Chimeric 14-3-3 proteins for unravelling interactions with intrinsically disordered partners

C Terminus ◽

Transient Interactions ◽

Partner Proteins

ABSTRACTIn eukaryotes, several proteins act as “hubs”, integrating signals from a variety of interacting partners that bind to the hub through intrinsically disordered regions. Not surprisingly, one of the major hubs, the 14-3-3 protein, that plays wide-ranging roles in cellular processes, has been linked with a number of disorders including neurodegenerative diseases and cancer. A partner protein usually binds with its phosphopeptide accommodated in an amphipathic groove (AG) of 14-3-3, a promising platform for therapeutic intervention. Protein plasticity in the groove allows to accommodate a range of phosphopeptides with different sequences. So far, in spite of mammoth effort, accurate structural information has been derived only for few 14-3-3 complexes with phosphopeptide-containing proteins or various short synthetic peptides. The progress has been prevented by intrinsic disorder of partner proteins and, in case of transient interactions, by the low affinity of phosphopeptides. We reasoned that these problems could be resolved by using chimeric 14-3-3 proteins with incorporated peptide sequences. We tested this hypothesis and found that such chimeric proteins are easy to design, express, purify and crystallize. We show that when attached to the C terminus of 14-3-3 via an optimal linker, peptides become stoichiometrically phosphorylated by protein kinase A during bacterial co-expression. We determined crystal structures for complexes of chimeric 14-3-3 protein fused with three different peptides. In most of the cases, the phosphopeptide is bound inside the AG, providing invaluable information on its interaction with the protein. This approach can reinvigorate studies of 14-3-3 protein complexes, including those with otherwise challenging low affinity phosphopeptides. Furthermore, 14-3-3-phosphopeptide chimeras can be useful for the design of novel biosensors for in vitro and in vivo imaging experiments.

Molecular and Biochemical Techniques for Deciphering p53-MDM2 Regulatory Mechanisms

Biomolecules ◽

10.3390/biom11010036 ◽

2020 ◽

Vol 11 (1) ◽

pp. 36

Author(s):

Konstantinos Karakostis ◽

Ignacio López ◽

Ana M. Peña-Balderas ◽

Robin Fåhareus ◽

Vanesa Olivares-Illana

Keyword(s):

Protein Complexes ◽

Post Translational Modifications ◽

Advantages And Disadvantages ◽

Multiple Partners ◽

Allosteric Interactions ◽

Spatio Temporal

The p53 and Mouse double minute 2 (MDM2) proteins are hubs in extensive networks of interactions with multiple partners and functions. Intrinsically disordered regions help to adopt function-specific structural conformations in response to ligand binding and post-translational modifications. Different techniques have been used to dissect interactions of the p53-MDM2 pathway, in vitro, in vivo, and in situ each having its own advantages and disadvantages. This review uses the p53-MDM2 to show how different techniques can be employed, illustrating how a combination of in vitro and in vivo techniques is highly recommended to study the spatio-temporal location and dynamics of interactions, and to address their regulation mechanisms and functions. By using well-established techniques in combination with more recent advances, it is possible to rapidly decipher complex mechanisms, such as the p53 regulatory pathway, and to demonstrate how protein and nucleotide ligands in combination with post-translational modifications, result in inter-allosteric and intra-allosteric interactions that govern the activity of the protein complexes and their specific roles in oncogenesis. This promotes elegant therapeutic strategies that exploit protein dynamics to target specific interactions.

Molecular determinants of phase separation forDrosophilaDNA replication licensing factors

10.1101/2021.04.12.439485 ◽

2021 ◽

Author(s):

Matthew W. Parker ◽

Jonchee Kao ◽

Alvin Huang ◽

James M. Berger ◽

Michael R. Botchan

Keyword(s):

Phase Separation ◽

Self Assembly ◽

A Priori ◽

Initiation Factor ◽

Sequence Composition ◽

Partner Proteins

ABSTRACTLiquid-liquid phase separation (LLPS) of intrinsically disordered regions (IDRs) in proteins can drive the formation of membraneless compartments in cells. Phase-separated structures enrich for specific partner proteins and exclude others. We have shown that the IDRs of metazoan DNA replication initiators drive DNA-dependent phase separationin vitroand chromosome bindingin vivo, and that initiator condensates selectively recruit specific partner proteins. How initiator IDRs facilitate LLPS and maintain compositional specificity is unknown. UsingD. melanogaster (Dm)Cdt1 as a model initiation factor, we show that phase separation results from a synergy between electrostatic DNA-bridging interactions and hydrophobic inter-IDR contacts. Both sets of interactions depend on sequence composition (but not sequence order), are resistant to 1,6- hexanediol, and do not depend on aromaticity. These findings demonstrate that distinct sets of interactions drive self-assembly and condensate specificity across different phase-separating systems and advance efforts to predict IDR LLPS propensity and specificitya priori.

Molecular determinants of phase separation for Drosophila DNA replication licensing factors

eLife ◽

10.7554/elife.70535 ◽

2021 ◽

Vol 10 ◽

Author(s):

Matthew W Parker ◽

Jonchee A Kao ◽

Alvin Huang ◽

James M Berger ◽

Michael R Botchan

Keyword(s):

Phase Separation ◽

Dna Replication ◽

A Priori ◽

Initiation Factor ◽

Sequence Composition ◽

Partner Proteins

Liquid-liquid phase separation (LLPS) of intrinsically disordered regions (IDRs) in proteins can drive the formation of membraneless compartments in cells. Phase-separated structures enrich for specific partner proteins and exclude others. Previously, we showed that the IDRs of metazoan DNA replication initiators drive DNA-dependent phase separation in vitro and chromosome binding in vivo, and that initiator condensates selectively recruit replication-specific partner proteins (Parker et al., 2019). How initiator IDRs facilitate LLPS and maintain compositional specificity is unknown. Here, using D. melanogaster (Dm) Cdt1 as a model initiation factor, we show that phase separation results from a synergy between electrostatic DNA-bridging interactions and hydrophobic inter-IDR contacts. Both sets of interactions depend on sequence composition (but not sequence order), are resistant to 1,6-hexanediol, and do not depend on aromaticity. These findings demonstrate that distinct sets of interactions drive condensate formation and specificity across different phase-separating systems and advance efforts to predict IDR LLPS propensity and partner selection a priori.

Watching cellular machinery in action, one molecule at a time

The Journal of Cell Biology ◽

10.1083/jcb.201610025 ◽

2016 ◽

Vol 216 (1) ◽

pp. 41-51 ◽

Cited By ~ 22

Author(s):

Enrico Monachino ◽

Lisanne M. Spenkelink ◽

Antoine M. van Oijen

Keyword(s):

Dynamic Behavior ◽

Single Molecule ◽

Protein Complexes ◽

Imaging Techniques ◽

Ensemble Averaging ◽

Cellular Processes ◽

Cellular Machinery

Single-molecule manipulation and imaging techniques have become important elements of the biologist’s toolkit to gain mechanistic insights into cellular processes. By removing ensemble averaging, single-molecule methods provide unique access to the dynamic behavior of biomolecules. Recently, the use of these approaches has expanded to the study of complex multiprotein systems and has enabled detailed characterization of the behavior of individual molecules inside living cells. In this review, we provide an overview of the various force- and fluorescence-based single-molecule methods with applications both in vitro and in vivo, highlighting these advances by describing their applications in studies on cytoskeletal motors and DNA replication. We also discuss how single-molecule approaches have increased our understanding of the dynamic behavior of complex multiprotein systems. These methods have shown that the behavior of multicomponent protein complexes is highly stochastic and less linear and deterministic than previously thought. Further development of single-molecule tools will help to elucidate the molecular dynamics of these complex systems both inside the cell and in solutions with purified components.

Single-embryo phosphoproteomics reveals the importance of intrinsic disorder in cell cycle dynamics

10.1101/2021.08.29.458076 ◽

2021 ◽

Author(s):

Juan Manuel Valverde ◽

Geronimo Dubra ◽

Henk van den Toorn ◽

Guido van Mierlo ◽

Michiel Vermeulen ◽

...

Keyword(s):

Cell Cycle ◽

Intrinsically Disordered Proteins ◽

Protein Complexes ◽

Disordered Proteins ◽

Cell Cycles ◽

Egg Extracts ◽

Cell Cycle Dynamics

Switch-like cyclin-dependent kinase (CDK)-1 activation is thought to underlie the abruptness of mitotic onset, but how CDKs can simultaneously phosphorylate many diverse substrates is unknown, and direct evidence for such phosphorylation dynamics in vivo is lacking. Here, we analysed protein phosphorylation states in single Xenopus embryos throughout synchronous cell cycles. Over a thousand phosphosites were dynamic in vivo, and assignment of cell cycle phases using egg extracts revealed hundreds of S-phase phosphorylations. Targeted phosphoproteomics in single embryos showed switch-like mitotic phosphorylation of diverse protein complexes. The majority of cell cycle-regulated phosphosites occurred in CDK consensus motifs, and 72% located to intrinsically disordered regions. Dynamically phosphorylated proteins, and documented substrates of cell cycle kinases, are significantly more disordered than phosphoproteins in general. Furthermore, 30-50% are components of membraneless organelles. Our results suggest that phosphorylation of intrinsically disordered proteins by cell cycle kinases, particularly CDKs, allows switch-like mitotic cellular reorganisation.

Coilin oligomerization and remodeling by Nopp140 reveals a hybrid assembly mechanism for Cajal bodies

10.1101/2021.08.01.454609 ◽

2021 ◽

Author(s):

Edward Courchaine ◽

Martin Machyna ◽

Korinna Straube ◽

Sarah Sauyet ◽

Jade Enright ◽

...

Keyword(s):

Mutational Analysis ◽

Protein Complexes ◽

Scaffolding Protein ◽

Single Amino Acid ◽

Cajal Bodies ◽

Assembly Mechanism ◽

Functional Components

Cajal bodies (CBs) are ubiquitous nuclear membraneless organelles (MLOs) that promote efficient biogenesis of RNA-protein complexes. Depletion of the CB scaffolding protein coilin is lethal for vertebrate embryogenesis, making CBs a strong model for understanding the structure and function of MLOs. Although it is assumed that CBs form through biomolecular condensation, the biochemical and biophysical principles that govern CB dynamics have eluded study. Here, we identify features of the coilin protein that drive CB assembly and shape. Focusing on coilin's N-terminal domain (NTD), we discovered its unexpected capacity for oligomerization in vivo. Single amino acid mutational analysis of coilin revealed distinct molecular interactions required for oligomerization and binding to the Nopp140 ligand, which facilitates CB assembly. We demonstrate that the intrinsically disordered regions of Nopp140 have substantial condensation properties and suggest that Nopp140 binding thereby remodels stable coilin oligomers to form a particle that recruits other functional components.

Oligomerization of the Human Adenosine A2A Receptor is Driven by the Intrinsically Disordered C-terminus

10.1101/2020.12.21.423144 ◽

2020 ◽

Cited By ~ 1

Author(s):

Khanh D. Q. Nguyen ◽

Michael Vigers ◽

Eric Sefah ◽

Susanna Seppälä ◽

Jennifer P. Hoover ◽

...

Keyword(s):

Electrostatic Interactions ◽

Hydrophobic Interactions ◽

Interaction Sites ◽

C Terminus ◽

A2a Receptor ◽

Multiple Interaction ◽

Adenosine A2a

ABSTRACTG protein-coupled receptors (GPCRs) have long been shown to exist as oligomers with functional properties distinct from those of the monomeric counterparts, but the driving factors of GPCR oligomerization remain relatively unexplored. In this study, we focus on the human adenosine A2A receptor (A2AR), a model GPCR that forms oligomers both in vitro and in vivo. Combining experimental and computational approaches, we discover that the intrinsically disordered C-terminus of A2AR drives the homo-oligomerization of the receptor. The formation of A2AR oligomers declines progressively and systematically with the shortening of the C-terminus. Multiple interaction sites and types are responsible for A2AR oligomerization, including disulfide linkages, hydrogen bonds, electrostatic interactions, and hydrophobic interactions. These interactions are enhanced by depletion interactions along the C-terminus, forming a tunable network of bonds that allow A2AR oligomers to adopt multiple interfaces. This study uncovers the disordered C-terminus as a prominent driving factor for the oligomerization of a GPCR, offering important guidance for structure-function studies of A2AR and other GPCRs.

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 ◽

Intrinsically Disordered Protein ◽

Protein 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.

Unstructured mRNAs form multivalent RNA-RNA interactions to generate TIS granule networks

10.1101/2020.02.14.949503 ◽

2020 ◽

Cited By ~ 4

Author(s):

Weirui Ma ◽

Gang Zhen ◽

Wei Xie ◽

Christine Mayr

Keyword(s):

Endoplasmic Reticulum ◽

Protein Synthesis ◽

Complex Formation ◽

Major Site ◽

Protein Complex Formation ◽

Unstructured Regions

SummaryThe TIS granule network is a constitutively expressed membraneless organelle that concentrates mRNAs with AU-rich elements and interacts with the major site of protein synthesis, the rough endoplasmic reticulum. Most known biomolecular condensates are sphere-like, but TIS granules have a mesh-like morphology. Through in vivo and in vitro reconstitution experiments we discovered that this shape is generated by extensive intermolecular RNA-RNA interactions. They are mostly accomplished by mRNAs with large unstructured regions in their 3′UTRs that we call intrinsically disordered regions (IDRs). As AU-rich RNA is a potent chaperone that suppresses protein aggregation and is overrepresented in mRNAs with IDRs, our data suggests that TIS granules concentrate mRNAs that assist protein folding. In addition, the proximity of translating mRNAs in TIS granule networks may enable co-translational protein complex formation.

Novel molecular aspects of the CRISPR backbone protein ‘Cas7’ from cyanobacteria

Biochemical Journal ◽

10.1042/bcj20200026 ◽

2020 ◽

Vol 477 (5) ◽

pp. 971-983 ◽

Cited By ~ 1

Author(s):

Prakash Kalwani ◽

Devashish Rath ◽

Anand Ballal

Keyword(s):

Rna Recognition Motif ◽

Type I ◽

Mutant Proteins ◽

Oligomeric Form ◽

Photosynthetic Organism ◽

Cyanobacterium Anabaena

The cyanobacterium Anabaena PCC 7120 shows the presence of Type I-D CRISPR system that can potentially confer adaptive immunity. The Cas7 protein (Alr1562), which forms the backbone of the type I-D surveillance complex, was characterized from Anabaena. Alr1562, showed the presence of the non-canonical RNA recognition motif and two intrinsically disordered regions (IDRs). When overexpressed in E. coli, the Alr1562 protein was soluble and could be purified by affinity chromatography, however, deletion of IDRs rendered Alr1562 completely insoluble. The purified Alr1562 was present in the dimeric or a RNA-associated higher oligomeric form, which appeared as spiral structures under electron microscope. With RNaseA and NaCl treatment, the higher oligomeric form converted to the lower oligomeric form, indicating that oligomerization occurred due to the association of Alr1562 with RNA. The secondary structure of both these forms was largely similar, resembling that of a partially folded protein. The dimeric Alr1562 was more prone to temperature-dependent aggregation than the higher oligomeric form. In vitro, the Alr1562 bound more specifically to a minimal CRISPR unit than to the non-specific RNA. Residues required for binding of Alr1562 to RNA, identified by protein modeling-based approaches, were mutated for functional validation. Interestingly, these mutant proteins, showing reduced ability to bind RNA were predominantly present in dimeric form. Alr1562 was detected with specific antiserum in Anabaena, suggesting that the type I-D system is expressed and may be functional in vivo. This is the first report that describes the characterization of a Cas protein from any photosynthetic organism.