Altered assembly paths mitigate interference among paralogous complexes.

Protein complexes are fundamental to all cellular processes, so understanding their evolutionary history and assembly processes is important. Gene duplication followed by divergence is considered a primary mechanism for diversifying protein complexes. Nonetheless, to what extent assembly of present-day paralogous complexes has been constrained by their long evolutionary pathways and how cross-complex interference is avoided remain unanswered questions. Subunits of protein complexes are often stabilized upon complex formation, whereas unincorporated subunits are degraded. How such cooperative stability influences protein complex assembly also remains unclear. Here, we demonstrate that subcomplexes determined by cooperative stabilization interactions serve as building blocks for protein complex assembly. We further develop a protein stability-guided method to compare the assembly processes of paralogous complexes in cellulo. Our findings support that oligomeric state and the structural organization of paralogous complexes can be maintained even if their assembly processes are rearranged. Our results indicate that divergent assembly processes by paralogous complexes not only enable the complexes to evolve new functions, but also reinforce their segregation by establishing incompatibility against deleterious hybrid assemblies.

Studying Biomolecular Protein Complexes via Origami and 3D-Printed Models.

Living organisms are constructed from proteins that assemble into biomolecular complexes, each with a unique shape and function. Our knowledge about the structure-activity relationship of these complexes is still limited, mainly because of their small size, complex structure, fast processes, and changing environment. Furthermore, the constraints of current microscopic tools and the difficulty in applying molecular dynamic simulations to capture the dynamic response of biomolecular complexes and long-term phenomena call for new supplementary tools and approaches that can help bridge this gap. In this paper, we present an approach to comparing biomolecular and origami hierarchical structures and apply it to comparing bacterial microcompartments (BMCs) with spiral-based origami models. Our first analysis compares proteins that assemble the BMC with an origami model called "flasher", which is the unit cell of an assembled origami model. Then, the BMC structure is compared with the assembled origami model and based on the similarity, a physical scaled-up origami model, which is analogous to the BMC, is constructed. The origami model is translated into a computer-aided design model and manufactured via 3D-printing technology. Finite element analysis and physical experiments of the origami model and 3D-printed parts reveal trends in the mechanical response of the icosahedron, which is constructed from tiled-chiral elements. The chiral elements rotate as the icosahedron expands and we deduce that it allows the BMC to open gates for transmembrane passage of materials.

Improved protein complex prediction with AlphaFold-multimer by denoising the MSA profile.

Structure prediction of protein complexes has improved significantly with AlphaFold2 and AlphaFold-multimer (AFM), but only 60% of dimers are accurately predicted. Here, we learn a bias to the MSA representation that improves the predictions by performing gradient descent through the AFM network. We demonstrate the performance on seven difficult targets from CASP15 and increase the average MMscore to 0.76 compared to 0.63 with AFM. We evaluate the procedure on 487 protein complexes where AFM fails and obtain an increased success rate (MMscore>0.75) of 33% on these difficult targets. Our protocol, AFProfile, provides a way to direct predictions towards a defined target function guided by the MSA. We expect gradient descent over the MSA to be useful for different tasks.

Maturation and Assembly of mTOR Complexes by the HSP90-R2TP-TTT Chaperone System: Molecular Insights and Mechanisms.

The mechanistic target of rapamycin (mTOR) is a master regulator of cell growth and metabolism, integrating environmental signals to regulate anabolic and catabolic processes, regulating lipid synthesis, growth factor-induced cell proliferation, cell survival, and migration. These activities are performed as part of two distinct complexes, mTORC1 and mTORC2, each with specific roles. mTORC1 and mTORC2 are elaborated dimeric structures formed by the interaction of mTOR with specific partners. mTOR functions only as part of these large complexes, but their assembly and activation require a dedicated and sophisticated chaperone system. mTOR folding and assembly are temporarily separated with the TELO2-TTI1-TTI2 (TTT) complex assisting the cotranslational folding of mTOR into a native conformation. Matured mTOR is then transferred to the R2TP complex for assembly of active mTORC1 and mTORC2 complexes. R2TP works in concert with the HSP90 chaperone to promote the incorporation of additional subunits to mTOR and dimerization. This review summarizes our current knowledge on how the HSP90-R2TP-TTT chaperone system facilitates the maturation and assembly of active mTORC1 and mTORC2 complexes, discussing interactions, structures, and mechanisms.

Mitigating the Blurring Effect of CryoEM Averaging on a Flexible and Highly Symmetric Protein Complex through Sub-Particle Reconstruction.

Many macromolecules are inherently flexible as a feature of their structure and function. During single-particle CryoEM processing, flexible protein regions can be detrimental to high-resolution reconstruction as signals from thousands of particles are averaged together. This "blurring" effect can be difficult to overcome and is possibly more pronounced when averaging highly symmetric complexes. Approaches to mitigating flexibility during CryoEM processing are becoming increasingly critical as the technique advances and is applied to more dynamic proteins and complexes. Here, we detail the use of sub-particle averaging and signal subtraction techniques to precisely target and resolve flexible DARPin protein attachments on a designed tetrahedrally symmetric protein scaffold called DARP14. Particles are first aligned as full complexes, and then the symmetry is reduced by alignment and focused refinement of the constituent subunits. The final reconstructions we obtained were vastly improved over the fully symmetric reconstructions, with observable secondary structure and side-chain placement. Additionally, we were also able to reconstruct the core region of the scaffold to 2.7 Å. The data processing protocol outlined here is applicable to other dynamic and symmetric protein complexes, and our improved maps could allow for new structure-guided variant designs of DARP14.

Microscale measurements of protein complexes from single cells.

Proteins execute numerous cell functions in concert with one another in protein-protein interactions (PPI). While essential in each cell, such interactions are not identical from cell to cell. Instead, PPI heterogeneity contributes to cellular phenotypic heterogeneity in health and diseases such as cancer. Understanding cellular phenotypic heterogeneity thus requires measurements of properties of PPIs such as abundance, stoichiometry, and kinetics at the single-cell level. Here, we review recent, exciting progress in single-cell PPI measurements. Novel technology in this area is enabled by microscale and microfluidic approaches that control analyte concentration in timescales needed to outpace PPI disassembly kinetics. We describe microscale innovations, needed technical capabilities, and methods poised to be adapted for single-cell analysis in the near future.

Cohesin-Dependent Loop Extrusion: Molecular Mechanics and Role in Cell Physiology.

The most prominent representatives of multisubunit SMC complexes, cohesin and condensin, are best known as structural components of mitotic chromosomes. It turned out that these complexes, as well as their bacterial homologues, are molecular motors, the ATP-dependent movement of these complexes along DNA threads leads to the formation of DNA loops. In recent years, we have witnessed an avalanche-like accumulation of data on the process of SMC dependent DNA looping, also known as loop extrusion. This review briefly summarizes the current understanding of the place and role of cohesin-dependent extrusion in cell physiology and presents a number of models describing the potential molecular mechanism of extrusion in a most compelling way. We conclude the review with a discussion of how the capacity of cohesin to extrude DNA loops may be mechanistically linked to its involvement in sister chromatid cohesion.

Isolation and Partial Characterization of Novel, Structurally Uniform (Hfq6)n≥8 Assemblies Carrying Accessory Transcription and Translation Factors.

In growing E. coli cells, the transcription-translation complexes (TTCs) form characteristic foci; however, the exact molecular composition of these superstructures is not known with certainty. Herein, we report that, during our recently developed "fast" procedures for purification of E. coli RNA polymerase (RP), a fraction of the RP's α/RpoA subunits is displaced from the core RP complexes and copurifies with multiprotein superstructures carrying the nucleic acid-binding protein Hfq and the ribosomal protein S6. We show that the main components of these large multiprotein assemblies are fixed protein copy-number (Hfq6)n≥8 complexes; these complexes have a high level of structural uniformity and are distinctly unlike the previously described (Hfq6)n "head-to-tail" polymers. We describe purification of these novel, structurally uniform (Hfq6)n≥8 complexes to near homogeneity and show that they also contain small nonprotein molecules and accessory S6. We demonstrate that Hfq, S6, and RP have similar solubility profiles and present evidence pointing to a role of the Hfq C-termini in superstructure formation. Taken together, our data offer new insights into the composition of the macromolecular assemblies likely acting as scaffolds for transcription complexes and ribosomes during bacterial cells' active growth.

Probing the Architecture of Multisubunit Protein Complexes with In-line Disulfide Reduction and Native MS Analysis.

Native mass spectrometry (MS) continues to enjoy growing popularity as a means of providing a wealth of information on noncovalent biopolymer assemblies ranging from composition and binding stoichiometry to characterization of the topology of these assemblies. The latter frequently relies on supplementing MS measurements with limited fragmentation of the noncovalent complexes in the gas phase to identify the pairs of neighboring subunits. While this approach has met with much success in the past two decades, its implementation remains difficult (and the success record relatively modest) within one class of noncovalent assemblies: protein complexes in which at least one binding partner has multiple subunits cross-linked by disulfide bonds. We approach this problem by inducing chemical reduction of disulfide bonds under nondenaturing conditions in solution followed by native MS analysis with online buffer exchange to remove unconsumed reagents that are incompatible with the electrospray ionization process. While this approach works well with systems comprised of thiol-linked subunits that remain stable upon reduction of the disulfide bridges (such as immunoglobulins), chemical reduction frequently gives rise to species that are unstable (prone to aggregation). This problem is circumvented by taking advantage of the recently introduced cross-path reactive chromatography platform (XPRC), which allows the disulfide reduction to be carried out in-line, thereby minimizing the loss of metastable protein subunits and their noncovalent complexes with the binding partners prior to MS analysis. The feasibility of this approach is demonstrated using hemoglobin complexes with haptoglobin 1-1, a glycoprotein consisting of four polypeptide chains cross-linked by disulfide bonds.

Protocol to test for the formation of ternary protein complexes in vivo or in vitro using a two-step immunoprecipitation approach.

Co-immunoprecipitation (coIP) is an experimental technique to study protein-protein interactions (PPIs). However, single-step coIP can only be used to identify the interaction between two proteins and does not solve the interaction testing of ternary complexes. Here, we present a protocol to test for the formation of ternary protein complexes in vivo or in vitro using a two-step coIP approach. We describe steps for cell culture and transfection, elution of target proteins, and two-step coIP including western blot analyses. For complete details on the use and execution of this protocol, please refer to Li et al.1.

Structural proteomics of a bacterial mega membrane protein complex: FtsH-HflK-HflC.

Recent advances in mass spectrometry (MS) yielding sensitive and accurate measurements along with developments in software tools have enabled the characterization of complex systems routinely. Thus, structural proteomics and cross-linking mass spectrometry (XL-MS) have become a useful method for structural modeling of protein complexes. Here, we utilized commonly used XL-MS software tools to elucidate the protein interactions within a membrane protein complex containing FtsH, HflK, and HflC, over-expressed in E. coli. The MS data were processed using MaxLynx, MeroX, MS Annika, xiSEARCH, and XlinkX software tools. The number of identified inter- and intra-protein cross-links varied among software. Each interaction was manually checked using the raw MS and MS/MS data and distance restraints to verify inter- and intra-protein cross-links. A total of 37 inter-protein and 148 intra-protein cross-links were determined in the FtsH-HflK-HflC complex. The 59 of them were new interactions on the lacking region of recently published structures. These newly identified interactions, when combined with molecular docking and structural modeling, present opportunities for further investigation. The results provide valuable information regarding the complex structure and function to decipher the intricate molecular mechanisms underlying the FtsH-HflK-HflC complex.

Structure-Based Protein Assembly Simulations Including Various Binding Sites and Conformations.

Many biological functions are mediated by large complexes formed by multiple proteins and other cellular macromolecules. Recent progress in experimental structure determination, as well as in integrative modeling and protein structure prediction using deep learning approaches, has resulted in a rapid increase in the number of solved multiprotein assemblies. However, the assembly process of large complexes from their components is much less well-studied. We introduce a rapid computational structure-based (SB) model, GoCa, that allows to follow the assembly process of large multiprotein complexes based on a known native structure. Beyond existing SB Go̅-type models, it distinguishes between intra- and intersubunit interactions, allowing us to include coupled folding and binding. It accounts automatically for the permutation of identical subunits in a complex and allows the definition of multiple minima (native) structures in the case of proteins that undergo global transitions during assembly. The model is successfully tested on several multiprotein complexes. The source code of the GoCa program including a tutorial is publicly available on Github: https://github.com/ZachariasLab/GoCa. We also provide a web source that allows users to quickly generate the necessary input files for a GoCa simulation: https://goca.t38webservices.nat.tum.de.

Mechanism of single-stranded DNA annealing by RAD52-RPA complex.

RAD52 is important for the repair of DNA double-stranded breaks1,2, mitotic DNA synthesis3-5 and alternative telomere length maintenance6,7. Central to these functions, RAD52 promotes the annealing of complementary single-stranded DNA (ssDNA)8,9 and provides an alternative to BRCA2/RAD51-dependent homologous recombination repair10. Inactivation of RAD52 in homologous-recombination-deficient BRCA1- or BRCA2-defective cells is synthetically lethal11,12, and aberrant expression of RAD52 is associated with poor cancer prognosis13,14. As a consequence, RAD52 is an attractive therapeutic target against homologous-recombination-deficient breast, ovarian and prostate cancers15-17. Here we describe the structure of RAD52 and define the mechanism of annealing. As reported previously18-20, RAD52 forms undecameric (11-subunit) ring structures, but these rings do not represent the active form of the enzyme. Instead, cryo-electron microscopy and biochemical analyses revealed that ssDNA annealing is driven by RAD52 open rings in association with replication protein-A (RPA). Atomic models of the RAD52-ssDNA complex show that ssDNA sits in a positively charged channel around the ring. Annealing is driven by the RAD52 N-terminal domains, whereas the C-terminal regions modulate the open-ring conformation and RPA interaction. RPA associates with RAD52 at the site of ring opening with critical interactions occurring between the RPA-interacting domain of RAD52 and the winged helix domain of RPA2. Our studies provide structural snapshots throughout the annealing process and define the molecular mechanism of ssDNA annealing by the RAD52-RPA complex.

Protocol for studying topological DNA interactions by purified fission yeast condensin.

To understand the transition from interphase chromatin into well-shaped chromosomes during cell divisions, we need to understand the biochemical activities of the contributing proteins. Here, we present a protocol to investigate how the ring-shaped condensin complex sequentially and topologically entraps two DNA substrates. We describe the steps to prepare purified Schizosaccharomyces pombe condensin, as well as bulk biochemical assays to monitor the first and second DNA capture reactions. This protocol may facilitate further investigations of these essential genome organizers. For complete details on the use and execution of this protocol, please refer to Tang et al.1.

Structure and interactions of the endogenous human Commander complex.

The Commander complex, a 16-protein assembly, plays multiple roles in cell homeostasis, cell cycle and immune response. It consists of copper-metabolism Murr1 domain proteins (COMMD1-10), coiled-coil domain-containing proteins (CCDC22 and CCDC93), DENND10 and the Retriever subcomplex (VPS26C, VPS29 and VPS35L), all expressed ubiquitously in the body and linked to various diseases. Here, we report the structure and key interactions of the endogenous human Commander complex by cryogenic-electron microscopy and mass spectrometry-based proteomics. The complex consists of a stable core of COMMD1-10 and an effector containing DENND10 and Retriever, scaffolded together by CCDC22 and CCDC93. We establish the composition of Commander and reveal major interaction interfaces. These findings clarify its roles in intracellular transport, and uncover a strong association with cilium assembly, and centrosome and centriole functions.

Targeting DCAF5 suppresses SMARCB1-mutant cancer by stabilizing SWI/SNF.

Whereas oncogenes can potentially be inhibited with small molecules, the loss of tumour suppressors is more common and is problematic because the tumour-suppressor proteins are no longer present to be targeted. Notable examples include SMARCB1-mutant cancers, which are highly lethal malignancies driven by the inactivation of a subunit of SWI/SNF (also known as BAF) chromatin-remodelling complexes. Here, to generate mechanistic insights into the consequences of SMARCB1 mutation and to identify vulnerabilities, we contributed 14 SMARCB1-mutant cell lines to a near genome-wide CRISPR screen as part of the Cancer Dependency Map Project1-3. We report that the little-studied gene DDB1-CUL4-associated factor 5 (DCAF5) is required for the survival of SMARCB1-mutant cancers. We show that DCAF5 has a quality-control function for SWI/SNF complexes and promotes the degradation of incompletely assembled SWI/SNF complexes in the absence of SMARCB1. After depletion of DCAF5, SMARCB1-deficient SWI/SNF complexes reaccumulate, bind to target loci and restore SWI/SNF-mediated gene expression to levels that are sufficient to reverse the cancer state, including in vivo. Consequently, cancer results not from the loss of SMARCB1 function per se, but rather from DCAF5-mediated degradation of SWI/SNF complexes. These data indicate that therapeutic targeting of ubiquitin-mediated quality-control factors may effectively reverse the malignant state of some cancers driven by disruption of tumour suppressor complexes.

Structural basis for intra- and intermolecular interactions on RAD9 subunit of 9-1-1 checkpoint clamp implies functional 9-1-1 regulation by RHINO.

Eukaryotic DNA clamp is a trimeric protein featuring a toroidal ring structure that binds DNA on the inside of the ring and multiple proteins involved in DNA transactions on the outside. Eukaryotes have two types of DNA clamps: the replication clamp PCNA and the checkpoint clamp RAD9-RAD1-HUS1 (9-1-1). 9-1-1 activates the ATR-CHK1 pathway in DNA damage checkpoint, regulating cell cycle progression. Structure of 9-1-1 consists of two moieties: a hetero-trimeric ring formed by PCNA-like domains of three subunits and an intrinsically disordered C-terminal region of the RAD9 subunit, called RAD9 C-tail. The RAD9 C-tail interacts with the 9-1-1 ring and disrupts the interaction between 9-1-1 and DNA, suggesting a negative regulatory role for this intramolecular interaction. In contrast, RHINO, a 9-1-1 binding protein, interacts with both RAD1 and RAD9 subunits, positively regulating checkpoint activation by 9-1-1. This study presents a biochemical and structural analysis of intra- and inter-molecular interactions on the 9-1-1 ring. Biochemical analysis indicates that RAD9 C-tail binds to the hydrophobic pocket on the PCNA-like domain of RAD9, implying that the pocket is involved in multiple protein-protein interactions. The crystal structure of the 9-1-1 ring in complex with a RHINO peptide reveals that RHINO binds to the hydrophobic pocket of RAD9, shedding light on the RAD9-binding motif. Additionally, the study proposes a structural model of the 9-1-1-RHINO quaternary complex. Together, these findings provide functional insights into the intra- and inter-molecular interactions on the front side of RAD9, elucidating the roles of RAD9 C-tail and RHINO in checkpoint activation.

New insights into GATOR2-dependent interactions and its conformational changes in amino acid sensing.

Eukaryotic cells coordinate growth under different environmental conditions via mechanistic target of rapamycin complex 1 (mTORC1). In the amino-acid-sensing signalling pathway, the GATOR2 complex, containing five evolutionarily conserved subunits (WDR59, Mios, WDR24, Seh1L and Sec13), is required to regulate mTORC1 activity by interacting with upstream CASTOR1 (arginine sensor) and Sestrin2 (leucine sensor and downstream GATOR1 complex). GATOR2 complex utilizes ß-propellers to engage with CASTOR1, Sestrin2 and GATOR1, removal of these ß-propellers results in substantial loss of mTORC1 capacity. However, structural information regarding the interface between amino acid sensors and GATOR2 remains elusive. With the recent progress of the AI-based tool AlphaFold2 (AF2) for protein structure prediction, structural models were predicted for Sentrin2-WDR24-Seh1L and CASTOR1-Mios ß-propeller. Furthermore, the effectiveness of relevant residues within the interface was examined using biochemical experiments combined with molecular dynamics (MD) simulations. Notably, fluorescence resonance energy transfer (FRET) analysis detected the structural transition of GATOR2 in response to amino acid signals, and the deletion of Mios ß-propeller severely impeded that change at distinct arginine levels. These findings provide structural perspectives on the association between GATOR2 and amino acid sensors and can facilitate future research on structure determination and function.

The PfRCR complex bridges malaria parasite and erythrocyte during invasion.

The symptoms of malaria occur during the blood stage of infection, when parasites invade and replicate within human erythrocytes. The PfPCRCR complex1, containing PfRH5 (refs. 2,3), PfCyRPA, PfRIPR, PfCSS and PfPTRAMP, is essential for erythrocyte invasion by the deadliest human malaria parasite, Plasmodium falciparum. Invasion can be prevented by antibodies3-6 or nanobodies1 against each of these conserved proteins, making them the leading blood-stage malaria vaccine candidates. However, little is known about how PfPCRCR functions during invasion. Here we present the structure of the PfRCR complex7,8, containing PfRH5, PfCyRPA and PfRIPR, determined by cryogenic-electron microscopy. We test the hypothesis that PfRH5 opens to insert into the membrane9, instead showing that a rigid, disulfide-locked PfRH5 can mediate efficient erythrocyte invasion. We show, through modelling and an erythrocyte-binding assay, that PfCyRPA-binding antibodies5 neutralize invasion through a steric mechanism. We determine the structure of PfRIPR, showing that it consists of an ordered, multidomain core flexibly linked to an elongated tail. We also show that the elongated tail of PfRIPR, which is the target of growth-neutralizing antibodies6, binds to the PfCSS-PfPTRAMP complex on the parasite membrane. A modular PfRIPR is therefore linked to the merozoite membrane through an elongated tail, and its structured core presents PfCyRPA and PfRH5 to interact with erythrocyte receptors. This provides fresh insight into the molecular mechanism of erythrocyte invasion and opens the way to new approaches in rational vaccine design.

Establishment of dsDNA-dsDNA interactions by the condensin complex.

Condensin is a structural maintenance of chromosomes (SMC) complex family member thought to build mitotic chromosomes by DNA loop extrusion. However, condensin variants unable to extrude loops, yet proficient in chromosome formation, were recently described. Here, we explore how condensin might alternatively build chromosomes. Using bulk biochemical and single-molecule experiments with purified fission yeast condensin, we observe that individual condensins sequentially and topologically entrap two double-stranded DNAs (dsDNAs). Condensin loading transitions through a state requiring DNA bending, as proposed for the related cohesin complex. While cohesin then favors the capture of a second single-stranded DNA (ssDNA), second dsDNA capture emerges as a defining feature of condensin. We provide complementary in vivo evidence for DNA-DNA capture in the form of condensin-dependent chromatin contacts within, as well as between, chromosomes. Our results support a "diffusion capture" model in which condensin acts in mitotic chromosome formation by sequential dsDNA-dsDNA capture.