Structural Biology

Structural Motif ◽

Post Translational Modifications ◽

Exon 1 ◽

Molecular Therapeutic

The 3,144–amino acid huntingtin protein (HTT) folds in water into a structure consisting of compact, organized domains interspersed with intrinsically disordered protein (IDP) elements. The IDPs function as sites of post-translational modifications and proteolysis as well as in targeting, binding, and aggregation. Although the dominant structural motif of HTT is the α‎-helix–rich HEAT repeat, the expanded polyglutamine (polyQ) toxicity responsible for Huntington’s disease is most likely played out within intrinsically disordered HTT exon 1–like fragments consisting of the 16– to 17–amino acid N-terminal HTTNT segment, the polyQ segment, and a proline-rich segment. The physical behavior of HTT exon 1 fragments is dominated by interactive, polyQ repeat length–dependent structural transitions responsible for membrane and protein–protein interactions and the formation of tetramers, higher oligomers, amyloid fibrils, and inclusions. Understanding the basis of this solution behavior may be the key to disease mechanisms and molecular therapeutic strategies.

Intrinsic Disorder in Tetratricopeptide Repeat Proteins

International Journal of Molecular Sciences ◽

10.3390/ijms21103709 ◽

2020 ◽

Vol 21 (10) ◽

pp. 3709 ◽

Cited By ~ 1

Author(s):

Nathan W. Van Bibber ◽

Cornelia Haerle ◽

Roy Khalife ◽

Bin Xue ◽

Vladimir N. Uversky

Keyword(s):

Protein Interactions ◽

Posttranslational Modifications ◽

Tandem Repeats ◽

Intrinsic Disorder ◽

Systematic Analysis ◽

Tetratricopeptide Repeats ◽

Tetratricopeptide Repeat Proteins

Among the realm of repeat containing proteins that commonly serve as “scaffolds” promoting protein-protein interactions, there is a family of proteins containing between 2 and 20 tetratricopeptide repeats (TPRs), which are functional motifs consisting of 34 amino acids. The most distinguishing feature of TPR domains is their ability to stack continuously one upon the other, with these stacked repeats being able to affect interaction with binding partners either sequentially or in combination. It is known that many repeat-containing proteins are characterized by high levels of intrinsic disorder, and that many protein tandem repeats can be intrinsically disordered. Furthermore, it seems that TPR-containing proteins share many characteristics with hybrid proteins containing ordered domains and intrinsically disordered protein regions. However, there has not been a systematic analysis of the intrinsic disorder status of TPR proteins. To fill this gap, we analyzed 166 human TPR proteins to determine the degree to which proteins containing TPR motifs are affected by intrinsic disorder. Our analysis revealed that these proteins are characterized by different levels of intrinsic disorder and contain functional disordered regions that are utilized for protein-protein interactions and often serve as targets of various posttranslational modifications.

Affinity versus specificity in coupled binding and folding reactions

Protein Engineering Design and Selection ◽

10.1093/protein/gzz020 ◽

2019 ◽

Author(s):

Stefano Gianni ◽

Per Jemth

Keyword(s):

Protein Interactions ◽

Intrinsically Disordered Proteins ◽

High Specificity ◽

Disordered Proteins ◽

Intrinsically Disordered Regions ◽

Interaction Interface ◽

Disordered Regions

Abstract Intrinsically disordered protein regions may fold upon binding to an interaction partner. It is often argued that such coupled binding and folding enables the combination of high specificity with low affinity. The basic tenet is that an unfavorable folding equilibrium will make the overall binding weaker while maintaining the interaction interface. While theoretically solid, we argue that this concept may be misleading for intrinsically disordered proteins. In fact, experimental evidence suggests that interactions of disordered regions usually involve extended conformations. In such cases, the disordered region is exceptionally unlikely to fold into a bound conformation in the absence of its binding partner. Instead, these disordered regions can bind to their partners in multiple different conformations and then fold into the native bound complex, thus, if anything, increasing the affinity through folding. We concede that (de)stabilization of native structural elements such as helices will modulate affinity, but this could work both ways, decreasing or increasing the stability of the complex. Moreover, experimental data show that intrinsically disordered binding regions display a range of affinities and specificities dictated by the particular side chains and length of the disordered region and not necessarily by the fact that they are disordered. We find it more likely that intrinsically disordered regions are common in protein–protein interactions because they increase the repertoire of binding partners, providing an accessible route to evolve interactions rather than providing a stability–affinity trade-off.

The disordered boundary of the cell: emerging properties of membrane-bound intrinsically disordered proteins

BioMolecular Concepts ◽

10.1515/bmc-2019-0003 ◽

2019 ◽

Vol 10 (1) ◽

pp. 25-36 ◽

Cited By ~ 1

Author(s):

Irrem-Laareb Mohammad ◽

Borja Mateos ◽

Miquel Pons

Keyword(s):

Lipid Bilayers ◽

Protein Interactions ◽

Intrinsically Disordered Proteins ◽

Disordered Proteins ◽

High Concentrations ◽

The Individual ◽

Confined Environment

AbstractWe define the disordered boundary of the cell (DBC) as the system formed by membrane tethered intrinsically disordered protein regions, dynamically coupled to the underlying membrane.The emerging properties of the DBC makes it a global system of study, which cannot be understood from the individual properties of their components. Similarly, the properties of lipid bilayers cannot be understood from just the sum of the properties of individual lipid molecules.The highly anisotropic confined environment, restricting the position and orientation of interacting sites, is affecting the properties of individual disordered proteins. In fact, the collective effect caused by high concentrations of disordered proteins extend beyond the sum of individual effects.Examples of emerging properties of the DBC include enhanced protein-protein interactions, protein-driven phase separations, Z-compartmentalization, and protein modulated electrostatics.

Dynamics, Conformational Entropy, and Frustration in Protein–Protein Interactions Involving an Intrinsically Disordered Protein Domain

ACS Chemical Biology ◽

10.1021/acschembio.7b01105 ◽

2018 ◽

Vol 13 (5) ◽

pp. 1218-1227 ◽

Cited By ~ 16

Author(s):

Ida Lindström ◽

Jakob Dogan

Keyword(s):

Protein Interactions ◽

Protein Domain ◽

Conformational Entropy ◽

Disordered Protein

Proteome-scale amino-acid resolution footprinting of protein-binding sites in the intrinsically disordered regions of the human proteome

10.1101/2021.04.13.439572 ◽

2021 ◽

Author(s):

Caroline Benz ◽

Muhammad Ali ◽

Izabella Krystkowiak ◽

Leandro Simonetti ◽

Ahmed Sayadi ◽

...

Keyword(s):

Amino Acid ◽

Protein Interactions ◽

Human Proteome ◽

Cell Physiology ◽

Linear Motif ◽

Intrinsically Disordered Regions ◽

Wide Range ◽

Disordered Regions

Specific protein-protein interactions are central to all processes that underlie cell physiology. Numerous studies using a wide range of experimental approaches have identified tens of thousands of human protein-protein interactions. However, many interactions remain to be discovered, and low affinity, conditional and cell type-specific interactions are likely to be disproportionately under-represented. Moreover, for most known protein-protein interactions the binding regions remain uncharacterized. We previously developed proteomic peptide phage display (ProP-PD), a method for simultaneous proteome-scale identification of short linear motif (SLiM)-mediated interactions and footprinting of the binding region with amino acid resolution. Here, we describe the second-generation human disorderome (HD2), an optimized ProP-PD library that tiles all disordered regions of the human proteome and allows the screening of ~1,000,000 overlapping peptides in a single binding assay. We define guidelines for how to process, filter and rank the results and provide PepTools, a toolkit for annotation and analysis of identified hits. We uncovered 2,161 interaction pairs for 35 known SLiM-binding domains and confirmed a subset of 38 interactions by biophysical or cell-based assays. Finally, we show how the amino acid resolution binding site information can be used to pinpoint functionally important disease mutations and phosphorylation events in intrinsically disordered regions of the human proteome. The HD2 ProP-PD library paired with PepTools represents a powerful pipeline for unbiased proteome-wide discovery of SLiM-based interactions.

Coordination of RNA and protein condensation by the P granule protein MEG-3

10.1101/2020.10.15.340570 ◽

2020 ◽

Author(s):

Helen Schmidt ◽

Andrea Putnam ◽

Dominique Rasoloson ◽

Geraldine Seydoux

Keyword(s):

Protein Interactions ◽

C Elegans ◽

Disordered Protein ◽

C Terminus ◽

Necessary And Sufficient

ABSTRACTGerm granules are RNA-protein condensates in germ cells. The mechanisms that drive germ granule assembly are not fully understood. MEG-3 is an intrinsically-disordered protein required for germ (P) granule assembly in C. elegans. MEG-3 forms gel-like condensates on liquid condensates assembled by PGL proteins. MEG-3 is related to the GCNA family and contains an N-terminal disordered region (IDR) and a predicted ordered C-terminus featuring an HMG-like motif (HMGL). Using in vitro and in vivo experiments, we find the MEG-3 C-terminus is necessary and sufficient to build MEG-3/PGL co-condensates independent of RNA. The HMGL domain is required for high affinity MEG-3/PGL binding in vitro and for assembly of MEG-3/PGL co-condensates in vivo. The MEG-3 IDR binds RNA in vitro and is required but not sufficient to recruit RNA to P granules. Our findings suggest that P granule assembly depends in part on protein-protein interactions that drive condensation independent of RNA.

Peptide array–based interactomics

Analytical and Bioanalytical Chemistry ◽

10.1007/s00216-021-03367-8 ◽

2021 ◽

Author(s):

Daniel Perez Hernandez ◽

Gunnar Dittmar

Keyword(s):

Amino Acid ◽

Protein Interactions ◽

Large Scale ◽

New Technologies ◽

Membrane Surface ◽

Post Translational Modifications ◽

Short Linear Motifs ◽

Binding Partners ◽

Linear Motifs

AbstractThe analysis of protein-protein interactions (PPIs) is essential for the understanding of cellular signaling. Besides probing PPIs with immunoprecipitation-based techniques, peptide pull-downs are an alternative tool specifically useful to study interactome changes induced by post-translational modifications. Peptides for pull-downs can be chemically synthesized and thus offer the possibility to include amino acid exchanges and post-translational modifications (PTMs) in the pull-down reaction. The combination of peptide pull-down and analysis of the binding partners with mass spectrometry offers the direct measurement of interactome changes induced by PTMs or by amino acid exchanges in the interaction site. The possibility of large-scale peptide synthesis on a membrane surface opened the possibility to systematically analyze interactome changes for mutations of many proteins at the same time. Short linear motifs (SLiMs) are amino acid patterns that can mediate protein binding. A significant number of SLiMs are located in regions of proteins, which are lacking a secondary structure, making the interaction motifs readily available for binding reactions. Peptides are particularly well suited to study protein interactions, which are based on SLiM-mediated binding. New technologies using arrayed peptides for interaction studies are able to identify SLIM-based interaction and identify the interaction motifs. Graphical abstract

Rational discovery of antimetastatic agents targeting the intrinsically disordered region of MBD2

Science Advances ◽

10.1126/sciadv.aav9810 ◽

2019 ◽

Vol 5 (11) ◽

pp. eaav9810 ◽

Cited By ~ 6

Author(s):

Min Young Kim ◽

Insung Na ◽

Ji Sook Kim ◽

Seung Han Son ◽

Sungwoo Choi ◽

...

Keyword(s):

Protein Interactions ◽

Drug Targets ◽

Cancer Metastasis ◽

Epithelial Mesenchymal Transition ◽

Tumor Model ◽

Mesenchymal Transition ◽

Intrinsically Disordered

Although intrinsically disordered protein regions (IDPRs) are commonly engaged in promiscuous protein-protein interactions (PPIs), using them as drug targets is challenging due to their extreme structural flexibility. We report a rational discovery of inhibitors targeting an IDPR of MBD2 that undergoes disorder-to-order transition upon PPI and is critical for the regulation of the Mi-2/NuRD chromatin remodeling complex (CRC). Computational biology was essential for identifying target site, searching for promising leads, and assessing their binding feasibility and off-target probability. Molecular action of selected leads inhibiting the targeted PPI of MBD2 was validated in vitro and in cell, followed by confirming their inhibitory effects on the epithelial-mesenchymal transition of various cancer cells. Identified lead compounds appeared to potently inhibit cancer metastasis in a murine xenograft tumor model. These results constitute a pioneering example of rationally discovered IDPR-targeting agents and suggest Mi-2/NuRD CRC and/or MBD2 as a promising target for treating cancer metastasis.