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Biophysical Characterization and Molecular Engineering of Multidomain Proteins 3.0
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Biophysical Characterization and Molecular Engineering of Multidomain Proteins 3.0

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Biophysics".

Deadline for manuscript submissions: closed (25 December 2023) | Viewed by 11399

Special Issue Editors

Prof. Dr. Koichi Kato

E-Mail Website
Guest Editor
Department of Creative Research, Biomolecular Organization Research Group, Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences, Okazaki 444-8787, Japan
Interests: biomolecular ordering; glycobiophysics; biomolecular NMR spectroscopy
Special Issues, Collections and Topics in MDPI journals
Prof. Dr. Takayuki Uchihashi

E-Mail Website
Guest Editor
Laboratory of Biomolecular Dynamics and Function, Department of Physics, Nagoya University, Nagoya 464-8602, Japan
Interests: single-molecule biophysics; protein dynamics; protein assembly; atomic force microscopy; optical microscopy
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

This Special Issue is the continuation of our previous Special Issues "Biophysical Characterization and Molecular Engineering of Multidomain Proteins" and “Biophysical Characterization and Molecular Engineering of Multidomain Proteins 2.0”

Most proteins working in living systems consist of evolutionarily acquired multiple domains, which cooperate with one another and exhibit synergistic actions, thereby exerting sophisticated functions typified by allosteric regulations. Structural proteomics, in conjunction with bioinformatics, have achieved the systematic classification and prediction of tertiary structures of individual domains as globular structural units. However, it remains challenging to delineate or predict the overall conformations of multidomain proteins, primarily because of their dynamic properties. In this class of proteins, the globular domains are connected through flexible linkers and, consequently, are mobile to a greater or lesser extent, which enables variable spatial arrangements of the domains, depending on their cognate ligands or binding partners, as well as solution conditions such as pH. Therefore, to elucidate the mechanisms underlying multidomain protein functions, applications of experimental and theoretical methods are necessary in order to provide dynamic views of domain–domain interactions. This line of approach will offer a structural basis for the design and engineering of multidomain proteins.

As the guest editors of this Special Issue, titled “Biophysical Characterization and Molecular Engineering of Multidomain Proteins 3.0”, in IJMS, we welcome contributions from various research fields, including biophysics, bioinformatics, biomolecular engineering, and molecular phylogenetics. Formats for submissions include original research reports, reviews/mini-reviews, perspectives/opinions, and methodology articles.

Prof. Dr. Koichi Kato
Prof. Dr. Takayuki Uchihashi
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. There is an Article Processing Charge (APC) for publication in this open access journal. For details about the APC please see here. Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Published Papers (7 papers)

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Research

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13 pages, 5519 KiB  
Article
Interactions of the N- and C-Terminal SH3 Domains of Drosophila Drk with the Proline-Rich Peptides from Sos and Dos
by Pooppadi Maxin Sayeesh, Mayumi Iguchi, Yusuke Suemoto, Jin Inoue, Kohsuke Inomata, Teppei Ikeya and Yutaka Ito
Int. J. Mol. Sci. 2023, 24(18), 14135; https://doi.org/10.3390/ijms241814135 - 15 Sep 2023
Viewed by 891
Abstract
Drk, a homologue of human GRB2 in Drosophila, receives signals from outside the cells through the interaction of its SH2 domain with the phospho-tyrosine residues in the intracellular regions of receptor tyrosine kinases (RTKs) such as Sevenless, and transduces the signals downstream [...] Read more.
Drk, a homologue of human GRB2 in Drosophila, receives signals from outside the cells through the interaction of its SH2 domain with the phospho-tyrosine residues in the intracellular regions of receptor tyrosine kinases (RTKs) such as Sevenless, and transduces the signals downstream through the association of its N- and C-terminal SH3 domains (Drk-NSH3 and Drk-CSH3, respectively) with proline-rich motifs (PRMs) in Son of Sevenless (Sos) or Daughter of Sevenless (Dos). Isolated Drk-NSH3 exhibits a conformational equilibrium between the folded and unfolded states, while Drk-CSH3 adopts only a folded confirmation. Drk interacts with PRMs of the PxxPxR motif in Sos and the PxxxRxxKP motif in Dos. Our previous study has shown that Drk-CSH3 can bind to Sos, but the interaction between Drk-NSH3 and Dos has not been investigated. To assess the affinities of both SH3 domains towards Sos and Dos, we conducted NMR titration experiments using peptides derived from Sos and Dos. Sos-S1 binds to Drk-NSH3 with the highest affinity, strongly suggesting that the Drk-Sos multivalent interaction is initiated by the binding of Sos-S1 and NSH3. Our results also revealed that the two Sos-derived PRMs clearly favour NSH3 for binding, whereas the two Dos-derived PRMs show almost similar affinity for NSH3 and CSH3. We have also performed docking simulations based on the chemical shift perturbations caused by the addition of Sos- and Dos-derived peptides. Finally, we discussed the various modes in the interactions of Drk with Sos/Dos. Full article
(This article belongs to the Special Issue Biophysical Characterization and Molecular Engineering of Multidomain Proteins 3.0)
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15 pages, 3406 KiB  
Article
Structures of the Insecticidal Toxin Complex Subunit XptA2 Highlight Roles for Flexible Domains
by Cole L. Martin, David W. Chester, Christopher D. Radka, Lurong Pan, Zhengrong Yang, Rachel C. Hart, Elad M. Binshtein, Zhao Wang, Lisa Nagy, Lawrence J. DeLucas and Stephen G. Aller
Int. J. Mol. Sci. 2023, 24(17), 13221; https://doi.org/10.3390/ijms241713221 - 25 Aug 2023
Viewed by 1243
Abstract
The Toxin Complex (Tc) superfamily consists of toxin translocases that contribute to the targeting, delivery, and cytotoxicity of certain pathogenic Gram-negative bacteria. Membrane receptor targeting is driven by the A-subunit (TcA), which comprises IgG-like receptor binding domains (RBDs) at the surface. To better [...] Read more.
The Toxin Complex (Tc) superfamily consists of toxin translocases that contribute to the targeting, delivery, and cytotoxicity of certain pathogenic Gram-negative bacteria. Membrane receptor targeting is driven by the A-subunit (TcA), which comprises IgG-like receptor binding domains (RBDs) at the surface. To better understand XptA2, an insect specific TcA secreted by the symbiont X. nematophilus from the intestine of entomopathogenic nematodes, we determined structures by X-ray crystallography and cryo-EM. Contrary to a previous report, XptA2 is pentameric. RBD-B exhibits an indentation from crystal packing that indicates loose association with the shell and a hotspot for possible receptor binding or a trigger for conformational dynamics. A two-fragment XptA2 lacking an intact linker achieved the folded pre-pore state like wild type (wt), revealing no requirement of the linker for protein folding. The linker is disordered in all structures, and we propose it plays a role in dynamics downstream of the initial pre-pore state. Full article
(This article belongs to the Special Issue Biophysical Characterization and Molecular Engineering of Multidomain Proteins 3.0)
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11 pages, 2043 KiB  
Article
Molecular Design of FRET Probes Based on Domain Rearrangement of Protein Disulfide Isomerase for Monitoring Intracellular Redox Status
by Maho Yagi-Utsumi, Haruko Miura, Christian Ganser, Hiroki Watanabe, Methanee Hiranyakorn, Tadashi Satoh, Takayuki Uchihashi, Koichi Kato, Kei-ichi Okazaki and Kazuhiro Aoki
Int. J. Mol. Sci. 2023, 24(16), 12865; https://doi.org/10.3390/ijms241612865 - 16 Aug 2023
Viewed by 1157
Abstract
Multidomain proteins can exhibit sophisticated functions based on cooperative interactions and allosteric regulation through spatial rearrangements of the multiple domains. This study explored the potential of using multidomain proteins as a basis for Förster resonance energy transfer (FRET) biosensors, focusing on protein disulfide [...] Read more.
Multidomain proteins can exhibit sophisticated functions based on cooperative interactions and allosteric regulation through spatial rearrangements of the multiple domains. This study explored the potential of using multidomain proteins as a basis for Förster resonance energy transfer (FRET) biosensors, focusing on protein disulfide isomerase (PDI) as a representative example. PDI, a well-studied multidomain protein, undergoes redox-dependent conformational changes, enabling the exposure of a hydrophobic surface extending across the b’ and a’ domains that serves as the primary binding site for substrates. Taking advantage of the dynamic domain rearrangements of PDI, we developed FRET-based biosensors by fusing the b’ and a’ domains of thermophilic fungal PDI with fluorescent proteins as the FRET acceptor and donor, respectively. Both experimental and computational approaches were used to characterize FRET efficiency in different redox states. In vitro and in vivo evaluations demonstrated higher FRET efficiency of this biosensor in the oxidized form, reflecting the domain rearrangement and its responsiveness to intracellular redox environments. This novel approach of exploiting redox-dependent domain dynamics in multidomain proteins offers promising opportunities for designing innovative FRET-based biosensors with potential applications in studying cellular redox regulation and beyond. Full article
(This article belongs to the Special Issue Biophysical Characterization and Molecular Engineering of Multidomain Proteins 3.0)
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12 pages, 2405 KiB  
Article
Mutational and Environmental Effects on the Dynamic Conformational Distributions of Lys48-Linked Ubiquitin Chains
by Methanee Hiranyakorn, Maho Yagi-Utsumi, Saeko Yanaka, Naoya Ohtsuka, Norie Momiyama, Tadashi Satoh and Koichi Kato
Int. J. Mol. Sci. 2023, 24(7), 6075; https://doi.org/10.3390/ijms24076075 - 23 Mar 2023
Cited by 1 | Viewed by 1207
Abstract
In multidomain proteins, individual domains connected by flexible linkers are dynamically rearranged upon ligand binding and sensing changes in environmental factors, such as pH and temperature. Here, we characterize dynamic domain rearrangements of Lys48-linked ubiquitin (Ub) chains as models of multidomain proteins in [...] Read more.
In multidomain proteins, individual domains connected by flexible linkers are dynamically rearranged upon ligand binding and sensing changes in environmental factors, such as pH and temperature. Here, we characterize dynamic domain rearrangements of Lys48-linked ubiquitin (Ub) chains as models of multidomain proteins in which molecular surfaces mediating intermolecular interactions are involved in intramolecular domain–domain interactions. Using NMR and other biophysical techniques, we characterized dynamic conformational interconversions of diUb between open and closed states regarding solvent exposure of the hydrophobic surfaces of each Ub unit, which serve as binding sites for various Ub-interacting proteins. We found that the hydrophobic Ub-Ub interaction in diUb was reinforced by cysteine substitution of Lys48 of the distal Ub unit because of interaction between the cysteinyl thiol group and the C-terminal segment of the proximal Ub unit. In contrast, the replacement of the isopeptide linker with an artificial ethylenamine linker minimally affected the conformational distributions. Furthermore, we demonstrated that the mutational modification allosterically impacted the exposure of the most distal Ub unit in triUb. Thus, the conformational interconversion of Ub chains offers a unique design framework in Ub-based protein engineering not only for developing biosensing probes but also for allowing new opportunities for the allosteric regulation of multidomain proteins. Full article
(This article belongs to the Special Issue Biophysical Characterization and Molecular Engineering of Multidomain Proteins 3.0)
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19 pages, 3025 KiB  
Article
Deciphering the Binding of the Nuclear Localization Sequence of Myc Protein to the Nuclear Carrier Importin α3
by Bruno Rizzuti, Juan L. Iovanna and José L. Neira
Int. J. Mol. Sci. 2022, 23(23), 15333; https://doi.org/10.3390/ijms232315333 - 05 Dec 2022
Viewed by 1532
Abstract
The oncoprotein Myc is a transcription factor regulating global gene expression and modulating cell proliferation, apoptosis, and metabolism. Myc has a nuclear localization sequence (NLS) comprising residues Pro320 to Asp328, to allow for nuclear translocation. We designed a peptide comprising such region and [...] Read more.
The oncoprotein Myc is a transcription factor regulating global gene expression and modulating cell proliferation, apoptosis, and metabolism. Myc has a nuclear localization sequence (NLS) comprising residues Pro320 to Asp328, to allow for nuclear translocation. We designed a peptide comprising such region and the flanking residues (Ala310-Asn339), NLS-Myc, to study, in vitro and in silico, the ability to bind importin α3 (Impα3) and its truncated species (ΔImpα3) depleted of the importin binding domain (IBB), by using fluorescence, circular dichroism (CD), biolayer interferometry (BLI), nuclear magnetic resonance (NMR), and molecular simulations. NLS-Myc interacted with both importin species, with affinity constants of ~0.5 µM (for Impα3) and ~60 nM (for ΔImpα3), as measured by BLI. The molecular simulations predicted that the anchoring of NLS-Myc took place in the major binding site of Impα3 for the NLS of cargo proteins. Besides clarifying the conformational behavior of the isolated NLS of Myc in solution, our results identified some unique properties in the binding of this localization sequence to the nuclear carrier Impα3, such as a difference in the kinetics of its release mechanism depending on the presence or absence of the IBB domain. Full article
(This article belongs to the Special Issue Biophysical Characterization and Molecular Engineering of Multidomain Proteins 3.0)
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11 pages, 2923 KiB  
Article
Utilization of AlphaFold2 to Predict MFS Protein Conformations after Selective Mutation
by Qingjie Xiao, Mengxue Xu, Weiwei Wang, Tingting Wu, Weizhe Zhang, Wenming Qin and Bo Sun
Int. J. Mol. Sci. 2022, 23(13), 7235; https://doi.org/10.3390/ijms23137235 - 29 Jun 2022
Cited by 7 | Viewed by 2359
Abstract
The major facilitator superfamily (MFS) is the largest secondary transporter family and is responsible for transporting a broad range of substrates across the biomembrane. These proteins are involved in a series of conformational changes during substrate transport. To decipher the transport mechanism, it [...] Read more.
The major facilitator superfamily (MFS) is the largest secondary transporter family and is responsible for transporting a broad range of substrates across the biomembrane. These proteins are involved in a series of conformational changes during substrate transport. To decipher the transport mechanism, it is necessary to obtain structures of these different conformations. At present, great progress has been made in predicting protein structure based on coevolutionary information. In this study, AlphaFold2 was used to predict different conformational structures for 69 MFS transporters of E. coli after the selective mutation of residues at the interface between the N- and C-terminal domains. The predicted structures for these mutants had small RMSD values when compared to structures obtained using X-ray crystallography, which indicates that AlphaFold2 predicts the structure of MSF transporters with high accuracy. In addition, different conformations of other transporter family proteins have been successfully predicted based on mutation methods. This study provides a structural basis to study the transporting mechanism of the MFS transporters and a method to probe dynamic conformation changes of transporter family proteins when performing their function. Full article
(This article belongs to the Special Issue Biophysical Characterization and Molecular Engineering of Multidomain Proteins 3.0)
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Review

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19 pages, 5869 KiB  
Review
State-of-the-Art Molecular Dynamics Simulation Studies of RNA-Dependent RNA Polymerase of SARS-CoV-2
by Shoichi Tanimoto, Satoru G. Itoh and Hisashi Okumura
Int. J. Mol. Sci. 2022, 23(18), 10358; https://doi.org/10.3390/ijms231810358 - 08 Sep 2022
Cited by 4 | Viewed by 2180
Abstract
Molecular dynamics (MD) simulations are powerful theoretical methods that can reveal biomolecular properties, such as structure, fluctuations, and ligand binding, at the level of atomic detail. In this review article, recent MD simulation studies on these biomolecular properties of the RNA-dependent RNA polymerase [...] Read more.
Molecular dynamics (MD) simulations are powerful theoretical methods that can reveal biomolecular properties, such as structure, fluctuations, and ligand binding, at the level of atomic detail. In this review article, recent MD simulation studies on these biomolecular properties of the RNA-dependent RNA polymerase (RdRp), which is a multidomain protein, of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are presented. Although the tertiary structures of RdRps in SARS-CoV-2 and SARS-CoV are almost identical, the RNA synthesis activity of RdRp of SARS-CoV is higher than SARS-CoV-2. Recent MD simulations observed a difference in the dynamic properties of the two RdRps, which may cause activity differences. RdRp is also a drug target for Coronavirus disease 2019 (COVID-19). Nucleotide analogs, such as remdesivir and favipiravir, are considered to be taken up by RdRp and inhibit RNA replication. Recent MD simulations revealed the recognition mechanism of RdRp for these drug molecules and adenosine triphosphate (ATP). The ligand-recognition ability of RdRp decreases in the order of remdesivir, favipiravir, and ATP. As a typical recognition process, it was found that several lysine residues of RdRp transfer these ligand molecules to the binding site such as a “bucket brigade.” This finding will contribute to understanding the mechanism of the efficient ligand recognition by RdRp. In addition, various simulation studies on the complexes of SARS-CoV-2 RdRp with several nucleotide analogs are reviewed, and the molecular mechanisms by which these compounds inhibit the function of RdRp are discussed. The simulation studies presented in this review will provide useful insights into how nucleotide analogs are recognized by RdRp and inhibit the RNA replication. Full article
(This article belongs to the Special Issue Biophysical Characterization and Molecular Engineering of Multidomain Proteins 3.0)
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