Fuzzy regions in an intrinsically disordered protein impair protein-protein interactions

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.

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Functional dissection of an intrinsically disordered protein: Understanding the roles of different domains of Knr4 protein in protein-protein interactions

Protein Science ◽

10.1002/pro.418 ◽

2010 ◽

Vol 19 (7) ◽

pp. 1376-1385 ◽

Cited By ~ 9

Author(s):

Adilia Dagkessamanskaia ◽

Fabien Durand ◽

Vladimir N. Uversky ◽

Matteo Binda ◽

Frédéric Lopez ◽

...

Keyword(s):

Protein Interactions ◽

Intrinsically Disordered Protein ◽

Protein Protein Interactions ◽

Intrinsically Disordered ◽

Disordered Protein

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α-carboxysome formation is mediated by the multivalent and disordered protein CsoS2

10.1101/708164 ◽

2019 ◽

Cited By ~ 2

Author(s):

Luke M. Oltrogge ◽

Thawatchai Chaijarasphong ◽

Allen W. Chen ◽

Eric R. Bolin ◽

Susan Marqusee ◽

...

Keyword(s):

Protein Interactions ◽

Electrostatic Interactions ◽

Intrinsically Disordered Protein ◽

Peptide Sequence ◽

Protein Protein Interactions ◽

X Ray ◽

Intrinsically Disordered ◽

Disordered Protein ◽

High Concentrations ◽

Insight Into

AbstractCarboxysomes are bacterial microcompartments that function as the centerpiece of the bacterial CO2-concentrating mechanism, feeding high concentrations of CO2 to the enzyme Rubisco for fixation. The carboxysome self-assembles from thousands of individual proteins into icosahedral-like particles with a dense enzyme cargo encapsulated within a proteinaceous shell. In the case of the α-carboxysome, there is little molecular insight into protein-protein interactions which drive the assembly process. Here we show that the N-terminus of CsoS2, an intrinsically disordered protein found in the α-carboxysome, possesses a repeated peptide sequence that binds Rubisco. X-ray structural analysis of the peptide bound to Rubisco reveals a series of conserved electrostatic interactions that are only made with properly assembled hexadecameric Rubisco. Although biophysical measurements indicate this single interaction is weak, its implicit multivalency induces high-affinity binding through avidity. Taken together, our results indicate CsoS2 acts as an interaction hub to condense Rubisco and enable efficient α-carboxysome formation.

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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 ◽

Intrinsically Disordered Protein ◽

Intrinsic Disorder ◽

Protein Protein Interactions ◽

Systematic Analysis ◽

Tetratricopeptide Repeats ◽

Intrinsically Disordered ◽

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.

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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 ◽

Intrinsically Disordered Protein ◽

High Specificity ◽

Disordered Proteins ◽

Protein Protein Interactions ◽

Intrinsically Disordered ◽

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.

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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 ◽

Intrinsically Disordered Protein ◽

Disordered Proteins ◽

Protein Protein Interactions ◽

Intrinsically Disordered ◽

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.

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NMR Lineshape Analysis of Intrinsically Disordered Protein Interactions

Methods in Molecular Biology - Intrinsically Disordered Proteins ◽

10.1007/978-1-0716-0524-0_24 ◽

2020 ◽

pp. 477-504

Author(s):

Christopher A. Waudby ◽

John Christodoulou

Keyword(s):

Protein Interactions ◽

Intrinsically Disordered Protein ◽

Lineshape Analysis ◽

Intrinsically Disordered ◽

Disordered Protein

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Structural and Functional Studies Of The Intrinsically Disordered Protein AF9 In MLL-AF9 Leukemia

Blood ◽

10.1182/blood.v122.21.3762.3762 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3762-3762

Author(s):

Aravinda Kuntimaddi ◽

Alyson Lokken ◽

Shubin Zhang ◽

Jeremy Thorpe ◽

Benjamin Leach ◽

...

Keyword(s):

Protein Interactions ◽

Conflicts Of Interest ◽

Complex Structure ◽

Intrinsically Disordered Protein ◽

Hox Gene ◽

Functional Studies ◽

Intrinsically Disordered ◽

Disordered Protein ◽

Terminal Domain

Abstract Mixed lineage leukemias are characterized by the creation of a chimeric fusion protein where MLL is fused in frame to over 60 different fusion partners, leading to the disruption of HOX gene regulation. AF9 is one of the most common MLL fusion partners, and MLL-AF9 leukemia is acute and aggressive with a poor overall prognosis. The mechanism by which AF9 regulates normal transcription and contributes to dysregulated transcription is poorly understood. We have shown that the C-terminal domain of AF9 binds to four different proteins, two of which (Dot1L – an H3K79 methyltransferase and AF4 – which recruits P-TEFb), are transcriptional activators, whereas the other two (CBX8 – which is a part of the PRC1 repressive complex and BCOR- BCL6 corepressor), are generally transcriptional repressors, suggesting that AF9 acts as a protein signaling hub. We have previously shown that the C-terminal domain of AF9 is an intrinsically disordered protein (IDP) meaning that it is unstructured on its own but undergoes coupled folding and binding upon interacting with its binding partners1. Our goal is to use structure-function studies to further understand AF9 protein interactions and to elucidate which of these interactions with AF9 are critical for MLL-AF9 leukemogenesis. We have determined NMR solution structures of complexes of Dot1L and CBX8 with AF9. Both Dot1L and CBX8 complexes form mixed alpha-beta structures, similar to that of our previously solved AF4-AF9 complex structure where AF4, Dot1L, and CBX8 all have a consensus LXVXIXL sequence and form a ß-strand and an extended ß-turn. Intriguingly, Dot1L contains three separate binding motifs for AF9 interaction, including one repeat motif separated by seven amino acids. Our 15N-1H HSQC NMR experiments show that each of these three binding sites on Dot1L adopts a similar fold with AF9 yet has vastly different binding affinities with AF9. We have created several structure-guided mutations on AF9 that differentially block interactions of its binding partners. A single charge reversal mutation on AF9 that blocks all protein interactions with AF9 was introduced into MLL-AF9 in vitro colony forming assays, and was able to abrogate the serial replating capacity of MLL-AF9. Other mutations that selectively block the interaction of various proteins with AF9, show differential effects in colony forming assays and HOX gene expression suggesting the importance of certain AF9 protein interactions in MLL-AF9 leukemogenesis. 1. Leach, B. I. et al. Leukemia fusion target AF9 is an intrinsically disordered transcriptional regulator that recruits multiple partners via coupled folding and binding. Structure21, 176-183, doi:10.1016/j.str.2012.11.011 (2013). Disclosures: No relevant conflicts of interest to declare.

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