Characterization of the SF3B1-SUGP1 interface reveals how numerous cancer mutations cause mRNA missplicing.

The spliceosomal gene SF3B1 is frequently mutated in cancer. While it is known that SF3B1 hotspot mutations lead to loss of splicing factor SUGP1 from spliceosomes, the cancer-relevant SF3B1-SUGP1 interaction has not been characterized. To address this issue, we show by structural modeling that two regions flanking the SUGP1 G-patch make numerous contacts with the region of SF3B1 harboring hotspot mutations. Experiments confirmed that all the cancer-associated mutations in these regions, as well as mutations affecting other residues in the SF3B1-SUGP1 interface, not only weaken or disrupt the interaction but also alter splicing similarly to SF3B1 cancer mutations. Finally, structural modeling of a trimeric protein complex reveals that the SF3B1-SUGP1 interaction "loops out" the G-patch for interaction with the helicase DHX15. Our study thus provides an unprecedented molecular view of a protein complex essential for accurate splicing and also reveals that numerous cancer-associated mutations disrupt the critical SF3B1-SUGP1 interaction.

AI-assisted proofreading of RNA splicing.

RNA helicases orchestrate proofreading mechanisms that facilitate accurate intron removal from pre-mRNAs. How these activities are recruited to spliceosome/pre-mRNA complexes remains poorly understood. In this issue of Genes & Development, Zhang and colleagues (pp. 968-983) combine biochemical experiments with AI-based structure prediction methods to generate a model for the interaction between SF3B1, a core splicing factor essential for the recognition of the intron branchpoint, and SUGP1, a protein that bridges SF3B1 with the helicase DHX15. Interaction with SF3B1 exposes the G-patch domain of SUGP1, facilitating binding to and activation of DHX15. The model can explain the activation of cryptic 3' splice sites induced by mutations in SF3B1 or SUGP1 frequently found in cancer.

Using AlphaFold Multimer to discover interkingdom protein-protein interactions.

Structural prediction by artificial intelligence can be powerful new instruments to discover novel protein-protein interactions, but the community still grapples with the implementation, opportunities and limitations. Here, we discuss and re-analyse our in silico screen for novel pathogen-secreted inhibitors of immune hydrolases to illustrate the power and limitations of structural predictions. We discuss strategies of curating sequences, including controls, and reusing sequence alignments and highlight important limitations caused by different platforms, sequence depth and computing times. We hope these experiences will support similar interactomic screens by the research community.

Improved the heterodimer protein complex prediction with protein language models.

AlphaFold-Multimer has greatly improved the protein complex structure prediction, but its accuracy also depends on the quality of the multiple sequence alignment (MSA) formed by the interacting homologs (i.e. interologs) of the complex under prediction. Here we propose a novel method, ESMPair, that can identify interologs of a complex using protein language models. We show that ESMPair can generate better interologs than the default MSA generation method in AlphaFold-Multimer. Our method results in better complex structure prediction than AlphaFold-Multimer by a large margin (+10.7% in terms of the Top-5 best DockQ), especially when the predicted complex structures have low confidence. We further show that by combining several MSA generation methods, we may yield even better complex structure prediction accuracy than Alphafold-Multimer (+22% in terms of the Top-5 best DockQ). By systematically analyzing the impact factors of our algorithm we find that the diversity of MSA of interologs significantly affects the prediction accuracy. Moreover, we show that ESMPair performs particularly well on complexes in eucaryotes.

Identification of type VI secretion system effector-immunity pairs using structural bioinformatics.

The type VI secretion system (T6SS) is an important mediator of microbe-microbe and microbe-host interactions. Gram-negative bacteria use the T6SS to inject T6SS effectors (T6Es), which are usually proteins with toxic activity, into neighboring cells. Antibacterial effectors have cognate immunity proteins that neutralize self-intoxication. Here, we applied novel structural bioinformatic tools to perform systematic discovery and functional annotation of T6Es and their cognate immunity proteins from a dataset of 17,920 T6SS-encoding bacterial genomes. Using structural clustering, we identified 517 putative T6E families, outperforming sequence-based clustering. We developed a logistic regression model to reliably quantify protein-protein interaction of new T6E-immunity pairs, yielding candidate immunity proteins for 231 out of the 517 T6E families. We used sensitive structure-based annotation which yielded functional annotations for 51% of the T6E families, again outperforming sequence-based annotation. Next, we validated four novel T6E-immunity pairs using basic experiments in E. coli. In particular, we showed that the Pfam domain DUF3289 is a homolog of Colicin M and that DUF943 acts as its cognate immunity protein. Furthermore, we discovered a novel T6E that is a structural homolog of SleB, a lytic transglycosylase, and identified a specific glutamate that acts as its putative catalytic residue. Overall, this study applies novel structural bioinformatic tools to T6E-immunity pair discovery, and provides an extensive database of annotated T6E-immunity pairs.

Protein complexes in cells by AI-assisted structural proteomics.

Accurately modeling the structures of proteins and their complexes using artificial intelligence is revolutionizing molecular biology. Experimental data enable a candidate-based approach to systematically model novel protein assemblies. Here, we use a combination of in-cell crosslinking mass spectrometry and co-fractionation mass spectrometry (CoFrac-MS) to identify protein-protein interactions in the model Gram-positive bacterium Bacillus subtilis. We show that crosslinking interactions prior to cell lysis reveals protein interactions that are often lost upon cell lysis. We predict the structures of these protein interactions and others in the SubtiWiki database with AlphaFold-Multimer and, after controlling for the false-positive rate of the predictions, we propose novel structural models of 153 dimeric and 14 trimeric protein assemblies. Crosslinking MS data independently validates the AlphaFold predictions and scoring. We report and validate novel interactors of central cellular machineries that include the ribosome, RNA polymerase, and pyruvate dehydrogenase, assigning function to several uncharacterized proteins. Our approach uncovers protein-protein interactions inside intact cells, provides structural insight into their interaction interfaces, and is applicable to genetically intractable organisms, including pathogenic bacteria.

Prediction of protein assemblies by structure sampling followed by interface-focused scoring.

Proteins often function as part of permanent or transient multimeric complexes, and understanding function of these assemblies requires knowledge of their three-dimensional structures. While the ability of AlphaFold to predict structures of individual proteins with unprecedented accuracy has revolutionized structural biology, modeling structures of protein assemblies remains challenging. To address this challenge, we developed a protocol for predicting structures of protein complexes involving model sampling followed by scoring focused on the subunit-subunit interaction interface. In this protocol, we diversified AlphaFold models by varying construction and pairing of multiple sequence alignments as well as increasing the number of recycles. In cases when AlphaFold failed to assemble a full protein complex or produced unreliable results, additional diverse models were constructed by docking of monomers or subcomplexes. All the models were then scored using a newly developed method, VoroIF-jury, which relies only on structural information. Notably, VoroIF-jury is independent of AlphaFold self-assessment scores and therefore can be used to rank models originating from different structure prediction methods. We tested our protocol in CASP15 and obtained top results, significantly outperforming the standard AlphaFold-Multimer pipeline. Analysis of our results showed that the accuracy of our assembly models was capped mainly by structure sampling rather than model scoring. This observation suggests that better sampling, especially for the antibody-antigen complexes, may lead to further improvement. Our protocol is expected to be useful for modeling and/or scoring protein assemblies.

Multi-level analysis of intrinsically disordered protein docking methods.

Intrinsically Disordered Proteins (IDPs) are a class of proteins in which at least some region of the protein does not possess any stable structure in solution in the physiological condition but may adopt an ordered structure upon binding to a globular receptor. These IDP-receptor complexes are thus subject to protein complex modeling in which computational techniques are applied to accurately reproduce the IDP ligand-receptor interactions. This often exists in the form of protein docking, in which the 3D structures of both the subunits are known, but the position of the ligand relative to the receptor is not. Here, we evaluate the performance of three IDP-receptor modeling tools with metrics that characterize the IDP-receptor interface at various resolutions. We show that all three methods are able to properly identify the general binding site, as identified by lower resolution metrics, but begin to struggle with higher resolution metrics that capture biophysical interactions.

A Model of the Full-Length Cytokinin Receptor: New Insights and Prospects.

Cytokinins (CK) are one of the most important classes of phytohormones that regulate a wide range of processes in plants. A CK receptor, a sensor hybrid histidine kinase, was discovered more than 20 years ago, but the structural basis for its signaling is still a challenge for plant biologists. To date, only two fragments of the CK receptor structure, the sensory module and the receiver domain, were experimentally resolved. Some other regions were built up by molecular modeling based on structures of proteins homologous to CK receptors. However, in the long term, these data have proven insufficient for solving the structure of the full-sized CK receptor. The functional unit of CK receptor is the receptor dimer. In this article, a molecular structure of the dimeric form of the full-length CK receptor based on AlphaFold Multimer and ColabFold modeling is presented for the first time. Structural changes of the receptor upon interacting with phosphotransfer protein are visualized. According to mathematical simulation and available data, both types of dimeric receptor complexes with hormones, either half- or fully liganded, appear to be active in triggering signals. In addition, the prospects of using this and similar models to address remaining fundamental problems of CK signaling were outlined.

The interaction between NLRP1 and oxidized TRX1 involves a transient disulfide bond.

NLRP1 is an innate immune receptor that detects pathogen-associated signals, assembles into a multiprotein structure called an inflammasome, and triggers a proinflammatory form of cell death called pyroptosis. We previously discovered that the oxidized, but not the reduced, form of thioredoxin-1 directly binds to NLRP1 and represses inflammasome formation. However, the molecular basis for NLRP1's selective association with only the oxidized form of TRX1 has not yet been established. Here, we leveraged AlphaFold-Multimer, site-directed mutagenesis, thiol-trapping experiments, and mass spectrometry to reveal that a specific cysteine residue (C427 in humans) on NLRP1 forms a transient disulfide bond with oxidized TRX1. Overall, this work demonstrates how NLRP1 monitors the cellular redox state, further illuminating an unexpected connection between the intracellular redox potential and the innate immune system.

Insights into AlphaFold's breakthrough in neurodegenerative diseases.

Neurodegenerative diseases (ND) are disorders of the central nervous system (CNS) characterized by impairment in neurons' functions, and complete loss, leading to memory loss, and difficulty in learning, language, and movement processes. The most common among these NDs are Alzheimer's disease (AD) and Parkinson's disease (PD), although several other disorders also exist. These are frontotemporal dementia (FTD), amyotrophic lateral syndrome (ALS), Huntington's disease (HD), and others; the major pathological hallmark of NDs is the proteinopathies, either of amyloid-ß (Aß), tauopathies, or synucleinopathies. Aggregation of proteins that do not undergo normal configuration, either due to mutations or through some disturbance in cellular pathway contributes to the diseases. Artificial Intelligence (AI) and deep learning (DL) have proven to be successful in the diagnosis and treatment of various congenital diseases. DL approaches like AlphaFold (AF) are a major leap towards success in CNS disorders. This 3D protein geometry modeling algorithm developed by DeepMind has the potential to revolutionize biology. AF has the potential to predict 3D-protein confirmation at an accuracy level comparable to experimentally predicted one, with the additional advantage of precisely estimating protein interactions. This breakthrough will be beneficial to identify diseases' advancement and the disturbance of signaling pathways stimulating impaired functions of proteins. Though AlphaFold has solved a major problem in structural biology, it cannot predict membrane proteins-a beneficial approach for drug designing.

Structural Modeling of T9SS Outer Membrane Proteins and Their Complexes.

The type 9 secretion system (T9SS) is a recently discovered machinery that both transports cargo proteins across the Gram-negative bacterial outer membrane and attaches them to lipopolysaccharides on the extracellular surface. Outer membrane proteins (OMPs) are key components of the T9SS and are involved in both steps. In this chapter, we describe a method for the in silico modeling of T9SS OMPs and their complexes, and model validation. This is useful when the production of recombinant OMPs is difficult, and these protocols can also be applied to OMP complexes outside of the T9SS.