bims-indpro 2022-04-03 papers

bims-indpro

Biomed News

on Intrinsically disordered proteins

Issue of 2022‒04‒03
eight papers selected by
Sara Mingu
Johannes Gutenberg University

The dynamic properties of a nuclear coactivator binding domain are evolutionarily conserved.
A large disordered region confers a wide spanning volume to vertebrate Suppressor of Fused as shown in a trans-species solution study.
Quantification of Entropic Excluded Volume Effects Driving Crowding-Induced Collapse and Folding of a Disordered Protein.
Structures of the Wild-Type and S59L Mutant CHCHD10 Proteins Important in Amyotrophic Lateral Sclerosis-Frontotemporal Dementia.
How Glutamate Promotes Liquid-liquid Phase Separation and DNA Binding Cooperativity of E. coli SSB Protein.
Deep learning in prediction of intrinsic disorder in proteins.
Intrinsically disordered N-terminal domain (NTD) of p53 interacts with mitochondrial PTP regulator Cyclophilin D.
Acquired disorder and asymmetry in a domain-swapped model for γ-crystallin aggregation.

Commun Biol. 2022 Mar 30. 5(1): 286

The dynamic properties of a nuclear coactivator binding domain are evolutionarily conserved.

Elin Karlsson, Frieda A Sorgenfrei, Eva Andersson, Jakob Dogan, Per Jemth, Celestine N Chi.

Evolution of proteins is constrained by their structure and function. While there is a consensus that the plasticity of intrinsically disordered proteins relaxes the structural constraints on evolution there is a paucity of data on the molecular details of these processes. The Nuclear Coactivator Binding Domain (NCBD) from CREB-binding protein is a protein interaction domain, which contains a hydrophobic core but is not behaving as a typical globular domain, and has been described as 'molten-globule like'. The highly dynamic properties of NCBD makes it an interesting model system for evolutionary structure-function investigation of intrinsically disordered proteins. We have here compared the structure and biophysical properties of an ancient version of NCBD present in a bilaterian animal ancestor living around 600 million years ago with extant human NCBD. Using a combination of NMR spectroscopy, circular dichroism and kinetics we show that although NCBD has increased its thermodynamic stability, it has retained its dynamic biophysical properties in the ligand-free state in the evolutionary lineage leading from the last common bilaterian ancestor to humans. Our findings suggest that the dynamic properties of NCBD have been maintained by purifying selection and thus are important for its function, which includes mediating several distinct protein-protein interactions.

DOI: https://doi.org/10.1038/s42003-022-03217-y
J Struct Biol. 2022 Mar 29. pii: S1047-8477(22)00023-5. [Epub ahead of print] 107853

A large disordered region confers a wide spanning volume to vertebrate Suppressor of Fused as shown in a trans-species solution study.

Staëlle Makamte, Aurélien Thureau, Amira Jabrani, Annick Paquelin, Anne Plessis, Mathieu Sanial, Olga Rudenko, Francesco Oteri, Marc Baaden, Valérie Biou.

  Hedgehog (Hh) pathway inhibition by the conserved protein Suppressor of Fused (SuFu) is crucial to vertebrate development. By constrast, SuFu loss-of-function mutant has little effect in drosophila. Previous publications showed that the crystal structures of human and drosophila SuFu consist of two ordered domains that are capable of breathing motions upon ligand binding. However, the crystal structure of human SuFu does not give information about twenty N-terminal residues (IDR1) and an eighty-residue-long region predicted as disordered (IDR2) in the C-terminus, whose function is important for the pathway repression. These two intrinsically disordered regions (IDRs) are species-dependent. To obtain information about the IDR regions, we studied full-length SuFu's structure in solution, both with circular dichroism and small angle X-ray scattering, comparing drosophila, zebrafish and human species, to better understand this considerable difference. Our studies show that, in spite of similar crystal structures restricted to ordered domains, drosophila and vertebrate SuFu have very different structures in solution. The IDR2 of vertebrates spans a large area, thus enabling it to reach for partners and be accessible for post-translational modifications. Furthermore, we show that the IDR2 region is highly conserved within phyla but varies in length and sequence, with insects having a shorter disordered region while that of vertebrates is broad and mobile. This major variation may explain the different phenotypes observed upon SuFu removal.

Keywords:  Hedgehog signal; intrinsically disordered protein; small-angle X-ray scattering

DOI:  https://doi.org/10.1016/j.jsb.2022.107853
J Phys Chem Lett. 2022 Mar 31. 3112-3120

Quantification of Entropic Excluded Volume Effects Driving Crowding-Induced Collapse and Folding of a Disordered Protein.

Divya Rajendran, Shrutarshi Mitra, Hiroyuki Oikawa, Kulkarni Madhurima, Ashok Sekhar, Satoshi Takahashi, Athi N Naganathan.

We investigate the conformational properties of the intrinsically disordered DNA-binding domain of CytR in the presence of the polymeric crowder polyethylene glycol (PEG). Integrating circular dichroism, nuclear magnetic resonance, and single-molecule Förster resonance energy transfer measurements, we demonstrate that disordered CytR populates a well-folded minor conformation in its native ensemble, while the unfolded ensemble collapses and folds with an increase in crowder density independent of the crowder size. Employing a statistical-mechanical model, the effective reduction in the accessible conformational space of a residue in the unfolded state is estimated to be 10% at 300 mg/mL PEG8000, relative to dilute conditions. The experimentally consistent PEG-temperature phase diagram thus constructed reveals that entropic effects can stabilize disordered CytR by 10 kJ mol-1, driving the equilibrium toward folded conformations under physiological conditions. Our work highlights the malleable conformational landscape of CytR, the presence of a folded conformation in the disordered ensemble, and proposes a scaling relation for quantifying excluded volume effects on protein stability.

DOI: https://doi.org/10.1021/acs.jpclett.2c00316
ACS Chem Neurosci. 2022 Mar 29.

Structures of the Wild-Type and S59L Mutant CHCHD10 Proteins Important in Amyotrophic Lateral Sclerosis-Frontotemporal Dementia.

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

  The S59L genetic mutation of the mitochondrial coiled-coil-helix-coiled-coil-helix domain-containing protein 10 (CHCHD10) is involved in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The wild-type and mutant forms of this protein contain intrinsically disordered regions, and their structural characterization has been facing challenges. Here, for the first time in the literature, we present the structural ensemble properties of the wild-type and S59L mutant form of CHCHD10 in an aqueous solution environment at the atomic level with dynamics. Even though available experiments suggested that the S59L mutation may not change the structure of the CHCHD10 protein, our structural analysis clearly shows that the structure of this protein is significantly affected by the S59L mutation. We present here the secondary structure components with their abundances per residue, the tertiary structure properties, the free energy surfaces based on the radius of gyration and end-to-end distance values, the Ramachandran plots, the quantity of intramolecular hydrogen bonds, and the principal component analysis results. These results may be crucial in designing more efficient treatment for ALS and FTD diseases.

Keywords:  CHCHD10; S59L genetic mutation; amyotrophic lateral sclerosis-frontotemporal dementia; wild-type

DOI:  https://doi.org/10.1021/acschemneuro.2c00011
J Mol Biol. 2022 Mar 26. pii: S0022-2836(22)00136-X. [Epub ahead of print] 167562

How Glutamate Promotes Liquid-liquid Phase Separation and DNA Binding Cooperativity of E. coli SSB Protein.

Alexander G Kozlov, Xian Cheng, Hongshan Zhang, Min Kyung Shinn, Elizabeth Weiland, Binh Nguyen, Irina A Shkel, Emily Zytkiewicz, Ilya J Finkelstein, M Thomas Record, Timothy M Lohman.

  E. coli single-stranded-DNA binding protein (EcSSB) displays nearest-neighbor (NN) and non-nearest-neighbor (NNN)) cooperativity in binding ssDNA during genome maintenance. NNN cooperativity requires the intrinsically-disordered linkers (IDL) of the C-terminal tails. Potassium glutamate (KGlu), the primaryE. colisalt, promotes NNN-cooperativity, while KCl inhibits it. We find that KGlu promotes compaction of a single polymeric SSB-coated ssDNA beyond what occurs in KCl, indicating a link of compaction to NNN-cooperativity. EcSSB also undergoes liquid-liquid phase separation (LLPS), inhibited by ssDNA binding. We find that LLPS, like NNN-cooperativity, is promoted by increasing [KGlu] in the physiological range, while increasing [KCl] and/or deletion of the IDL eliminate LLPS, indicating similar interactions in both processes. From quantitative determinations of interactions of KGlu and KCl with protein model compounds,we deduce that the opposing effects of KGlu and KCl on SSB LLPS and cooperativity arise from their opposite interactions with amide groups. KGlu interacts unfavorably with the backbone (especially Gly) and side chain amide groups of the IDL, promoting amide-amide interactions in LLPS and NNN-cooperativity. By contrast, KCl interacts favorably with these amide groups and therefore inhibits LLPS and NNN-cooperativity. These results highlight the importance of salt interactions in regulating the propensity of proteins to undergo LLPS.

Keywords:  DNA replication; biomolecular condensates; liquid-liquid phase separation; salt effects; single molecule DNA collapse

DOI:  https://doi.org/10.1016/j.jmb.2022.167562
Comput Struct Biotechnol J. 2022 ;20 1286-1294

Deep learning in prediction of intrinsic disorder in proteins.

Bi Zhao, Lukasz Kurgan.

  Intrinsic disorder prediction is an active area that has developed over 100 predictors. We identify and investigate a recent trend towards the development of deep neural network (DNN)-based methods. The first DNN-based method was released in 2013 and since 2019 deep learners account for majority of the new disorder predictors. We find that the 13 currently available DNN-based predictors are diverse in their topologies, sizes of their networks and the inputs that they utilize. We empirically show that the deep learners are statistically more accurate than other types of disorder predictors using the blind test dataset from the recent community assessment of intrinsic disorder predictions (CAID). We also identify several well-rounded DNN-based predictors that are accurate, fast and/or conveniently available. The popularity, favorable predictive performance and architectural flexibility suggest that deep networks are likely to fuel the development of future disordered predictors. Novel hybrid designs of deep networks could be used to adequately accommodate for diversity of types and flavors of intrinsic disorder. We also discuss scarcity of the DNN-based methods for the prediction of disordered binding regions and the need to develop more accurate methods for this prediction.

Keywords:  BRNN, Bidirectional recurrent neural networks; CAID, Critical Assessment of Intrinsic Protein Disorder; CASP, Critical Assessment of Structure Prediction; CNN, Convolutional neural networks; DNN, Deep neural network; Deep learning; Deep neural networks; Disordered binding regions; Disordered regions; FFNN, Feed forward neural networks; IDP, Intrinsically disordered protein; IDR, Intrinsically disordered region; Intrinsic disorder; Prediction

DOI:  https://doi.org/10.1016/j.csbj.2022.03.003
J Mol Biol. 2022 Mar 24. pii: S0022-2836(22)00126-7. [Epub ahead of print] 167552

Intrinsically disordered N-terminal domain (NTD) of p53 interacts with mitochondrial PTP regulator Cyclophilin D.

Jing Zhao, Xinyue Liu, Alan Blayney, Yumeng Zhang, Lauren Gandy, Paige Olivia Mirsky, Nathan Smith, Fuming Zhang, Robert J Linhardt, Jianhan Chen, Christopher Baines, Stewart N Loh, Chunyu Wang.

  Mitochondrial permeability transition pore (mPTP) plays crucial roles in cell death in a variety of diseases, including ischemia/reperfusion injury in heart attack and stroke, neurodegenerative conditions, and cancer. To date, cyclophilin D is the only confirmed component of mPTP. Under stress, p53 can translocate into mitochondria and interact with CypD, triggering necrosis and cell growth arrest. However, the molecular details of p53/CypD interaction are still poorly understood. Previously, several studies reported that p53 interacts with CypD through its DNA-binding domain (DBD). However, using surface plasmon resonance (SPR), we found that both NTD-DBD, NTD and NTD (1-70) bind to CypD at ∼μM KD. In solution NMR, NTD binds CypD with μM affinity and mimics the pattern of FLp53 binding in chemical shift perturbation. In contrast, neither solution NMR nor fluorescence anisotropy detected DBD binding to CypD. Thus, instead of DBD, NTD is the major CypD binding site on p53. NMR titration and MD simulation revealed that NTD binds CypD with broad and dynamic interfaces dominated by electrostatic interactions. NTD 20-70 was further identified as the minimal binding region for CypD interaction, and two NTD fragments, D1 (residues 22-44) and D2 (58-70), can each bind CypD with mM affinity. Our detailed biophysical characterization of the dynamic interface between NTD and CypD provides novel insights on the p53-dependent mPTP opening and drug discovery targeting NTD/CypD interface in diseases.

Keywords:  Cyclophilin D; MD simulation; NMR; mitochondrial permeability transition pore; p53

DOI:  https://doi.org/10.1016/j.jmb.2022.167552
J Mol Biol. 2022 Mar 24. pii: S0022-2836(22)00133-4. [Epub ahead of print] 167559

Acquired disorder and asymmetry in a domain-swapped model for γ-crystallin aggregation.

Vatsala Sagar, Graeme Wistow.

  Misfolding and aggregation of proteins occur in many pathological states. Because of the inherent disorder involved, these processes are difficult to study. We attempted to capture aggregation intermediates of γ S-crystallin, a highly stable, internally symmetrical monomeric protein, by crystallization under mildly acidic and oxidizing conditions. Here we describe novel oligomerization through strained domain-swapping and partial intermolecular disulfide formation. This forms an octamer built from asymmetric tetramers, each of which comprises an asymmetric pair of twisted, domain-swapped dimers. Each tetramer shows patterns of acquired disorder among subunits, ranging from local loss of secondary structure to regions of intrinsic disorder. The octamer ring is tied together by partial intermolecular disulfide bonds, which may contribute to strain and disorder in the octamer. Oligomerization in this structure is self-limited by the distorted octamer ring. In a more heterogeneous environment, the disordered regions could serve as seeds for cascading interactions with other proteins. Indeed, solubilized protein from crystals retain many features observed in the crystal and are prone to further oligomerization and precipitation. This structure illustrates modes of loss of organized structure and aggregation that are relevant for cataract and for other disorders involving deposition of formerly well-folded proteins.

Keywords:  Domain-swapping; aggregation; asymmetry; disorder; unfolding

DOI:  https://doi.org/10.1016/j.jmb.2022.167559