bims-indpro 2023-01-15 papers

bims-indpro

Biomed News

on Intrinsically disordered proteins

Issue of 2023–01–15
thirteen papers selected by
Sara Mingu, Johannes Gutenberg University

The deubiquitinase OTUD1 noncanonically suppresses Akt activation through its N-terminal intrinsically disordered region.
An intrinsically disordered antimicrobial peptide dendrimer from stereorandomized virtual screening.
Site-directed double monoubiquitination of the repeat domain of the amyloid-forming protein tau impairs self-assembly and coacervation.
Modeling of flexible membrane-bound biomolecular complexes for solution small-angle scattering.
The effect of KTKEGV repeat motif and intervening ATVA sequence on α-synuclein solubility and assembly.
The cynosure of CtBP: evolution of a bilaterian transcriptional corepressor.
Effects of the Jokela Type of Spinal Muscular Atrophy-Related G66V Mutation on the Structural Ensemble Characteristics of CHCHD10.
Conformational Search for the Building Block of Proteins Based on the Gradient Gravitational Search Algorithm (ConfGGS) Using Force Fields: CHARMM, AMBER, and OPLS-AA.
Decoding of the ubiquitin code for clearance of colliding ribosomes by the RQT complex.
Molecular Determinants of Fibrillation in a Viral Amyloidogenic Domain from Combined Biochemical and Biophysical Studies.
Structural and dynamical insights into SilE silver binding from combined analytical probes.
β-Sheet Structure Formation within Binary Blends of Two Spider Silk Related Peptides.
DeepPRObind: Modular deep learner that accurately predicts structure and disorder-annotated protein binding residues.

Cell Rep. 2022 Dec 28. pii: S2211-1247(22)01815-0. [Epub ahead of print]42(1): 111916

The deubiquitinase OTUD1 noncanonically suppresses Akt activation through its N-terminal intrinsically disordered region.

Guanlan Fan, Fan Wang, Yurou Chen, Qian Zheng, Jie Xiong, Qiongying Lv, Kejia Wu, Jiaqiang Xiong, Lei Wei, Dongqing Li, Jiachen Zhang, Wei Zhang, Feng Li.

  Akt is commonly activated and serves as a valuable target in human cancer. In this study, OTUD1 is identified as an Akt-associated protein and is downregulated upon Akt activation. Ectopic OTUD1 inhibits Akt phosphorylation; however, its deubiquitinase activity contributes only slightly to this effect. A short peptide (OUN-36) located in the OTUD1 N-terminal intrinsically disordered region strongly binds to the Akt PH domain. The residues in the PH domain, which are required for PtdIns(3,4,5)P3 recognition, are also essential for OUN-36 binding. OUN-36 preferentially inhibits Akt-hyperactive tumor cells' proliferation and interferes with Akt cell membrane localization, presumably by disrupting PH domain-PIP3 interaction. Importantly, OUN-36-based therapy efficiently abrogates Akt feedback reactivation in response to MK-2206 treatment and sensitizes cancer cells to chemotherapy and immunotherapy. We therefore show a mechanism by which OTUD1 modulates Akt activity and suggest a potential peptide-based cancer therapeutic strategy implemented by targeting the Akt PH domain.

Keywords:  Akt signaling pathway; CP: Cancer; CP: Molecular biology; targeted therapies; tumour-suppressor protein OTUD1

DOI:  https://doi.org/10.1016/j.celrep.2022.111916
Cell Rep Phys Sci. 2022 Dec 21. 3(12): 101161

An intrinsically disordered antimicrobial peptide dendrimer from stereorandomized virtual screening.

Xingguang Cai, Markus Orsi, Alice Capecchi, Thilo Köhler, Christian van Delden, Sacha Javor, Jean-Louis Reymond.

  Membrane-disruptive amphiphilic antimicrobial peptides behave as intrinsically disordered proteins by being unordered in water and becoming α-helical in contact with biological membranes. We recently discovered that synthesizing the α-helical antimicrobial peptide dendrimer L-T25 ((KL)8(KKL)4(KLL)2 KKLL) using racemic amino acids to form stereorandomized sr-T25, an analytically pure mixture of all possible diastereoisomers of L-T25, preserved antibacterial activity but abolished hemolysis and cytotoxicity, pointing to an intrinsically disordered antibacterial conformation and an α-helical cytotoxic conformation. In this study, to identify non-toxic intrinsically disordered homochiral antimicrobial peptide dendrimers (AMPDs), we surveyed sixty-three sr-analogs of sr-T25 selected by virtual screening. One of the analogs, sr-X18 ((KL)8(KLK)4(KLL)2 KLLL), lost antibacterial activity as L-enantiomer and became hemolytic due to α-helical folding. By contrast, the L- and D-enantiomers of sr-X22 ((KL)8(KL)4(KKLL)2 KLKK) were equally antibacterial, non-hemolytic, and non-toxic, implying an intrinsically disordered bioactive conformation. Screening stereorandomized libraries may be generally useful to identify or optimize intrinsically disordered bioactive peptides.

Keywords:  antimicrobial peptides; dendrimers; intrinsically disordered proteins; membrane disruption; multi-drug resistant bacteria; multidrug resistance; solid-phase peptide synthesis; stereorandomization; virtual screening

DOI:  https://doi.org/10.1016/j.xcrp.2022.101161
Bioorg Chem. 2023 Jan 07. pii: S0045-2068(23)00007-X. [Epub ahead of print]132 106347

Site-directed double monoubiquitination of the repeat domain of the amyloid-forming protein tau impairs self-assembly and coacervation.

Daniele Trivellato, Fulvio Floriani, Carlo Giorgio Barracchia, Francesca Munari, Mariapina D'Onofrio, Michael Assfalg.

  In Alzheimer's disease and related disorders called tauopathies, the microtubule-associated protein tau accumulates in the brain in the form of amyloid-like supramolecular filaments. As an intrinsically disordered protein, tau undergoes many post-translational modifications, including ubiquitination. Alterations to the levels of ubiquitination of tau have been observed at various stages of neurodegenerative conditions. We focus on proteoform-specific interrogations to obtain mechanistic insight into the effects of ubiquitination on disease-related conformational transitions of tau. Single and double ubiquitination of tau at residues Lys311 and Lys317 is strongly associated with pathological conditions. In this study, we leveraged disulfide-directed chemistry to install ubiquitin at one or both of those positions in the isolated microtubule-binding repeat domain of tau. We obtained homogeneously modified tau proteins and observed that they retained disordered character in solution. We found that ubiquitination in position 317 (with or without ubiquitination in position 311) impaired the formation of ordered fibrillar structures via oligomeric intermediates. Since the transition to fibrillar species may proceed via an alternative condensation pathway involving liquid droplet intermediates, we further tested the ability of the ubiquitinated proteoforms to phase separate. Single monoubiquitinated tau species were able to coacervate, however no liquid droplets were observed for the double ubiquitinated form. Taken together, the data indicate that double ubiquitination in the third repeat of tau disfavors the formation of amyloid aggregates by distinct mechanisms, suggesting that the presence of ubiquitinated residues 311 and 317 in insoluble tau may result from modifications in advanced stages of aggregation. These findings contribute to our understanding of the influence of site-specific ubiquitination on the pathological conformational transitions of a prototypical intrinsically disordered protein.

Keywords:  Amyloid; Biomolecular condensate; Chemoselective ligation; Neurodegeneration; Post-translational modification; Protein aggregation; Tau protein; Ubiquitination

DOI:  https://doi.org/10.1016/j.bioorg.2023.106347
J Colloid Interface Sci. 2022 Dec 10. pii: S0021-9797(22)02169-5. [Epub ahead of print]635 611-621

Modeling of flexible membrane-bound biomolecular complexes for solution small-angle scattering.

Abigail Barclay, Birthe B Kragelund, Lise Arleth, Martin Cramer Pedersen.

  Recent advances in protein expression protocols, sample handling, and experimental set up of small-angle scattering experiments have allowed users of the technique to structurally investigate biomolecules of growing complexity and structural disorder. Notable examples include intrinsically disordered proteins, multi-domain proteins and membrane proteins in suitable carrier systems. Here, we outline a modeling scheme for calculating the scattering profiles from such complex samples. This kind of modeling is necessary for structural information to be refined from the corresponding data. The scheme bases itself on a hybrid of classical form factor based modeling and the well-known spherical harmonics-based formulation of small-angle scattering amplitudes. Our framework can account for flexible domains alongside other structurally elaborate components of the molecular system in question. We demonstrate the utility of this modeling scheme through a recent example of a structural model of the growth hormone receptor membrane protein in a phospholipid bilayer nanodisc which is refined against experimental SAXS data. Additionally we investigate how the scattering profiles from the complex would appear under different scattering contrasts. For each contrast situation we discuss what structural information is contained and the related consequences for modeling of the data.

Keywords:  Intrinsically disordered protein; Membrane protein; Modeling; Phospholipid bilayer nanodisc; Small-angle X-ray scattering; Small-angle neutron scattering

DOI:  https://doi.org/10.1016/j.jcis.2022.12.024
J Neurochem. 2023 Jan 10.

The effect of KTKEGV repeat motif and intervening ATVA sequence on α-synuclein solubility and assembly.

Lisa Brontesi, Thibaut Imberdis, Nagendran Ramalingam, Ulf Dettmer.

  Alpha-synuclein (αS), the key protein in Parkinson's disease, is typically described as an intrinsically disordered protein. Consistent with this notion, several context-dependent folding states may coexist inside neurons. Unfolded soluble monomers, helical monomers at membranes and helical multimers (soluble or at membranes) have all been reported and may be in an equilibrium with each other. We previously found that αS can be stabilized in its membrane-associated monomeric form by genetically increasing the hydrophobicity of the membrane-embedded half of the αS helix. αS amphipathic helix formation at membranes is governed by up to nine 11-amino acid repeats with the core motif KTKEGV. However, this repeat is only imperfectly conserved, e.g., it consists of KAKEGV in repeat #1, KTKEQV in repeat #5, and AVVTGV in the poorly conserved repeat #6. Here we explored the effect of perfecting the αS core repeat to nine times KTKEGV ("9KV") and found by sequential protein extraction that this engineered mutant accumulates in the cytosolic phase of neural cells. Intact-cell crosslinking trapped a part of the cytosolic portion at multimeric positions (30, 60, 80, 100 kDa). Thus, compared to wild-type αS, αS 9KV seems less prone to populating the membrane-associated monomeric form. Removing the 'ATVA' intervening amino-acid sequence between repeats 4 and 5 slightly increased cytosolic localization, while adding 'ATVA' in between all repeats 1-7 caused αS to be trapped as a monomer in membrane fractions. Our results contribute to an ongoing debate on the dynamic structure of αS, highlighting that wild-type αS is unlikely to be fully multimeric/monomeric or fully cytosolic/membrane-associated in cells, but protein engineering can create αS variants that preferentially adopt a certain state. Overall, the imperfect nature of the KTKEGV repeat motifs and the presence of ATVA in between repeats 4 and 5 seem to prevent a strong cytosolic localization of αS, and thus play a major role in the protein's ability to dynamically populate cytosolic vs. membrane-associated and monomeric vs. multimeric states.

Keywords:  Alpha-synuclein; Parkinson's disease; membrane binding; multimer; neurotoxicity; tetramer

DOI:  https://doi.org/10.1111/jnc.15763
Mol Biol Evol. 2023 Jan 10. pii: msad003. [Epub ahead of print]

The cynosure of CtBP: evolution of a bilaterian transcriptional corepressor.

Ana-Maria Raicu, Dhruva Kadiyala, Madeline Niblock, Aanchal Jain, Yahui Yang, Kalynn M Bird, Kayla Bertholf, Akshay Seenivasan, Mohammad Siddiq, David N Arnosti.

Evolution of sequence-specific transcription factors clearly drives lineage-specific innovations, but less is known about how changes in the central transcriptional machinery may contribute to evolutionary transformations. In particular, transcriptional regulators are rich in intrinsically disordered regions that appear to be magnets for evolutionary innovation. The C-terminal Binding Protein (CtBP) is a transcriptional corepressor derived from an ancestral lineage of alpha hydroxyacid dehydrogenases; it is found in mammals and invertebrates, and features a core NAD-binding domain as well as an unstructured C-terminus (CTD) of unknown function. CtBP can act on promoters and enhancers to repress transcription through chromatin-linked mechanisms. Our comparative phylogenetic study shows that CtBP is a bilaterian innovation whose CTD of about 100 residues is present in almost all orthologs. CtBP CTDs contain conserved blocks of residues and retain a predicted disordered property, despite having variations in the primary sequence. Interestingly, the structure of the C-terminus has undergone radical transformation independently in certain lineages including flatworms and nematodes. Also contributing to CTD diversity is the production of myriad alternative RNA splicing products, including the production of "short" tailless forms of CtBP in Drosophila. Additional diversity stems from multiple gene duplications in vertebrates, where up to five CtBP orthologs have been observed. Vertebrate lineages show fewer major modifications in the unstructured CTD, possibly because gene regulatory constraints of the vertebrate body plan place specific constraints on this domain. Our study highlights the rich regulatory potential of this previously unstudied domain of a central transcriptional regulator.

DOI: https://doi.org/10.1093/molbev/msad003
Proteins. 2023 Jan 10.

Effects of the Jokela Type of Spinal Muscular Atrophy-Related G66V Mutation on the Structural Ensemble Characteristics of CHCHD10.

Hakan Alici, Vladimir N Uversky, David E Kang, Junga Alexa Woo, Orkid Coskuner-Weber.

  The G66V pathological variant of the coiled-coil-helix-coiled-coil-helix domain-containing protein 10 (CHCHD10), mitochondrial, plays a role in Jokela type spinal muscular atrophy. The wild- and G66V mutant-type CHCHD10 proteins contain intrinsically disordered regions, and therefore, their structural ensemble studies have been experiencing difficulties using conventional tools. Here, we show our results regarding the first characterization of the structural ensemble characteristics of the G66V mutant form of CHCHD10 and the first comparison of these characteristics with the structural ensemble properties of wild-type CHCHD10. We find that the structural properties, potential of mean force surfaces, and principal component analysis show stark differences between these two proteins. These results are important for a better pathology, biochemistry and structural biology understanding of CHCHD10 and its G66V genetic variant and it is likely that these reported structural properties are important for designing more efficient treatments for the Jokela type of spinal muscular atrophy disease.

Keywords:  CHCHD10; G66V; Jokela type spinal muscular atrophy; bioinformatics; genetics; multiple run molecular dynamics simulations; structural properties

DOI:  https://doi.org/10.1002/prot.26463
J Chem Inf Model. 2023 Jan 10.

Conformational Search for the Building Block of Proteins Based on the Gradient Gravitational Search Algorithm (ConfGGS) Using Force Fields: CHARMM, AMBER, and OPLS-AA.

Rojalin Pradhan, Sibarama Panigrahi, Prabhat K Sahu.

Proteins are linear polymers built from a repertoire of 20 different amino acids, which are considered building blocks of proteins. The diversity and versatility of these 20 building blocks with regard to their conformations are key to adopting three-dimensional structures that facilitate proteins to undergo important mechanistic biological processes in living systems. The present investigation reports a conformational search of 20 different amino acids, building blocks of proteins, using three different force fields, CHARMM, AMBER, and OPLS-AA, implemented in the gradient gravitational search algorithm. The search technique (ConfGGS) includes the contribution from both bonded and nonbonded terms using Cartesian coordinates. The efficiency of such conformational searches has also been compared with other optimization algorithms: DE/Best, DE/Rand, and PSO algorithms with respect to computational time and accuracy based on the minimum number of iteration steps and computed lowest mean absolute error (MAE) and mean standard deviation (MSD) values for dihedral angles of respective near-optimal structures. Moreover, the ConfGGS technique has also been extended to an ordered protein fragment (PQITL) extracted from HIV-1 protease (PDB ID: 1YTH), an intrinsically disordered protein fragment, i.e., an amyloid-forming segment (AVVTGVTAV), from the NAC domain of Parkinson's disease protein α-synuclein, residues 69-77 (PDB ID: 4RIK), the experimental NMR atomic-resolution structure of α-synuclein fibrils (PDB ID: 2N0A), and a disulfide bond-containing protein fragment sequence (PCYGWPVCY), residues 59-67 (PDB ID: 6Y4F) toward structure prediction as a close homologue compared with experimental accuracy, using the CHARMM force field. The MolProbity validation results for the protein fragment (PQITL) obtained by ConfGGS/CHARMM are in better agreement with the native protein fragment structure of HIV-1 protease (PDB ID: 1YTH). Furthermore, the computed results have also been compared with the coordinates obtained from the AlphaFold network.

DOI: https://doi.org/10.1021/acs.jcim.2c01398
Nat Commun. 2023 Jan 10. 14(1): 79

Decoding of the ubiquitin code for clearance of colliding ribosomes by the RQT complex.

Yoshitaka Matsuo, Takayuki Uchihashi, Toshifumi Inada.

The collision sensor Hel2 specifically recognizes colliding ribosomes and ubiquitinates the ribosomal protein uS10, leading to noncanonical subunit dissociation by the ribosome-associated quality control trigger (RQT) complex. Although uS10 ubiquitination is essential for rescuing stalled ribosomes, its function and recognition steps are not fully understood. Here, we show that the RQT complex components Cue3 and Rqt4 interact with the K63-linked ubiquitin chain and accelerate the recruitment of the RQT complex to the ubiquitinated colliding ribosome. The CUE domain of Cue3 and the N-terminal domain of Rqt4 bind independently to the K63-linked ubiquitin chain. Their deletion abolishes ribosomal dissociation mediated by the RQT complex. High-speed atomic force microscopy (HS-AFM) reveals that the intrinsically disordered regions of Rqt4 enable the expansion of the searchable area for interaction with the ubiquitin chain. These findings provide mechanistic insight into the decoding of the ubiquitin code for clearance of colliding ribosomes by the RQT complex.

DOI: https://doi.org/10.1038/s41467-022-35608-4
Int J Mol Sci. 2022 Dec 26. pii: 399. [Epub ahead of print]24(1):

Molecular Determinants of Fibrillation in a Viral Amyloidogenic Domain from Combined Biochemical and Biophysical Studies.

Juliet F Nilsson, Hakima Baroudi, Frank Gondelaud, Giulia Pesce, Christophe Bignon, Denis Ptchelkine, Joseph Chamieh, Hervé Cottet, Andrey V Kajava, Sonia Longhi.

  The Nipah and Hendra viruses (NiV and HeV) are biosafety level 4 human pathogens classified within the Henipavirus genus of the Paramyxoviridae family. In both NiV and HeV, the gene encoding the Phosphoprotein (P protein), an essential polymerase cofactor, also encodes the V and W proteins. These three proteins, which share an intrinsically disordered N-terminal domain (NTD) and have unique C-terminal domains (CTD), are all known to counteract the host innate immune response, with V and W acting by either counteracting or inhibiting Interferon (IFN) signaling. Recently, the ability of a short region within the shared NTD (i.e., PNT3) to form amyloid-like structures was reported. Here, we evaluated the relevance of each of three contiguous tyrosine residues located in a previously identified amyloidogenic motif (EYYY) within HeV PNT3 to the fibrillation process. Our results indicate that removal of a single tyrosine in this motif significantly decreases the ability to form fibrils independently of position, mainly affecting the elongation phase. In addition, we show that the C-terminal half of PNT3 has an inhibitory effect on fibril formation that may act as a molecular shield and could thus be a key domain in the regulation of PNT3 fibrillation. Finally, the kinetics of fibril formation for the two PNT3 variants with the highest and the lowest fibrillation propensity were studied by Taylor Dispersion Analysis (TDA). The results herein presented shed light onto the molecular mechanisms involved in fibril formation.

Keywords:  Congo Red; Hendra virus; Polyethylene glycol (PEG) precipitation assays; Small-Angle X-ray Scattering (SAXS); Taylor Dispersion Analysis (TDA); amyloid-like fibrils; intrinsically disordered proteins; negative staining Transmission Electron Microscopy (ns-TEM); paramyxoviruses

DOI:  https://doi.org/10.3390/ijms24010399
Phys Chem Chem Phys. 2023 Jan 09.

Structural and dynamical insights into SilE silver binding from combined analytical probes.

Yoan Monneau, Cyrielle Arrault, Coraline Duroux, Marie Martin, Fabien Chirot, Luke Mac Aleese, Marion Girod, Clothilde Comby-Zerbino, Agnès Hagège, Olivier Walker, Maggy Hologne.

Silver has been used for its antimicrobial properties to fight infection for thousands of years. Unfortunately, some Gram-negative bacteria have developed silver resistance causing the death of patients in a burn unit. The genes responsible for silver resistance have been designated as the sil operon. Among the proteins of the sil operon, SilE has been shown to play a key role in bacterial silver resistance. Based on the limited information available, it has been depicted as an intrinsically disordered protein that folds into helices upon silver ion binding. Herein, this work demonstrates that SilE is composed of 4 clearly identified helical segments in the presence of several silver ions. The combination of analytical and biophysical techniques (NMR spectroscopy, CD, SAXS, HRMS, CE-ICP-MS, and IM-MS) reveals that SilE harbors four strong silver binding sites among the eight sites available. We have also further evidenced that SilE does not adopt a globular structure but rather samples a large conformational space from elongated to more compact structures. This particular structural organization facilitates silver binding through much higher accessibility of the involved His and Met residues. These valuable results will advance our current understanding of the role of SilE in the silver efflux pump complex mechanism and will help in the future rational design of inhibitors to fight bacterial silver resistance.

DOI: https://doi.org/10.1039/d2cp04206a
Biomacromolecules. 2023 Jan 11.

β-Sheet Structure Formation within Binary Blends of Two Spider Silk Related Peptides.

Mirjam Hofmaier, Mikhail Malanin, Eva Bittrich, Sarah Lentz, Birgit Urban, Thomas Scheibel, Andreas Fery, Martin Müller.

Intrinsically disordered proteins (IDPs) play an important role in molecular biology and medicine because their induced folding can lead to so-called conformational diseases, where β-amyloids play an important role. Still, the molecular folding process into the different substructures, such as parallel/antiparallel or extended β-sheet/crossed β-sheet is not fully understood. The recombinant spider silk protein eADF4(Cx) consisting of repeating modules C, which are composed of a crystalline (pep-c) and an amorphous peptide sequence (pep-a), can be used as a model system for IDP since it can assemble into similar structures. In this work, blend films of the pep-c and pep-a sequences were investigated to modulate the β-sheet formation by varying the molar fraction of pep-c and pep-a. Dichroic Fourier-transform infrared spectroscopy (FTIR), circular dichroism, spectroscopic ellipsometry, atomic force microscopy, and IR nanospectroscopy were used to examine the secondary structure, the formation of parallel and antiparallel β-sheets, their orientation, and the microscopic roughness and phase formation within peptide blend films upon methanol post-treatment. New insights into the formation of filament-like structures in these silk blend films were obtained. Filament-like structures could be locally assigned to β-sheet-rich structures. Further, the antiparallel or parallel character and the orientation of the formed β-sheets could be clearly determined. Finally, the ideal ratio of pep-a and pep-c sequences found in the fibroin 4 of the major ampullate silk of spiders could also be rationalized by comparing the blend and spider silk protein systems.

DOI: https://doi.org/10.1021/acs.biomac.2c01266
J Mol Biol. 2023 Jan 05. pii: S0022-2836(23)00001-3. [Epub ahead of print] 167945

DeepPRObind: Modular deep learner that accurately predicts structure and disorder-annotated protein binding residues.

Fuhao Zhang, Min Li, Jian Zhang, Wenbo Shi, Lukasz Kurgan.

  Current sequence-based predictors of protein-binding residues (PBRs) belong to two distinct categories: structure-trained vs. intrinsic disorder-trained. Since disordered PBRs differ from structured PBRs in several ways, including ability to bind multiple partners by folding into different conformations and enrichment in different amino acids, the structure-trained and disorder-trained predictors were shown to provide inaccurate results for the other annotation type. A simple consensus-based solution that combines structure- and disorder-trained methods provides limited levels of predictive performance and generates relatively many cross-predictions, where residues that interact with other ligand types are predicted as PBRs. We address this unsolved problem by designing a novel and fast deep-learner, DeepPRObind, that relies on carefully designed modular convolutional architecture and uses innovative aggregate input features. Comparative empirical tests on a low-similarity test dataset reveal that DeepPRObind generates accurate predictions of structured and disordered PBRs and low amounts of cross-predictions, outperforming a comprehensive collection of 12 predictors of PBRs. Given the relatively low runtime of DeepPRObind (40 seconds per protein), we further validate its results based on an analysis of putative PBRs in the yeast proteome, confirming that interactions in disordered regions are enriched among hub proteins. We release DeepPRObind as a convenient web server at https://www.csuligroup.com/DeepPRObind/.

Keywords:  deep learner; intrinsic disorder; prediction; protein-binding residues; protein-protein interactions

DOI:  https://doi.org/10.1016/j.jmb.2023.167945