bims-indpro 2022-03-06 papers

bims-indpro

Biomed News

on Intrinsically disordered proteins

Issue of 2022–03–06
ten papers selected by
Sara Mingu, Johannes Gutenberg University

NMR of intrinsically disordered proteins: A note on the application of 15N-13Cα het-TOCSY mixing for 13Cα magnetisation transfers.
Preparation of Bioderived and Biodegradable Surfactants Based on an Intrinsically Disordered Protein Sequence.
Continuum dynamics and statistical correction of compositional heterogeneity in multivalent IDP oligomers resolved by single-particle EM.
Spatiotemporal modulations in heterotypic condensates of prion and α-synuclein control phase transitions and amyloid conversion.
Curious Case of MAD2 Protein: Diverse Folding Intermediates Leading to Alternate Native States.
Conformational dynamics promotes disordered regions from function-dispensable to essential in evolved site-specific DNA recombinases.
Mammalian nuclear speckles exhibit stable association with chromatin: a biochemical study.
TRPM5 Channel Binds Calcium-Binding Proteins Calmodulin and S100A1.
Generation of functional oligopeptides that promote osteogenesis based on unsupervised deep learning of protein IDRs.
Influence of the Dynamically Disordered N-Terminal Tail Domain on the Amyloid Core Structure of Human Y145Stop Prion Protein Fibrils.

J Magn Reson. 2022 Mar 01. pii: S1090-7807(22)00024-6. [Epub ahead of print]337 107166

NMR of intrinsically disordered proteins: A note on the application of 15N-13Cα het-TOCSY mixing for 13Cα magnetisation transfers.

Amit Kumar, Christoph Wiedemann, Peter Bellstedt, Ramadurai Ramachandran, Oliver Ohlenschläger.

  Intrinsically disordered proteins (IDPs) or protein regions represent functionally important biomolecules without unique structure. Their inherent flexibility prevents high-resolution structure determination by X-ray or cryo-EM methods. In contrast, NMR spectroscopy provides an extensive and still growing set of experimental approaches to obtain detailed information on structure and dynamics of IDPs. Here, it is experimentally demonstrated that 15N-13Cα band-selective heteronuclear cross-polarisation that has been successfully employed recently to achieve the efficient transfer of 15Nx magnetisation from amino acid residue 'i' to 'i + 1' and 'i - 1' residues in uniformly (15N,13C)-labelled intrinsically disordered proteins can also be applied to transfer, without significant relaxation losses, 13Cαx magnetisation from an amino acid residue to its neighbouring residues. The possibility to obtain in one-shot correlation spectra arising from the simultaneous transfer of 15Nx and 13Cαx magnetisations from an amino acid residue to neighbouring residues is also demonstrated.

Keywords:  (13)C(α) magnetisation; Assignment; Heteronuclear cross-polarisation; Intrinsically disordered region; NMR; α-synuclein

DOI:  https://doi.org/10.1016/j.jmr.2022.107166
Biomacromolecules. 2022 Mar 03.

Preparation of Bioderived and Biodegradable Surfactants Based on an Intrinsically Disordered Protein Sequence.

Sarah H Klass, Jamie M Gleason, Anthony O Omole, Bibiana Onoa, Carlos J Bustamante, Matthew B Francis.

Surfactants, block copolymers, and other types of micellar systems are used in a wide variety of biomedical and industrial processes. However, most commonly used surfactants are synthetically derived and pose environmental and toxicological concerns throughout their product life cycle. Because of this, bioderived and biodegradable surfactants are promising alternatives. For biosurfactants to be implemented industrially, they need to be produced on a large scale and also have tailorable properties that match those afforded by the polymerization of synthetic surfactants. In this paper, a scalable and versatile production method for biosurfactants based on a hydrophilic intrinsically disordered protein (IDP) sequence with a genetically engineered hydrophobic domain is used to study variables that impact their physicochemical and self-assembling properties. These amphiphilic sequences were found to self-assemble into micelles over a broad range of temperatures, pH values, and ionic strengths. To investigate the role of the IDP hydrophilic domain on self-assembly, variants with increased overall charges and systematically decreased IDP domain lengths were produced and examined for their sizes, morphologies, and critical micelle concentrations (CMCs). The results of these studies indicate that decreasing the length of the IDP domain and consequently the molecular weight and hydrophilic fraction leads to smaller micelles. In addition, significantly increasing the amount of charged residues in the hydrophilic IDP domain results in micelles of similar sizes but with higher CMC values. This represents an initial step in developing a quantitative model for the future engineering of biosurfactants based on this IDP sequence.

DOI: https://doi.org/10.1021/acs.biomac.2c00051
J Mol Biol. 2022 Mar 01. pii: S0022-2836(22)00094-8. [Epub ahead of print] 167520

Continuum dynamics and statistical correction of compositional heterogeneity in multivalent IDP oligomers resolved by single-particle EM.

Barmak Mostofian, Russell McFarland, Aidan Estelle, Jesse Howe, Elisar Barbar, Steve L Reichow, Daniel M Zuckerman.

  Multivalent intrinsically disordered protein (IDP) complexes are prevalent in biology and act in regulation of diverse processes, including transcription, signaling events, and the assembly and disassembly of complex macromolecular architectures. These systems pose significant challenges to structural investigation, due to continuum dynamics imparted by the IDP and compositional heterogeneity resulting from characteristic low-affinity interactions. Here, we developed a modular pipeline for automated single-particle electron microscopy (EM) distribution analysis of common but relatively understudied semi-ordered systems: 'beads-on-a-string' assemblies, composed of IDPs bound at multivalent sites to the ubiquitous ∼20kDacross-linking hub protein LC8. This approach quantifies conformational geometries and compositional heterogeneity on a single-particle basis, and statistically corrects spurious observations arising from random proximity of bound and unbound LC8. The statistical correction is generically applicable to oligomer characterization and not specific to our pipeline. Following validation, the approach was applied to the nuclear pore IDP Nup159 and the transcription factor ASCIZ. This analysis unveiled significant compositional and conformational diversity in both systems that could not be obtained from ensemble single particle EM class-averaging strategies, and new insights for exploring how these architectural properties might contribute to their physiological roles in supramolecular assembly and transcriptional regulation. We expect that this approach may be adopted to many other intrinsically disordered systems that have evaded traditional methods of structural characterization.

Keywords:  Intrinsically disordered proteins (IDP); LC8; electron microscopy (EM); multivalency; single-molecule

DOI:  https://doi.org/10.1016/j.jmb.2022.167520
Nat Commun. 2022 Mar 03. 13(1): 1154

Spatiotemporal modulations in heterotypic condensates of prion and α-synuclein control phase transitions and amyloid conversion.

Aishwarya Agarwal, Lisha Arora, Sandeep K Rai, Anamika Avni, Samrat Mukhopadhyay.

Biomolecular condensation via liquid-liquid phase separation of proteins and nucleic acids is associated with a range of critical cellular functions and neurodegenerative diseases. Here, we demonstrate that complex coacervation of the prion protein and α-synuclein within narrow stoichiometry results in the formation of highly dynamic, reversible, thermo-responsive liquid droplets via domain-specific electrostatic interactions between the positively-charged intrinsically disordered N-terminal segment of prion and the acidic C-terminal tail of α-synuclein. The addition of RNA to these coacervates yields multiphasic, vesicle-like, hollow condensates. Picosecond time-resolved measurements revealed the presence of transient electrostatic nanoclusters that are stable on the nanosecond timescale and can undergo breaking-and-making of interactions on slower timescales giving rise to a liquid-like behavior in the mesoscopic regime. The liquid-to-solid transition drives a rapid conversion of complex coacervates into heterotypic amyloids. Our results suggest that synergistic prion-α-synuclein interactions within condensates provide mechanistic underpinnings of their physiological role and overlapping neuropathological features.

DOI: https://doi.org/10.1038/s41467-022-28797-5
J Phys Chem B. 2022 Mar 01.

Curious Case of MAD2 Protein: Diverse Folding Intermediates Leading to Alternate Native States.

Catherine Ghosh, Biman Jana.

Anfinsen's dogma postulates that for one sequence there will be only one unique structure that is necessary for the functioning of the protein. However, over the years there have been a number of departures from this postulate. As far as function is considered, there are growing examples of proteins that "moonlight", perform multiple unrelated functions. With the discovery of intrinsically disordered proteins, morpheeins, chameleonic sequences, and metamorphic proteins that can switch folds, we have acquired a more nuanced understanding of protein folding and dynamics. Appearing to apparently contradict the classical folding paradigm, metamorphic proteins are considered exotic species. In this work, we have explored the free energy landscape and folding pathways of the metamorphic protein MAD2 which is an important component of the spindle checkpoint. It coexists in two alternate states: the inactive open state and the active closed state. Using a dual-basin structure-based model approach we have shown that a variety of intermediates and multiple pathways are available to MAD2 to fold into its alternate forms. This approach involves performing molecular dynamics simulations of coarse-grained models of MAD2 where the structural information regarding both of its native conformations is explicitly included in terms of their native contacts in the force field used. Detailed analyses have indicated that some of the contacts within the protein play a key role in determining which folding pathway will be selected and point to a probable long-range communication between the N and the C termini of the protein that seems to control its folding. Finally, our work also provides a rationale for the experimentally observed preference of the ΔC10 variant of MAD2 to exist in the open state.

DOI: https://doi.org/10.1021/acs.jpcb.2c00382
Comput Struct Biotechnol J. 2022 ;20 989-1001

Conformational dynamics promotes disordered regions from function-dispensable to essential in evolved site-specific DNA recombinases.

Carla Guillén-Pingarrón, Pedro M Guillem-Gloria, Anjali Soni, Gloria Ruiz-Gómez, Martina Augsburg, Frank Buchholz, Massimiliano Anselmi, M Teresa Pisabarro.

  Protein intrinsically disordered regions (IDRs) play pivotal roles in molecular recognition and regulatory processes through structural disorder-to-order transitions. To understand and exploit the distinctive functional implications of IDRs and to unravel the underlying molecular mechanisms, structural disorder-to-function relationships need to be deciphered. The DNA site-specific recombinase system Cre/loxP represents an attractive model to investigate functional molecular mechanisms of IDRs. Cre contains a functionally dispensable disordered N-terminal tail, which becomes indispensable in the evolved Tre/loxLTR recombinase system. The difficulty to experimentally obtain structural information about this tail has so far precluded any mechanistic study on its involvement in DNA recombination. Here, we use in vitro and in silico evolution data, conformational dynamics, AI-based folding simulations, thermodynamic stability calculations, mutagenesis and DNA recombination assays to investigate how evolution and the dynamic behavior of this IDR may determine distinct functional properties. Our studies suggest that partial conformational order in the N-terminal tail of Tre recombinase and its packing to a conserved hydrophobic surface on the protein provide thermodynamic stability. Based on our results, we propose a link between protein stability and function, offering new plausible atom-detailed mechanistic insights into disorder-function relationships. Our work highlights the potential of N-terminal tails to be exploited for regulation of the activity of Cre-like tyrosine-type SSRs, which merits future investigations and could be of relevance in future rational engineering for their use in biotechnology and genomic medicine.

Keywords:  AI-based folding; Evolution; Intrinsically disordered protein regions; Molecular dynamics; Structural disorder; Thermodynamic stability; site-specific DNA recombinase

DOI:  https://doi.org/10.1016/j.csbj.2022.01.010
Nucleus. 2022 Dec;13(1): 58-73

Mammalian nuclear speckles exhibit stable association with chromatin: a biochemical study.

Komal Raina, Basuthkar J Rao.

  Nuclear Speckles (NS) are phase-separated condensates of protein and RNA whose components dynamically coordinate RNA transcription, splicing, transport and DNA repair. NS, probed largely by imaging studies, remained historically well known as Interchromatin Granule Clusters, and biochemical properties, especially their association with Chromatin have been largely unexplored. In this study, we tested whether NS exhibit any stable association with chromatin and show that limited DNAse-1 nicking of chromatin leads to the collapse of NS into isotropic distribution or aggregates of constituent proteins without affecting other nuclear structures. Further biochemical probing revealed that NS proteins were tightly associated with chromatin, extractable only by high-salt treatment just like histone proteins. NS were also co-released with solubilised mono-dinucleosomal chromatin fraction following the MNase digestion of chromatin. We propose a model that NS-chromatin constitutes a "putative stable association" whose coupling might be subject to the combined regulation from both chromatin and NS changes.Abbreviations: NS: Nuclear speckles; DSB: double strand breaks; PTM: posttranslational modifications; DDR: DNA damage repair; RBP-RNA binding proteins; TAD: topologically associated domains; LCR: low complexity regions; IDR: intrinsically disordered regions.

Keywords:  DNase 1; Nuclear speckles; SR proteins; chromatin-association; speckle-associated chromatin

DOI:  https://doi.org/10.1080/19491034.2021.2024948
Biochemistry. 2022 Feb 28.

TRPM5 Channel Binds Calcium-Binding Proteins Calmodulin and S100A1.

Kristyna Bousova, Monika Zouharova, Petr Herman, Jiri Vymetal, Veronika Vetyskova, Katerina Jiraskova, Jiri Vondrasek.

Melastatin transient receptor potential (TRPM) channels belong to one of the most significant subgroups of the transient receptor potential (TRP) channel family. Here, we studied the TRPM5 member, the receptor exposed to calcium-mediated activation, resulting in taste transduction. It is known that most TRP channels are highly modulated through interactions with extracellular and intracellular agents. The binding sites for these ligands are usually located at the intracellular N- and C-termini of the TRP channels, and they can demonstrate the character of an intrinsically disordered protein (IDP), which allows such a region to bind various types of molecules. We explored the N-termini of TRPM5 and found the intracellular regions for calcium-binding proteins (CBPs) the calmodulin (CaM) and calcium-binding protein S1 (S100A1) by in vitro binding assays. Furthermore, molecular docking and molecular dynamics simulations (MDs) of the discovered complexes confirmed their known common binding interface patterns and the uniqueness of the basic residues present in the TRPM binding regions for CaM/S100A1.

DOI: https://doi.org/10.1021/acs.biochem.1c00647
Bone Res. 2022 Mar 01. 10(1): 23

Generation of functional oligopeptides that promote osteogenesis based on unsupervised deep learning of protein IDRs.

Mingxiang Cai, Baichuan Xiao, Fujun Jin, Xiaopeng Xu, Yuwei Hua, Junhui Li, Pingping Niu, Meijing Liu, Jiaqi Wu, Rui Yue, Yong Zhang, Zuolin Wang, Yongbiao Zhang, Xiaogang Wang, Yao Sun.

Deep learning (DL) is currently revolutionizing peptide drug development due to both computational advances and the substantial recent expansion of digitized biological data. However, progress in oligopeptide drug development has been limited, likely due to the lack of suitable datasets and difficulty in identifying informative features to use as inputs for DL models. Here, we utilized an unsupervised deep learning model to learn a semantic pattern based on the intrinsically disordered regions of ~171 known osteogenic proteins. Subsequently, oligopeptides were generated from this semantic pattern based on Monte Carlo simulation, followed by in vivo functional characterization. A five amino acid oligopeptide (AIB5P) had strong bone-formation-promoting effects, as determined in multiple mouse models (e.g., osteoporosis, fracture, and osseointegration of implants). Mechanistically, we showed that AIB5P promotes osteogenesis by binding to the integrin α5 subunit and thereby activating FAK signaling. In summary, we successfully established an oligopeptide discovery strategy based on a DL model and demonstrated its utility from cytological screening to animal experimental verification.

DOI: https://doi.org/10.1038/s41413-022-00193-1
Front Mol Biosci. 2022 ;9 841790

Influence of the Dynamically Disordered N-Terminal Tail Domain on the Amyloid Core Structure of Human Y145Stop Prion Protein Fibrils.

Zhe Qi, Krystyna Surewicz, Witold K Surewicz, Christopher P Jaroniec.

  The Y145Stop mutant of human prion protein (huPrP23-144) is associated with a familial prionopathy and provides a convenient in vitro model for investigating amyloid strains and cross-seeding barriers. huPrP23-144 fibrils feature a compact and relatively rigid parallel in-register β-sheet amyloid core spanning ∼30 C-terminal amino acid residues (∼112-141) and a large ∼90-residue dynamically disordered N-terminal tail domain. Here, we systematically evaluate the influence of this dynamic domain on the structure adopted by the huPrP23-144 amyloid core region, by investigating using magic-angle spinning solid-state nuclear magnetic resonance (NMR) spectroscopy a series of fibril samples formed by huPrP23-144 variants corresponding to deletions of large segments of the N-terminal tail. We find that deletion of the bulk of the N-terminal tail, up to residue 98, yields amyloid fibrils with native-like huPrP23-144 core structure. Interestingly, deletion of additional flexible residues in the stretch 99-106 located outside of the amyloid core yields shorter heterogenous fibrils with fingerprint NMR spectra that are clearly distinct from those for full-length huPrP23-144, suggestive of the onset of perturbations to the native structure and degree of molecular ordering for the core residues. For the deletion variant missing residues 99-106 we show that native huPrP23-144 core structure can be "restored" by seeding the fibril growth with preformed full-length huPrP23-144 fibrils.

Keywords:  amyloid; intrinsically disordered region/protein; magic angle spinning (MAS) solid-state NMR; octarepeat; prion

DOI:  https://doi.org/10.3389/fmolb.2022.841790