forms of SOD1 in various applications, the development
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Unraveling paralog‐specific Notch signaling through thermodynamics of ternary complex formation and transcriptional activation of chimeric receptors Protein Sci. (IF 8.0) Pub Date : 2024-03-21 Kristen M. Ramsey, Doug Barrick
Notch signaling in humans is mediated by four paralogous receptors that share conserved architectures and possess overlapping, yet non‐redundant functions. The receptors share a canonical activation pathway wherein upon extracellular ligand binding, the Notch intracellular domain (NICD) is cleaved from the membrane and translocates to the nucleus where its N‐terminal RBP‐j‐associated molecule (RAM)
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Liquid–liquid phase separation of α‐synuclein is highly sensitive to sequence complexity Protein Sci. (IF 8.0) Pub Date : 2024-03-21 Anindita Mahapatra, Robert W. Newberry
The Parkinson's‐associated protein α‐synuclein (α‐syn) can undergo liquid–liquid phase separation (LLPS), which typically leads to the formation of amyloid fibrils. The coincidence of LLPS and amyloid formation has complicated the identification of the molecular determinants unique to LLPS of α‐syn. Moreover, the lack of strategies to selectively perturb LLPS makes it difficult to dissect the biological
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Structural dynamics and functional cooperativity of human NQO1 by ambient temperature serial crystallography and simulations Protein Sci. (IF 8.0) Pub Date : 2024-03-19 Alice Grieco, Sergio Boneta, José A. Gavira, Angel L. Pey, Shibom Basu, Julien Orlans, Daniele de Sanctis, Milagros Medina, Jose Manuel Martin‐Garcia
The human NQO1 (hNQO1) is a flavin adenine nucleotide (FAD)‐dependent oxidoreductase that catalyzes the two‐electron reduction of quinones to hydroquinones, being essential for the antioxidant defense system, stabilization of tumor suppressors, and activation of quinone‐based chemotherapeutics. Moreover, it is overexpressed in several tumors, which makes it an attractive cancer drug target. To decipher
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Engineering diaryl alcohol dehydrogenase KpADH reveals importance of retaining hydration shell in organic solvent tolerance Protein Sci. (IF 8.0) Pub Date : 2024-03-19 Lu Zhang, Wei Dai, Shuo Rong, Ulrich Schwaneberg, Guochao Xu, Ye Ni
Alcohol dehydrogenases (ADHs) are synthetically important biocatalysts for the asymmetric synthesis of chiral alcohols. The catalytic performance of ADHs in the presence of organic solvents is often important since most prochiral ketones are highly hydrophobic. Here, the organic solvent tolerance of KpADH from Kluyveromyces polyspora was semi‐rationally evolved. Using tolerant variants obtained, meticulous
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Sensitive detection of SARS‐CoV‐2 main protease 3CLpro with an engineered ribonuclease zymogen Protein Sci. (IF 8.0) Pub Date : 2024-03-19 Evans C. Wralstad, Ronald T. Raines
Alongside vaccines and antiviral therapeutics, diagnostic tools are a crucial aid in combating the COVID‐19 pandemic caused by the etiological agent SARS‐CoV‐2. All common assays for infection rely on the detection of viral sub‐components, including structural proteins of the virion or fragments of the viral genome. Selective pressure imposed by human intervention of COVID‐19 can, however, induce viral
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Calcium mediated static and dynamic allostery in S100A12: Implications for target recognition by S100 proteins Protein Sci. (IF 8.0) Pub Date : 2024-03-19 Qian Wang, Christopher DiForte, Aleksey Aleshintsev, Gianna Elci, Shibani Bhattacharya, Angelo Bongiorno, Rupal Gupta
Structure and functions of S100 proteins are regulated by two distinct calcium binding EF hand motifs. In this work, we used solution‐state NMR spectroscopy to investigate the cooperativity between the two calcium binding sites and map the allosteric changes at the target binding site. To parse the contribution of the individual calcium binding events, variants of S100A12 were designed to selectively
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Biochemical and structural characterization reveals Rv3400 codes for β‐phosphoglucomutase in Mycobacterium tuberculosis Protein Sci. (IF 8.0) Pub Date : 2024-03-19 Latika Singh, Subramanian Karthikeyan, Krishan Gopal Thakur
Mycobacterium tuberculosis (Mtb) adapt to various host environments and utilize a variety of sugars and lipids as carbon sources. Among these sugars, maltose and trehalose, also play crucial role in bacterial physiology and virulence. However, some key enzymes involved in trehalose and maltose metabolism in Mtb are not yet known. Here we structurally and functionally characterized a conserved hypothetical
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Classifying protein kinase conformations with machine learning Protein Sci. (IF 8.0) Pub Date : 2024-03-19 Ivan Reveguk, Thomas Simonson
Protein kinases are key actors of signaling networks and important drug targets. They cycle between active and inactive conformations, distinguished by a few elements within the catalytic domain. One is the activation loop, whose conserved DFG motif can occupy DFG‐in, DFG‐out, and some rarer conformations. Annotation and classification of the structural kinome are important, as different conformations
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Cell surface β‐lactamase recruitment: A facile selection to identify protein–protein interactions Protein Sci. (IF 8.0) Pub Date : 2024-03-19 Jordan A. Hinmon, Jade M. King, Latrina J. Mayo, Cierra R. Faries, Ya'hnis T. Lockett, David W. Crawford, Patrick C. Beardslee, Alexander Hendricks, Brian R. McNaughton
Protein–protein interactions (PPIs) are central to many cellular processes, and the identification of novel PPIs is a critical step in the discovery of protein therapeutics. Simple methods to identify naturally existing or laboratory evolved PPIs are therefore valuable research tools. We have developed a facile selection that links PPI‐dependent β‐lactamase recruitment on the surface of Escherichia
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RrA, an enzyme from Rhodospirillum rubrum, is a prototype of a new family of short‐chain L‐asparaginases Protein Sci. (IF 8.0) Pub Date : 2024-03-19 Di Zhang, Honorata Czapinska, Matthias Bochtler, Alexander Wlodawer, Jacek Lubkowski
L‐Asparaginases (ASNases) catalyze the hydrolysis of L‐Asn to L‐Asp and ammonia. Members of the ASNase family are used as drugs in the treatment of leukemia, as well as in the food industry. The protomers of bacterial ASNases typically contain 300–400 amino acids (typical class 1 ASNases). In contrast, the chain of ASNase from Rhodospirillum rubrum, reported here and referred to as RrA, consists of
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Design of AsLOV2 domain as a carrier of light‐induced dissociable FMN photosensitizer Protein Sci. (IF 8.0) Pub Date : 2024-03-19 Kristína Felčíková, Andrej Hovan, Marek Polák, Dmitry S. Loginov, Veronika Holotová, Carlos Díaz, Tibor Kožár, One‐Sun Lee, Rastislav Varhač, Petr Novák, Gregor Bánó, Erik Sedlák
Flavin mononucleotide (FMN) is a highly efficient photosensitizer (PS) yielding singlet oxygen (1O2). However, its 1O2 production efficiency significantly decreases upon isoalloxazine ring encapsulation into the protein matrix in genetically encoded photosensitizers (GEPS). Reducing isoalloxazine ring interactions with surrounding amino acids by protein engineering may increase 1O2 production efficiency
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Xplor‐NIH: Better parameters and protocols for NMR protein structure determination Protein Sci. (IF 8.0) Pub Date : 2024-03-19 Guillermo A. Bermejo, Nico Tjandra, G. Marius Clore, Charles D. Schwieters
The present work describes an update to the protein covalent geometry and atomic radii parameters in the Xplor‐NIH biomolecular structure determination package. In combination with an improved treatment of selected non‐bonded interactions between atoms three bonds apart, such as those involving methyl hydrogens, and a previously developed term that affects the system's gyration volume, the new parameters
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Transmembrane conformation of the envelope protein of an alpha coronavirus, NL63 Protein Sci. (IF 8.0) Pub Date : 2024-03-19 Iva Sučec, Yanina Pankratova, Mriganka Parasar, Mei Hong
The envelope (E) proteins of coronaviruses (CoVs) form cation‐conducting channels that are associated with the pathogenicity of these viruses. To date, high‐resolution structural information about these viroporins is limited to the SARS‐CoV E protein. To broaden our structural knowledge of other members of this family of viroporins, we now investigate the conformation of the E protein of the human
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Dual‐wield NTPases: A novel protein family mined from AlphaFold DB Protein Sci. (IF 8.0) Pub Date : 2024-03-19 Koya Sakuma, Ryotaro Koike, Motonori Ota
AlphaFold protein structure database (AlphaFold DB) archives a vast number of predicted models. We conducted systematic data mining against AlphaFold DB and discovered an uncharacterized P‐loop NTPase family. The structure of the protein family was surprisingly novel, showing an atypical topology for P‐loop NTPases, noticeable twofold symmetry, and two pairs of independent putative active sites. Our
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The dynamics of the flavin, NADPH, and active site loops determine the mechanism of activation of class B flavin‐dependent monooxygenases Protein Sci. (IF 8.0) Pub Date : 2024-03-19 Gustavo Pierdominici‐Sottile, Juliana Palma, María Leticia Ferrelli, Pablo Sobrado
Flavin‐dependent monooxygenases (FMOs) constitute a diverse enzyme family that catalyzes crucial hydroxylation, epoxidation, and Baeyer–Villiger reactions across various metabolic pathways in all domains of life. Due to the intricate nature of this enzyme family's mechanisms, some aspects of their functioning remain unknown. Here, we present the results of molecular dynamics computations, supplemented
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The structural landscape of the immunoglobulin fold by large‐scale de novo design Protein Sci. (IF 8.0) Pub Date : 2024-03-19 Jorge Roel‐Touris, Lourdes Carcelén, Enrique Marcos
De novo designing immunoglobulin‐like frameworks that allow for functional loop diversification shows great potential for crafting antibody‐like scaffolds with fully customizable structures and functions. In this work, we combined de novo parametric design with deep‐learning methods for protein structure prediction and design to explore the structural landscape of 7‐stranded immunoglobulin domains
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A cellulosomal double‐dockerin module from Clostridium thermocellum shows distinct structural and cohesin‐binding features Protein Sci. (IF 8.0) Pub Date : 2024-03-19 Chao Chen, Hongwu Yang, Sheng Dong, Cai You, Sarah Moraïs, Edward A. Bayer, Ya‐Jun Liu, Jinsong Xuan, Qiu Cui, Itzhak Mizrahi, Yingang Feng
Cellulosomes are intricate cellulose‐degrading multi‐enzymatic complexes produced by anaerobic bacteria, which are valuable for bioenergy development and biotechnology. Cellulosome assembly relies on the selective interaction between cohesin modules in structural scaffolding proteins (scaffoldins) and dockerin modules in enzymes. Although the number of tandem cohesins in the scaffoldins is believed
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Examining evolutionary scale modeling‐derived different‐dimensional embeddings in the antimicrobial peptide classification through a KNIME workflow Protein Sci. (IF 8.0) Pub Date : 2024-03-19 Karla L. Martínez‐Mauricio, César R. García‐Jacas, Greneter Cordoves‐Delgado
Molecular features play an important role in different bio‐chem‐informatics tasks, such as the Quantitative Structure–Activity Relationships (QSAR) modeling. Several pre‐trained models have been recently created to be used in downstream tasks, either by fine‐tuning a specific model or by extracting features to feed traditional classifiers. In this regard, a new family of Evolutionary Scale Modeling
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Assessing the mechanism of fast‐cycling cancer‐associated mutations of Rac1 small Rho GTPase Protein Sci. (IF 8.0) Pub Date : 2024-03-19 Angela Parise, Alessandra Magistrato
Rho‐GTPases proteins function as molecular switches alternating from an active to an inactive state upon Guanosine triphosphate (GTP) binding and hydrolysis to Guanosine diphosphate (GDP). Among them, Rac subfamily regulates cell dynamics, being overexpressed in distinct cancer types. Notably, these proteins are object of frequent cancer‐associated mutations at Pro29 (P29S, P29L, and P29Q). To assess
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Labile assembly of a tardigrade protein induces biostasis Protein Sci. (IF 8.0) Pub Date : 2024-03-19 S. Sanchez‐Martinez, K. Nguyen, S. Biswas, V. Nicholson, A. V. Romanyuk, J. Ramirez, S. Kc, A. Akter, C. Childs, E. K. Meese, E. T. Usher, G. M. Ginell, F. Yu, E. Gollub, M. Malferrari, F. Francia, G. Venturoli, E. W. Martin, F. Caporaletti, G. Giubertoni, S. Woutersen, S. Sukenik, D. N. Woolfson, A. S. Holehouse, T. C. Boothby
Tardigrades are microscopic animals that survive desiccation by inducing biostasis. To survive drying tardigrades rely on intrinsically disordered CAHS proteins, which also function to prevent perturbations induced by drying in vitro and in heterologous systems. CAHS proteins have been shown to form gels both in vitro and in vivo, which has been speculated to be linked to their protective capacity
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Engineering of IF1‐susceptive bacterial F1‐ATPase Protein Sci. (IF 8.0) Pub Date : 2024-03-19 Yuichiro C. Hatasaki, Ryohei Kobayashi, Ryo R. Watanabe, Mayu Hara, Hiroshi Ueno, Hiroyuki Noji
IF1, an inhibitor protein of mitochondrial ATP synthase, suppresses ATP hydrolytic activity of F1. One of the unique features of IF1 is the selective inhibition in mitochondrial F1 (MF1); it inhibits catalysis of MF1 but does not affect F1 with bacterial origin despite high sequence homology between MF1 and bacterial F1. Here, we aimed to engineer thermophilic Bacillus F1 (TF1) to confer the susceptibility
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Construction of a versatile fusion protein for targeted therapy and immunotherapy Protein Sci. (IF 8.0) Pub Date : 2024-03-19 Xiu‐Song Huang, Li‐Ting Yang, Ke Yang, Hang Zhou, Tuersunayi Abudureheman, Wei‐Wei Zheng, Kai‐Ming Chen, Cai‐Wen Duan
Antibody (Ab)‐based drugs have been widely used in targeted therapies and immunotherapies, leading to significant improvements in tumor therapy. However, the failure of Ab therapy due to the loss of target antigens or Ab modifications that affect its function limits its application. In this study, we expanded the application of antibodies (Abs) by constructing a fusion protein as a versatile tool for