bims-indpro Biomed News
on Intrinsically disordered proteins
Issue of 2022–03–13
eight papers selected by
Sara Mingu, Johannes Gutenberg University



  1. PLoS Comput Biol. 2022 Mar 11. 18(3): e1009911
      All proteomes contain both proteins and polypeptide segments that don't form a defined three-dimensional structure yet are biologically active-called intrinsically disordered proteins and regions (IDPs and IDRs). Most of these IDPs/IDRs lack useful functional annotation limiting our understanding of their importance for organism fitness. Here we characterized IDRs using protein sequence annotations of functional sites and regions available in the UniProt knowledgebase ("UniProt features": active site, ligand-binding pocket, regions mediating protein-protein interactions, etc.). By measuring the statistical enrichment of twenty-five UniProt features in 981 IDRs of 561 human proteins, we identified eight features that are commonly located in IDRs. We then collected the genetic variant data from the general population and patient-based databases and evaluated the prevalence of population and pathogenic variations in IDPs/IDRs. We observed that some IDRs tolerate 2 to 12-times more single amino acid-substituting missense mutations than synonymous changes in the general population. However, we also found that 37% of all germline pathogenic mutations are located in disordered regions of 96 proteins. Based on the observed-to-expected frequency of mutations, we categorized 34 IDRs in 20 proteins (DDX3X, KIT, RB1, etc.) as intolerant to mutation. Finally, using statistical analysis and a machine learning approach, we demonstrate that mutation-intolerant IDRs carry a distinct signature of functional features. Our study presents a novel approach to assign functional importance to IDRs by leveraging the wealth of available genetic data, which will aid in a deeper understating of the role of IDRs in biological processes and disease mechanisms.
    DOI:  https://doi.org/10.1371/journal.pcbi.1009911
  2. STAR Protoc. 2022 03 18. 3(1): 101200
      Comprehensive understanding of a protein's function depends on having reliable, sophisticated tools to study protein structural dynamics in physiologically-relevant conditions. Here, we present an effective, robust step-by-step protocol to monitor the structural dynamics (including hydration dynamics) of a protein utilizing various site-directed fluorescence (SDFL) approaches. This protocol should be widely applicable for studying soluble proteins, intrinsically-disordered proteins, and membrane proteins. For complete details on the use and execution of this protocol, please refer to Das et al. (2020), Das and Raghuraman (2021), and Chatterjee et al. (2021).
    Keywords:  Biophysics; Molecular/Chemical Probes; Protein Biochemistry; Structural Biology
    DOI:  https://doi.org/10.1016/j.xpro.2022.101200
  3. Cell Rep. 2022 Mar 08. pii: S2211-1247(22)00202-9. [Epub ahead of print]38(10): 110469
      Human cytomegalovirus (HCMV) replicates its DNA genome in specialized replication compartments (RCs) in the host cell nucleus. These membrane-less organelles originate as spherical structures and grow in size over time. However, the mechanism of RC biogenesis has remained understudied. Using live-cell imaging and photo-oligomerization, we show that a central component of RCs, the UL112-113 proteins, undergo liquid-liquid phase separation (LLPS) to form RCs in the nucleus. We show that the self-interacting domain and large intrinsically disordered regions of UL112-113 are required for LLPS. Importantly, viral DNA induces local clustering of these proteins and lowers the threshold for phase separation. The formation of phase-separated compartments around viral genomes is necessary to recruit the viral DNA polymerase for viral genome replication. Thus, HCMV uses its UL112-113 proteins to generate RCs around viral genomes by LLPS to ensure the formation of a pro-replicative environment.
    Keywords:  Herpesviridae; LLPS; human cytomegalovirus; human herpesvirus 5; liquid-liquid phase separation; live-cell imaging; membrane-less organelle; molecular virology; phase transition; replication compartment; viral replication
    DOI:  https://doi.org/10.1016/j.celrep.2022.110469
  4. Nat Commun. 2022 Mar 11. 13(1): 1278
      Yeast Cadmium Factor 1 (Ycf1) sequesters heavy metals and glutathione into the vacuole to counter cell stress. Ycf1 belongs to the ATP binding cassette C-subfamily (ABCC) of transporters, many of which are regulated by phosphorylation on intrinsically-disordered domains. The regulatory mechanism of phosphorylation is still poorly understood. Here, we report two cryo-EM structures of Ycf1 at 3.4 Å and 4.0 Å resolution in inward-facing open conformations that capture previously unobserved ordered states of the intrinsically disordered regulatory domain (R-domain). R-domain phosphorylation is clearly evident and induces a topology promoting electrostatic and hydrophobic interactions with Nucleotide Binding Domain 1 (NBD1) and the Lasso motif. These interactions stay constant between the structures and are related by rigid body movements of the NBD1/R-domain complex. Biochemical data further show R-domain phosphorylation reorganizes the Ycf1 architecture and is required for maximal ATPase activity. Together, we provide insights into how R-domains control ABCC transporter activity.
    DOI:  https://doi.org/10.1038/s41467-022-28811-w
  5. Biochem Biophys Res Commun. 2022 Feb 25. pii: S0006-291X(22)00300-X. [Epub ahead of print]603 13-20
      α-Synuclein (α-Syn) is an aggregation-prone protein whose accumulation in Lewy bodies leads to neurodegenerative diseases like Parkinson's disease (PD). Calcium plays a critical role in neurons, and calcium dysregulation is one of the risk factors of PD. It is known that Ca2+ interacts with α-Syn and affects its assembly. However, how Ca2+ regulates α-Syn aggregation remains unclear. Here, we reported that Ca2+ accelerates α-Syn amyloid aggregation through the modulation of protein phase separation. We observed that Ca2+ promotes the formation of α-Syn liquid droplets but does not change the protein fluidity inside the droplets. Further studies showed Ca2+-involved α-Syn droplets are still able to fuse. A metal chelator eliminated Ca2+-induced enlargement of α-Syn droplets, suggesting the influence of Ca2+ on α-Syn assembly could be reversed at the stage of liquid-liquid phase separation (LLPS). Interestingly, our data showed Ca2+ still promoted α-Syn phase separation in the presence of the lipid membranes. In addition, Ca2+/α-syn droplets could efficiently recruit lipid vesicles to the surface of these condensates. Our findings demonstrate that Ca2+ facilitates α-Syn phase separation to accelerate amyloid aggregation and pave the path for understanding the implications of Ca2+ in α-Syn accumulation and PD.
    Keywords:  Aggregation; Calcium; Droplet; Liquid-liquid phase separation (LLPS); α-Synuclein
    DOI:  https://doi.org/10.1016/j.bbrc.2022.02.097
  6. J Magn Reson. 2022 Feb 17. pii: S1090-7807(22)00027-1. [Epub ahead of print]337 107169
      Selective detection of biomolecules and their modifications in cells is essential for understanding cell functions and diseases. We have developed an NMR pulse sequence, Ac-FIND (Acetylation-FIltered aNd eDited), which uses isotope editing/filtering techniques for selective detection of protein acetylation. Acetylation of the N-terminus and lysine side chains by N-succinimidyl acetate was selectively observed for intrinsically disordered α-synuclein and well-ordered ubiquitin. Furthermore, when nonacetylated 13C/15N-enriched α-synuclein was introduced into live HEK293 cells, intracellular N-terminal acetylation of α-synuclein was detected by the appearance of a single peak using Ac-FIND. This work demonstrates the utility of NMR to detect a specific protein modification both in vitro and in live cells.
    Keywords:  Ac-FIND; In cell NMR; Isotope editing/filtering; Protein acetylation; α-Synuclein
    DOI:  https://doi.org/10.1016/j.jmr.2022.107169
  7. Nanoscale. 2022 Mar 08.
      The conversion of intrinsically disordered Tau to highly ordered amyloid aggregates is associated with a wide range of neurodegenerative diseases termed tauopathies. The presence of lipid bilayer membranes is a critical factor that accelerates the abnormal aggregation of Tau protein. However, the lipid membrane-induced conformational changes of Tau and the mechanism for the accelerated fibrillation remain elusive. In this study, single-molecule Förster resonance energy transfer (smFRET) and fluorescence correlation spectroscopy (FCS) were applied to detect the conformational changes and intermolecular interactions of full-length Tau in the presence of different concentrations of 1,2-dimyristoyl-sn-glycero-3-phosphatidylserine (DMPS) vesicles. The results show that the conformation of Tau becomes expanded with opening of the N-terminal and C-terminal domains of Tau upon binding to DMPS. At low DMPS concentrations, Tau forms oligomers with a partially extended conformation which facilitates the amyloid fibrillization process. At high DMPS concentrations, Tau monomer binds to lipid membranes in a fully expanded conformation at low density thus inhibiting intermolecular aggregation. Our study reveals the underlying mechanisms by which lipid membranes influence amyloid formation of Tau, providing a foundation for further understanding of the pathogenesis and physiology of the interplay between Tau protein and lipid membranes.
    DOI:  https://doi.org/10.1039/d1nr05960b
  8. Nat Commun. 2022 Mar 09. 13(1): 1223
      Trans-activation response DNA-binding protein of 43  kDa (TDP-43) regulates RNA processing and forms neuropathological aggregates in patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Investigating TDP-43 post-translational modifications, we discovered that K84 acetylation reduced nuclear import whereas K136 acetylation impaired RNA binding and splicing capabilities of TDP-43. Such failure of RNA interaction triggered TDP-43 phase separation mediated by the C-terminal low complexity domain, leading to the formation of insoluble aggregates with pathologically phosphorylated and ubiquitinated TDP-43. Introduction of acetyl-lysine at the identified sites via amber suppression confirmed the results from site-directed mutagenesis. K84-acetylated TDP-43 showed cytoplasmic mislocalization, and the aggregation propensity of K136-acetylated TDP-43 was confirmed. We generated antibodies selective for TDP-43 acetylated at these lysines, and found that sirtuin-1 can potently deacetylate K136-acetylated TDP-43 and reduce its aggregation propensity. Thus, distinct lysine acetylations modulate nuclear import, RNA binding and phase separation of TDP-43, suggesting regulatory mechanisms for TDP-43 pathogenesis.
    DOI:  https://doi.org/10.1038/s41467-022-28822-7