Uncovering domain motif interactions using high‐throughput protein–protein interaction detection methods

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Uncovering domain motif interactions using high-throughput protein–protein interaction detection methods

Protein–protein interactions (PPIs), often mediated by short linear motifs (SLiMs), shape cellular functions. This review provides an overview of SLiMs, and scrutinises current PPI detection techniques, highlighting their relevance to SLiM-mediated interactions and addressing challenges in detecting domain–motif interactions (DMIs). Case studies, like BioGrid database analysis, suggest high-throughput PPI methods as reliable sources for predicting DMIs, enriching our understanding of cellular dynamics.


Protein–protein interactions (PPIs) are often mediated by short linear motifs (SLiMs) in one protein and domain in another, known as domain–motif interactions (DMIs). During the past decade, SLiMs have been studied to find their role in cellular functions such as post-translational modifications, regulatory processes, protein scaffolding, cell cycle progression, cell adhesion, cell signalling and substrate selection for proteasomal degradation. This review provides a comprehensive overview of the current PPI detection techniques and resources, focusing on their relevance to capturing interactions mediated by SLiMs. We also address the challenges associated with capturing DMIs. Moreover, a case study analysing the BioGrid database as a source of DMI prediction revealed significant known DMI enrichment in different PPI detection methods. Overall, it can be said that current high-throughput PPI detection methods can be a reliable source for predicting DMIs.

Assessment of machine‐learning predictions for the Mediator complex subunit MED25 ACID domain interactions with transactivation domains

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Assessment of machine-learning predictions for the Mediator complex subunit MED25 ACID domain interactions with transactivation domains

In this study, we report a systematic assessment of AlphaFold performance to predict 9 different human MED25 ACID domain–transactivation domain (TAD) interfaces and evaluate the accuracy of the models through comparison with published and new experimental data. We also reveal a new interaction surface unique to plants by predicting 3 different Arabidopsis thaliana MED25 complexes.


The human Mediator complex subunit MED25 binds transactivation domains (TADs) present in various cellular and viral proteins using two binding interfaces, named H1 and H2, which are found on opposite sides of its ACID domain. Here, we use and compare deep learning methods to characterize human MED25–TAD interfaces and assess the predicted models to published experimental data. For the H1 interface, AlphaFold produces predictions with high-reliability scores that agree well with experimental data, while the H2 interface predictions appear inconsistent, preventing reliable binding modes. Despite these limitations, we experimentally assess the validity of MED25 interface predictions with the viral transcriptional activators Lana-1 and IE62. AlphaFold predictions also suggest the existence of a unique hydrophobic pocket for the Arabidopsis MED25 ACID domain.

Identification and characterization of nuclear localization signals in the circumsporozoite protein of Plasmodium falciparum

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Identification and characterization of nuclear localization signals in the circumsporozoite protein of Plasmodium falciparum

The circumsporozoite protein of Plasmodium falciparum (Pf-CSP) contains two nuclear localization signals (NLSs). The lysine and arginine residues in both NLSs are essential for Pf-CSP interaction with importin-α, aiding Pf-CSP's nuclear translocation. Both NLSs are vital for nuclear localization, with individual NLSs resulting in weaker localization. Pf-CSP also contains a distinct nuclear export signal motif that coordinates the protein nucleocytoplasmic shuttling mechanisms.


Secretory proteins of Plasmodium exhibit differential spatial and functional activity within the host cell nucleus. However, the nuclear localization signals (NLSs) for these proteins remain largely uncharacterized. In this study, we have identified and characterized two NLSs in the circumsporozoite protein of Plasmodium falciparum (Pf-CSP). Both NLSs in the Pf-CSP contain clusters of lysine and arginine residues essential for specific interactions with the conserved tryptophan and asparagine residues of importin-α, facilitating nuclear translocation of Pf-CSP. While the two NLSs of Pf-CSP function independently and are both crucial for nuclear localization, a single NLS of Pf-CSP leads to weak nuclear localization. These findings shed light on the mechanism of nuclear penetrability of secretory proteins of Plasmodium proteins.

Drosophila germ granules are assembled from protein components through different modes of competing interactions with the multi‐domain Tudor protein

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Drosophila germ granules are assembled from protein components through different modes of competing interactions with the multi-domain Tudor protein

Protein components of the germ granules, Aubergine (Aub) and Pyruvate Kinase (PyK), use different modes to associate with Tudor (Tud) protein, which contains 11 Tud domains. Aub binds to a single Tud domain and PyK requires two Tud domains. Aub and PyK compete for binding to Tud in vitro and form separate clusters within a granule in vivo, thereby minimizing the competition.


Membraneless organelles are RNA–protein assemblies which have been implicated in post-transcriptional control. Germ cells form membraneless organelles referred to as germ granules, which contain conserved proteins including Tudor domain-containing scaffold polypeptides and their partner proteins that interact with Tudor domains. Here, we show that in Drosophila, different germ granule proteins associate with the multi-domain Tudor protein using different numbers of Tudor domains. Furthermore, these proteins compete for interaction with Tudor in vitro and, surprisingly, partition to distinct and poorly overlapping clusters in germ granules in vivo. This partition results in minimization of the competition. Our data suggest that Tudor forms structurally different configurations with different partner proteins which dictate different biophysical properties and phase separation parameters within the same granule.

A two‐step mechanism for the binding of the HIV‐1 MPER epitope by the 10E8 antibody onto biosensor‐supported lipid bilayers

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A two-step mechanism for the binding of the HIV-1 MPER epitope by the 10E8 antibody onto biosensor-supported lipid bilayers

MPER-targeting antibodies display nearly pan-neutralizing activity against HIV. Elucidating the mechanisms of epitope recognition by these antibodies is paramount for developing preventive vaccines and antibody-based treatments. Here, we report that binding of 10E8 to the MPER helix epitope presented in the membrane microenvironment occurs in two steps: (i) engagement with the solvent-exposed MPER portion; and (ii) accommodation of the membrane surface.


HIV-1 antibodies targeting the carboxy-terminal area of the membrane-proximal external region (ctMPER) are close to exerting viral pan-neutralization. Here, we reconstituted the ctMPER epitope as the N-terminal extremity of the Env glycoprotein transmembrane domain helix and immobilized it onto biosensor-supported lipid bilayers. We assessed the binding mechanism of anti-MPER antibody 10E8 through Surface Plasmon Resonance, and found, through equilibrium and kinetic binding analyses as a function of bilayer thickness, peptide length, and paratope mutations, that 10E8 engages first with the epitope peptide (encounter), limited by ctMPER helix accessibility at the membrane surface, and then inserts into the lipid bilayer assisted by favorable Fab-membrane interactions (docking). This mechanistic information may help in devising new strategies to develop more efficient MPER-targeting vaccines.

Plasmodium falciparum J‐dot localized J domain protein A8iJp modulates the chaperone activity of human HSPA8

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Plasmodium falciparum J-dot localized J domain protein A8iJp modulates the chaperone activity of human HSPA8

A8iJp, a type-IV J domain protein of Plasmodium falciparum, gets truncated and trafficked into the host erythrocyte. The exported A8iJp gets localized to the lumen of the J-dots and can influence the chaperone activity of the human HSP70 chaperone HsHSPA8. Also, a subset of HsHSPA8 co-localizes with A8iJp within infected human erythrocytes. We suggest that A8iJp influences the functioning of HsHSPA8 during host erythrocyte remodeling.


Plasmodium falciparum renovates the host erythrocyte to survive during intraerythrocytic development. This renovation requires many parasite proteins to unfold and move outside the parasitophorous vacuolar membrane, and chaperone-regulated protein folding becomes essential for the exported proteins to function. We report on a type-IV J domain protein (JDP), PF3D7_1401100, which we found to be processed before export and trafficked inside the lumen of parasite-derived structures known as J-dots. We found this protein to have holdase activity, as well as stimulate the ATPase and aggregation suppression activity of the human HSP70 chaperone HsHSPA8; thus, we named it “HSPA8-interacting J protein” (A8iJp). Moreover, we found a subset of HsHSPA8 to co-localize with A8iJp inside the infected human erythrocyte. Our results suggest that A8iJp modulates HsHSPA8 chaperone activity and may play an important role in host erythrocyte renovation.

Immune dysregulation in long COVID

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Nature Immunology, Published online: 08 April 2024; doi:10.1038/s41590-024-01795-z

Profiling of plasma proteins in individuals with COVID-19 shows that complement activation and myeloid inflammation are major pathways in the pathogenesis of long COVID and identifies distinct profiles of immune dysregulation in individuals with long COVID, highlighting the heterogeneous and diverse nature of this disease.