bims-indpro 2022-09-04 papers

bims-indpro

Biomed News

on Intrinsically disordered proteins

Issue of 2022–09–04
ten papers selected by
Sara Mingu, Johannes Gutenberg University

Guide to studying intrinsically disordered proteins by high-speed atomic force microscopy.
Recognizing the Binding Pattern and Dissociation Pathways of the p300 Taz2-p53 TAD2 Complex.
Investigating the conformational dynamics of Zika virus NS4B protein.
Contributions of the N-terminal intrinsically disordered region of the severe acute respiratory syndrome coronavirus 2 nucleocapsid protein to RNA-induced phase separation.
Mapping the per-residue surface electrostatic potential of CAPRIN1 along its phase-separation trajectory.
IDPConformerGenerator: A Flexible Software Suite for Sampling the Conformational Space of Disordered Protein States.
Functional analysis of the N-terminal region of acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis.
LncRNAs divide and rule: The master regulators of phase separation.
Percolation transition prescribes protein size-specific barrier to passive transport through the nuclear pore complex.
Exploring Structural Flexibility and Stability of α-Synuclein by the Landau-Ginzburg-Wilson Approach.

Methods. 2022 Aug 30. pii: S1046-2023(22)00184-0. [Epub ahead of print]

Guide to studying intrinsically disordered proteins by high-speed atomic force microscopy.

Noriyuki Kodera, Toshio Ando.

Intrinsically disordered proteins (IDPs) are partially or entirely disordered. Their intrinsically disordered regions (IDRs) dynamically explore a wide range of structural space by their highly flexible nature. Due to this distinct feature largely different from structured proteins, conventional structural analyses relying on ensemble averaging is unsuitable for characterizing the dynamic structure of IDPs. Therefore, single-molecule measurement tools have been desired in IDP studies. High-speed atomic force microscopy (HS-AFM) is a unique tool that allows us to directly visualize single biomolecules at 2-3 nm lateral and ∼0.1 nm vertical spatial resolution, and at sub-100 ms temporal resolution under near physiological conditions, without any chemical labeling. HS-AFM has been successfully used not only to characterize the shape and motion of IDP molecules but also to visualize their function-related dynamics. In this article, after reviewing the principle and current performances of HS-AFM, we describe experimental considerations in the HS-AFM imaging of IDPs and methods to quantity molecular features from captured images. Finally, we outline recent HS-AFM imaging studies of IDPs.

DOI: https://doi.org/10.1016/j.ymeth.2022.08.008
JACS Au. 2022 Aug 22. 2(8): 1935-1945

Recognizing the Binding Pattern and Dissociation Pathways of the p300 Taz2-p53 TAD2 Complex.

Tongtong Li, Stefano Motta, Amy O Stevens, Shenghan Song, Emily Hendrix, Alessandro Pandini, Yi He.

The dynamic association and dissociation between proteins are the basis of cellular signal transduction. This process becomes much more complicated if one or both interaction partners are intrinsically disordered because intrinsically disordered proteins can undergo disorder-to-order transitions upon binding to their partners. p53, a transcription factor with disordered regions, plays significant roles in many cellular signaling pathways. It is critical to understand the binding/unbinding mechanism involving these disordered regions of p53 at the residue level to reveal how p53 performs its biological functions. Here, we studied the dissociation process of the intrinsically disordered N-terminal transactivation domain 2 (TAD2) of p53 and the transcriptional adaptor zinc-binding 2 (Taz2) domain of transcriptional coactivator p300 using a combination of classical molecular dynamics, steered molecular dynamics, self-organizing maps, and time-resolved force distribution analysis (TRFDA). We observed two different dissociation pathways with different probabilities. One dissociation pathway starts from the TAD2 N-terminus and propagates to the α-helix and finally the C-terminus. The other dissociation pathway is in the opposite order. Subsequent TRFDA results reveal that key residues in TAD2 play critical roles. Besides the residues in agreement with previous experimental results, we also highlighted some other residues that play important roles in the disassociation process. In the dissociation process, non-native interactions were formed to partially compensate for the energy loss due to the breaking of surrounding native interactions. Moreover, our statistical analysis results of other experimentally determined complex structures involving either Taz2 or TAD2 suggest that the binding of the Taz2-TAD2 complex is mainly governed by the binding site of Taz2, which includes three main binding regions. Therefore, the complexes involving Taz2 may follow similar binding/unbinding behaviors, which could be studied together to generate common principles.

DOI: https://doi.org/10.1021/jacsau.2c00358
Virology. 2022 Aug 21. pii: S0042-6822(22)00129-5. [Epub ahead of print]575 20-35

Investigating the conformational dynamics of Zika virus NS4B protein.

Taniya Bhardwaj, Prateek Kumar, Rajanish Giri.

  Zika virus (ZIKV) NS4B protein is a membranotropic multifunctional protein. Despite its versatile functioning, its topology and dynamics are not entirely understood. There is no X-ray or cryo-EM structure available for any flaviviral NS4B full-length protein. In this study, we have investigated the structural dynamics of full-length ZIKV NS4B protein through 3D structure models using molecular dynamics simulations and experimental techniques. Also, we employed a reductionist approach to understand the dynamics of NS4B protein where we studied its N-terminal (residues 1-38), C-terminal (residues 194-251), and cytosolic (residues 131-169) regions in isolation in addition to the full-length protein. Further, using a series of circular dichroism spectroscopic experiments, we validate the cytosolic region as an intrinsically disordered protein region. The microsecond-long all atoms molecular dynamics and replica-exchange simulations complement the experimental observations. Furthermore, we have also studied the NS4B proteins C-terminal regions of four other flaviviruses viz. DENV2, JEV, WNV, and YFV through microsecond simulations to characterize their behaviour in presence and absence of lipid membranes. There are significant differences observed in the conformations of other flavivirus NS4B C-terminal regions in comparison to ZIKV NS4B. Lastly, we have proposed a ZIKV NS4B protein model illustrating its putative topology consisting of various membrane-spanning and non-membranous regions.

Keywords:  AlphaFold2; Cytosolic region; Intrinsically disordered protein region; Membrane topology; Molecular dynamics simulations

DOI:  https://doi.org/10.1016/j.virol.2022.08.005
Protein Sci. 2022 Sep;31(9): e4409

Contributions of the N-terminal intrinsically disordered region of the severe acute respiratory syndrome coronavirus 2 nucleocapsid protein to RNA-induced phase separation.

Milan Zachrdla, Adriana Savastano, Alain Ibáñez de Opakua, Maria-Sol Cima-Omori, Markus Zweckstetter.

  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein is an essential structural component of mature virions, encapsulating the genomic RNA and modulating RNA transcription and replication. Several of its activities might be associated with the protein's ability to undergo liquid-liquid phase separation. NSARS-CoV-2 contains an intrinsically disordered region at its N-terminus (NTE) that can be phosphorylated and is affected by mutations found in human COVID-19 infections, including in the Omicron variant of concern. Here, we show that NTE deletion decreases the range of RNA concentrations that can induce phase separation of NSARS-CoV-2 . In addition, deletion of the prion-like NTE allows NSARS-CoV-2 droplets to retain their liquid-like nature during incubation. We further demonstrate that RNA-binding engages multiple parts of the NTE and changes NTE's structural properties. The results form the foundation to characterize the impact of N-terminal mutations and post-translational modifications on the molecular properties of the SARS-CoV-2 nucleocapsid protein. STATEMENT: The nucleocapsid protein of SARS-CoV-2 plays an important role in both genome packaging and viral replication upon host infection. Replication has been associated with RNA-induced liquid-liquid phase separation of the nucleocapsid protein. We present insights into the role of the N-terminal part of the nucleocapsid protein in the protein's RNA-mediated liquid-liquid phase separation.

Keywords:  NMR; RNA; SARS-CoV-2; liquid-liquid phase separation; nucleocapsid protein

DOI:  https://doi.org/10.1002/pro.4409
Proc Natl Acad Sci U S A. 2022 Sep 06. 119(36): e2210492119

Mapping the per-residue surface electrostatic potential of CAPRIN1 along its phase-separation trajectory.

Yuki Toyama, Atul Kaushik Rangadurai, Julie D Forman-Kay, Lewis E Kay.

  Electrostatic interactions and charge balance are important for the formation of biomolecular condensates involving proteins and nucleic acids. However, a detailed, atomistic picture of the charge distribution around proteins during the phase-separation process is lacking. Here, we use solution NMR spectroscopy to measure residue-specific near-surface electrostatic potentials (ϕENS) of the positively charged carboxyl-terminal intrinsically disordered 103 residues of CAPRIN1, an RNA-binding protein localized to membraneless organelles playing an important role in messenger RNA (mRNA) storage and translation. Measured ϕENS values have been mapped along the adenosine triphosphate (ATP)-induced phase-separation trajectory. In the absence of ATP, ϕENS values for the mixed state of CAPRIN1 are positive and large and progressively decrease as ATP is added. This is coupled to increasing interchain interactions, particularly between aromatic-rich and arginine-rich regions of the protein. Upon phase separation, CAPRIN1 molecules in the condensed phase are neutral (ϕENS [Formula: see text] 0 mV), with ∼five molecules of ATP associated with each CAPRIN1 chain. Increasing the ATP concentration further inverts the CAPRIN1 electrostatic potential, so that molecules become negatively charged, especially in aromatic-rich regions, leading to re-entrance into a mixed phase. Our results collectively show that a subtle balance between electrostatic repulsion and interchain attractive interactions regulates CAPRIN1 phase separation and provides insight into how nucleotides, such as ATP, can induce formation of and subsequently dissolve protein condensates.

Keywords:  ATP; biomolecular condensates; intrinsically disordered proteins; paramagnetic relaxation enhancement; solution NMR

DOI:  https://doi.org/10.1073/pnas.2210492119
J Phys Chem A. 2022 Aug 28.

IDPConformerGenerator: A Flexible Software Suite for Sampling the Conformational Space of Disordered Protein States.

João M C Teixeira, Zi Hao Liu, Ashley Namini, Jie Li, Robert M Vernon, Mickaël Krzeminski, Alaa A Shamandy, Oufan Zhang, Mojtaba Haghighatlari, Lei Yu, Teresa Head-Gordon, Julie D Forman-Kay.

The power of structural information for informing biological mechanisms is clear for stable folded macromolecules, but similar structure-function insight is more difficult to obtain for highly dynamic systems such as intrinsically disordered proteins (IDPs) which must be described as structural ensembles. Here, we present IDPConformerGenerator, a flexible, modular open-source software platform for generating large and diverse ensembles of disordered protein states that builds conformers that obey geometric, steric, and other physical restraints on the input sequence. IDPConformerGenerator samples backbone phi (φ), psi (ψ), and omega (ω) torsion angles of relevant sequence fragments from loops and secondary structure elements extracted from folded protein structures in the RCSB Protein Data Bank and builds side chains from robust Monte Carlo algorithms using expanded rotamer libraries. IDPConformerGenerator has many user-defined options enabling variable fractional sampling of secondary structures, supports Bayesian models for assessing the agreement of IDP ensembles for consistency with experimental data, and introduces a machine learning approach to transform between internal and Cartesian coordinates with reduced error. IDPConformerGenerator will facilitate the characterization of disordered proteins to ultimately provide structural insights into these states that have key biological functions.

DOI: https://doi.org/10.1021/acs.jpca.2c03726
FEBS Open Bio. 2022 Aug 24.

Functional analysis of the N-terminal region of acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis.

Kohei Sasamoto, Tomoki Himiyama, Kunihiko Moriyoshi, Takashi Ohmoto, Koichi Uegaki, Tsutomu Nakamura, Yoshiaki Nishiya.

  Acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis (TTE0866) has an N-terminal region (NTR; residues 23-135) between the signal sequence (residues 1-22) and the catalytic domain (residues 136-324), which is of unknown function. Our previous study revealed the crystal structure of the wild-type (WT) enzyme containing the NTR and the catalytic domain. Although the structure of the catalytic domain was successfully determined, that of the NTR was undetermined, as its electron density was unclear. In this study, we investigated the role of the NTR through functional and structural analyses of NTR truncation mutants. Based on sequence and secondary structure analyses, NTR was confirmed to be an intrinsically disordered region. The truncation of NTR significantly decreased the solubility of the proteins at low salt concentrations compared to that of the WT. The NTR-truncated mutant easily crystallized in a conventional buffer solution. The crystal exhibited crystallographic properties comparable to those of the WT crystals suitable for structural determination. These results suggest that NTR plays a role in maintaining the solubility and inhibiting the crystallization of the catalytic domain.

Keywords:  Caldanaerobacter; acetylxylan esterase; crystallization; intrinsically disordered region; protein solubility

DOI:  https://doi.org/10.1002/2211-5463.13476
Front Genet. 2022 ;13 930792

LncRNAs divide and rule: The master regulators of phase separation.

Kumaravel Somasundaram, Bhavana Gupta, Nishkarsh Jain, Samarjit Jana.

  Most of the human genome, except for a small region that transcribes protein-coding RNAs, was considered junk. With the advent of RNA sequencing technology, we know that much of the genome codes for RNAs with no protein-coding potential. Long non-coding RNAs (lncRNAs) that form a significant proportion are dynamically expressed and play diverse roles in physiological and pathological processes. Precise spatiotemporal control of their expression is essential to carry out various biochemical reactions inside the cell. Intracellular organelles with membrane-bound compartments are known for creating an independent internal environment for carrying out specific functions. The formation of membrane-free ribonucleoprotein condensates resulting in intracellular compartments is documented in recent times to execute specialized tasks such as DNA replication and repair, chromatin remodeling, transcription, and mRNA splicing. These liquid compartments, called membrane-less organelles (MLOs), are formed by liquid-liquid phase separation (LLPS), selectively partitioning a specific set of macromolecules from others. While RNA binding proteins (RBPs) with low complexity regions (LCRs) appear to play an essential role in this process, the role of RNAs is not well-understood. It appears that short nonspecific RNAs keep the RBPs in a soluble state, while longer RNAs with unique secondary structures promote LLPS formation by specifically binding to RBPs. This review will update the current understanding of phase separation, physio-chemical nature and composition of condensates, regulation of phase separation, the role of lncRNA in the phase separation process, and the relevance to cancer development and progression.

Keywords:  N6-methylAdenosine (m6A); RNA binding proteins; RNA granules; biomolecular condensates; intrinsically disordered region; lncRNA; multivalency; phase separation

DOI:  https://doi.org/10.3389/fgene.2022.930792
Nat Commun. 2022 Sep 01. 13(1): 5138

Percolation transition prescribes protein size-specific barrier to passive transport through the nuclear pore complex.

David Winogradoff, Han-Yi Chou, Christopher Maffeo, Aleksei Aksimentiev.

Nuclear pore complexes (NPCs) control biomolecular transport in and out of the nucleus. Disordered nucleoporins in the complex's pore form a permeation barrier, preventing unassisted transport of large biomolecules. Here, we combine coarse-grained simulations of experimentally derived NPC structures with a theoretical model to determine the microscopic mechanism of passive transport. Brute-force simulations of protein transport reveal telegraph-like behavior, where prolonged diffusion on one side of the NPC is interrupted by rapid crossings to the other. We rationalize this behavior using a theoretical model that reproduces the energetics and kinetics of permeation solely from statistics of transient voids within the disordered mesh. As the protein size increases, the mesh transforms from a soft to a hard barrier, enabling orders-of-magnitude reduction in permeation rate for proteins beyond the percolation size threshold. Our model enables exploration of alternative NPC architectures and sets the stage for uncovering molecular mechanisms of facilitated nuclear transport.

DOI: https://doi.org/10.1038/s41467-022-32857-1
J Phys Chem B. 2022 Sep 02.

Exploring Structural Flexibility and Stability of α-Synuclein by the Landau-Ginzburg-Wilson Approach.

Anatolii Korneev, Alexander Begun, Sergei Liubimov, Khatuna Kachlishvili, Alexander Molochkov, Antti J Niemi, Gia G Maisuradze.

α-Synuclein (αS) is the principal protein component of the Lewy body and Lewy neurite deposits that are found in the brains of the victims of one of the most prevalent neurodegenerative disorders, Parkinson's disease. αS can be qualified as a chameleon protein because of the large number of different conformations that it is able to adopt: it is disordered under physiological conditions in solution, in equilibrium with a minor α-helical tetrameric form in the cytoplasm, and is α-helical when bound to a cell membrane. Also, in vitro, αS forms polymorphic amyloid fibrils with unique arrangements of cross-β-sheet motifs. Therefore, it is of interest to elucidate the origins of the structural flexibility of αS and what makes αS stable in different conformations. We address these questions here by analyzing the experimental structures of the micelle-bound, tetrameric, and fibrillar αS in terms of a kink (heteroclinic standing wave solution) of a generalized discrete nonlinear Schrödinger equation. It is illustrated that without molecular dynamics simulations the kinks are capable of identifying the key residues causing structural flexibility of αS. Also, the stability of the experimental structures of αS is investigated by simulating heating/cooling trajectories using the Glauber algorithm. The findings are consistent with experiments.

DOI: https://doi.org/10.1021/acs.jpcb.2c04651