Development of a compact alkynyl-enrichable crosslinker for in-depth in-vivo crosslinking analysis

Chemical crosslinking coupled with mass spectrometry (CXMS) has emerged as a powerful technique to capture the dynamic information of protein complexes with high sensitivity, throughput and sample universality. To advance the study of in-vivo protein structures and protein-protein interactions on the large scale, a new alkynyl-enrichable crosslinker was developed with high efficiency of membrane penetration, reactivity and enrichment. The crosslinker was successfully used for in-vivo crosslinking of intact human cells, resulting in 6820 non-redundant crosslinks identified at a false discovery rate (FDR) of 1% using pLink 2.0, which 4898 (71.8%) of the cross-links were assigned as intraprotein and 1922 (28.2%) were interprotein links. To our knowledge, this is also the first time to realize the in-vivo crosslinking with a non-cleavable cross-linker for homo species cells.

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Human mitochondrial protein complexes revealed by large-scale coevolution analysis and deep learning-based structure modeling

10.1101/2021.09.14.460228 ◽

2021 ◽

Author(s):

Jimin Pei ◽

Jing Zhang ◽

Qian Cong

Keyword(s):

Deep Learning ◽

Protein Interactions ◽

Large Scale ◽

Protein Complexes ◽

Mitochondrial Protein ◽

Protein Structures ◽

Complex Structures ◽

Protein Protein Interactions ◽

Learning Methods ◽

Contact Probability

AbstractRecent development of deep-learning methods has led to a breakthrough in the prediction accuracy of 3-dimensional protein structures. Extending these methods to protein pairs is expected to allow large-scale detection of protein-protein interactions and modeling protein complexes at the proteome level. We applied RoseTTAFold and AlphaFold2, two of the latest deep-learning methods for structure predictions, to analyze coevolution of human proteins residing in mitochondria, an organelle of vital importance in many cellular processes including energy production, metabolism, cell death, and antiviral response. Variations in mitochondrial proteins have been linked to a plethora of human diseases and genetic conditions. RoseTTAFold, with high computational speed, was used to predict the coevolution of about 95% of mitochondrial protein pairs. Top-ranked pairs were further subject to the modeling of the complex structures by AlphaFold2, which also produced contact probability with high precision and in many cases consistent with RoseTTAFold. Most of the top ranked pairs with high contact probability were supported by known protein-protein interactions and/or similarities to experimental structural complexes. For high-scoring pairs without experimental complex structures, our coevolution analyses and structural models shed light on the details of their interfaces, including CHCHD4-AIFM1, MTERF3-TRUB2, FMC1-ATPAF2, ECSIT-NDUFAF1 and COQ7-COQ9, among others. We also identified novel PPIs (PYURF-NDUFAF5, LYRM1-MTRF1L and COA8-COX10) for several proteins without experimentally characterized interaction partners, leading to predictions of their molecular functions and the biological processes they are involved in.

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Interfacial Peptides as Affinity Modulating Agents of Protein-Protein Interactions

Biomolecules ◽

10.3390/biom12010106 ◽

2022 ◽

Vol 12 (1) ◽

pp. 106

Author(s):

Pavel V. Ershov ◽

Yuri V. Mezentsev ◽

Alexis S. Ivanov

Keyword(s):

Protein Interactions ◽

Targeted Delivery ◽

Protein Complexes ◽

Protein Protein Interactions ◽

Good Efficiency ◽

Cellular Targets ◽

Proteolytic Stability ◽

Clinically Significant

The identification of disease-related protein-protein interactions (PPIs) creates objective conditions for their pharmacological modulation. The contact area (interfaces) of the vast majority of PPIs has some features, such as geometrical and biochemical complementarities, “hot spots”, as well as an extremely low mutation rate that give us key knowledge to influence these PPIs. Exogenous regulation of PPIs is aimed at both inhibiting the assembly and/or destabilization of protein complexes. Often, the design of such modulators is associated with some specific problems in targeted delivery, cell penetration and proteolytic stability, as well as selective binding to cellular targets. Recent progress in interfacial peptide design has been achieved in solving all these difficulties and has provided a good efficiency in preclinical models (in vitro and in vivo). The most promising peptide-containing therapeutic formulations are under investigation in clinical trials. In this review, we update the current state-of-the-art in the field of interfacial peptides as potent modulators of a number of disease-related PPIs. Over the past years, the scientific interest has been focused on following clinically significant heterodimeric PPIs MDM2/p53, PD-1/PD-L1, HIF/HIF, NRF2/KEAP1, RbAp48/MTA1, HSP90/CDC37, BIRC5/CRM1, BIRC5/XIAP, YAP/TAZ–TEAD, TWEAK/FN14, Bcl-2/Bax, YY1/AKT, CD40/CD40L and MINT2/APP.

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FrustratometeR: an R-package to compute Local frustration in protein structures, point mutants and MD simulations

10.1101/2020.11.26.400432 ◽

2020 ◽

Author(s):

Atilio O. Rausch ◽

Maria I. Freiberger ◽

Cesar O. Leonetti ◽

Diego M. Luna ◽

Leandro G. Radusky ◽

...

Keyword(s):

Protein Interactions ◽

Large Scale ◽

Protein Structures ◽

Md Simulations ◽

R Package ◽

Protein Protein Interactions ◽

Large Scale Analysis ◽

Functional Aspects ◽

Catalytic Sites ◽

Polypeptide Chains

Once folded natural protein molecules have few energetic conflicts within their polypeptide chains. Many protein structures do however contain regions where energetic conflicts remain after folding, i.e. they have highly frustrated regions. These regions, kept in place over evolutionary and physiological timescales, are related to several functional aspects of natural proteins such as protein-protein interactions, small ligand recognition, catalytic sites and allostery. Here we present FrustratometeR, an R package that easily computes local energetic frustration on a personal computer or a cluster. This package facilitates large scale analysis of local frustration, point mutants and MD trajectories, allowing straightforward integration of local frustration analysis in to pipelines for protein structural analysis.Availability and implementation: https://github.com/proteinphysiologylab/frustratometeR

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Evolutionary diversification of protein–protein interactions by interface add-ons

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1707335114 ◽

2017 ◽

Vol 114 (40) ◽

pp. E8333-E8342 ◽

Cited By ~ 13

Author(s):

Maximilian G. Plach ◽

Florian Semmelmann ◽

Florian Busch ◽

Markus Busch ◽

Leonhard Heizinger ◽

...

Keyword(s):

Protein Interaction ◽

Protein Interactions ◽

Protein Complexes ◽

Evolutionary Strategy ◽

Structural Level ◽

Protein Protein Interactions ◽

Protein Protein Interaction ◽

Evolutionary Diversification

Cells contain a multitude of protein complexes whose subunits interact with high specificity. However, the number of different protein folds and interface geometries found in nature is limited. This raises the question of how protein–protein interaction specificity is achieved on the structural level and how the formation of nonphysiological complexes is avoided. Here, we describe structural elements called interface add-ons that fulfill this function and elucidate their role for the diversification of protein–protein interactions during evolution. We identified interface add-ons in 10% of a representative set of bacterial, heteromeric protein complexes. The importance of interface add-ons for protein–protein interaction specificity is demonstrated by an exemplary experimental characterization of over 30 cognate and hybrid glutamine amidotransferase complexes in combination with comprehensive genetic profiling and protein design. Moreover, growth experiments showed that the lack of interface add-ons can lead to physiologically harmful cross-talk between essential biosynthetic pathways. In sum, our complementary in silico, in vitro, and in vivo analysis argues that interface add-ons are a practical and widespread evolutionary strategy to prevent the formation of nonphysiological complexes by specializing protein–protein interactions.

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Recent advances in large-scale protein interactome mapping

F1000Research ◽

10.12688/f1000research.7629.1 ◽

2016 ◽

Vol 5 ◽

pp. 782 ◽

Cited By ~ 23

Author(s):

Virja Mehta ◽

Laura Trinkle-Mulcahy

Keyword(s):

Protein Interactions ◽

Large Scale ◽

Protein Complexes ◽

Specific Protein ◽

Quality Data ◽

Protein Protein Interactions ◽

Cellular Functions ◽

Biological Studies ◽

Protein Interactome ◽

Literature Curation

Protein-protein interactions (PPIs) underlie most, if not all, cellular functions. The comprehensive mapping of these complex networks of stable and transient associations thus remains a key goal, both for systems biology-based initiatives (where it can be combined with other ‘omics’ data to gain a better understanding of functional pathways and networks) and for focused biological studies. Despite the significant challenges of such an undertaking, major strides have been made over the past few years. They include improvements in the computation prediction of PPIs and the literature curation of low-throughput studies of specific protein complexes, but also an increase in the deposition of high-quality data from non-biased high-throughput experimental PPI mapping strategies into publicly available databases.

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Sequential digestion with Trypsin and Elastase in cross-linking/mass spectrometry

10.1101/450981 ◽

2018 ◽

Author(s):

Therese Dau ◽

Kapil Gupta ◽

Imre Berger ◽

Juri Rappsilber

Keyword(s):

Mass Spectrometry ◽

Protein Interactions ◽

Protein Structures ◽

Cross Linking ◽

Cleavage Sites ◽

Protein Modelling ◽

Protein Protein Interactions ◽

Important Approach ◽

Cross Links ◽

Sequential Digestion

ABSTRACTCross-linking/mass spectrometry has become an important approach for studying protein structures and protein-protein interactions. The amino acid composition of some protein regions impedes the detection of cross-linked residues, although it would yield invaluable information for protein modelling. Here, we report on a sequential digestion strategy with trypsin and elastase to penetrate regions with a low density of trypsin cleavage sites. We exploited intrinsic substrate recognition properties of elastase to specifically target larger tryptic peptides. Our application of this protocol to the TAF4-12 complex allowed us to identify cross-links in previously inaccessible regions.

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Reliable identification of protein-protein interactions by crosslinking mass spectrometry

10.1101/2020.05.25.114256 ◽

2020 ◽

Author(s):

Swantje Lenz ◽

Ludwig R. Sinn ◽

Francis J. O’Reilly ◽

Lutz Fischer ◽

Fritz Wegner ◽

...

Keyword(s):

Mass Spectrometry ◽

Protein Interactions ◽

Large Scale ◽

Protein Complexes ◽

Estimation Procedure ◽

Scale Analysis ◽

Protein Protein Interactions ◽

False Discovery ◽

Large Scale Analysis ◽

False Discovery Rate Estimation

Crosslinking mass spectrometry is widening its scope from structural analyzes of purified multi-protein complexes towards systems-wide analyzes of protein-protein interactions. Assessing the error in these large datasets is currently a challenge. Using a controlled large-scale analysis of Escherichia coli cell lysate, we demonstrate a reliable false-discovery rate estimation procedure for protein-protein interactions identified by crosslinking mass spectrometry.

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Circadian Interactomics: How Research Into Protein-Protein Interactions Beyond the Core Clock Has Influenced the Model of Circadian Timekeeping

Journal of Biological Rhythms ◽

10.1177/07487304211014622 ◽

2021 ◽

pp. 074873042110146

Author(s):

Alexander E. Mosier ◽

Jennifer M. Hurley

Keyword(s):

Circadian Clock ◽

Protein Interactions ◽

Large Scale ◽

Protein Complexes ◽

Model Organisms ◽

Protein Protein Interactions ◽

Macromolecular Complexes ◽

The Core ◽

Circadian Control ◽

Macromolecular Protein

The circadian clock is the broadly conserved, protein-based, timekeeping mechanism that synchronizes biology to the Earth’s 24-h light-dark cycle. Studies of the mechanisms of circadian timekeeping have placed great focus on the role that individual protein-protein interactions play in the creation of the timekeeping loop. However, research has shown that clock proteins most commonly act as part of large macromolecular protein complexes to facilitate circadian control over physiology. The formation of these complexes has led to the large-scale study of the proteins that comprise these complexes, termed here “circadian interactomics.” Circadian interactomic studies of the macromolecular protein complexes that comprise the circadian clock have uncovered many basic principles of circadian timekeeping as well as mechanisms of circadian control over cellular physiology. In this review, we examine the wealth of knowledge accumulated using circadian interactomics approaches to investigate the macromolecular complexes of the core circadian clock, including insights into the core mechanisms that impart circadian timing and the clock’s regulation of many physiological processes. We examine data acquired from the investigation of the macromolecular complexes centered on both the activating and repressing arm of the circadian clock and from many circadian model organisms.

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Modeling of Protein–Protein Interactions in Cytokinin Signal Transduction

International Journal of Molecular Sciences ◽

10.3390/ijms20092096 ◽

2019 ◽

Vol 20 (9) ◽

pp. 2096 ◽

Cited By ~ 5

Author(s):

Dmitry V. Arkhipov ◽

Sergey N. Lomin ◽

Yulia A. Myakushina ◽

Ekaterina M. Savelieva ◽

Dmitry I. Osolodkin ◽

...

Keyword(s):

Protein Interactions ◽

Hot Spots ◽

Protein Complexes ◽

Structural Features ◽

Signaling Proteins ◽

In Planta ◽

Protein Protein Interactions ◽

Response Regulators ◽

Protein Surfaces

The signaling of cytokinins (CKs), classical plant hormones, is based on the interaction of proteins that constitute the multistep phosphorelay system (MSP): catalytic receptors—sensor histidine kinases (HKs), phosphotransmitters (HPts), and transcription factors—response regulators (RRs). Any CK receptor was shown to interact in vivo with any of the studied HPts and vice versa. In addition, both of these proteins tend to form a homodimer or a heterodimeric complex with protein-paralog. Our study was aimed at explaining by molecular modeling the observed features of in planta protein–protein interactions, accompanying CK signaling. For this purpose, models of CK-signaling proteins’ structure from Arabidopsis and potato were built. The modeled interaction interfaces were formed by rather conserved areas of protein surfaces, complementary in hydrophobicity and electrostatic potential. Hot spots amino acids, determining specificity and strength of the interaction, were identified. Virtual phosphorylation of conserved Asp or His residues affected this complementation, increasing (Asp-P in HK) or decreasing (His-P in HPt) the affinity of interacting proteins. The HK–HPt and HPt–HPt interfaces overlapped, sharing some of the hot spots. MSP proteins from Arabidopsis and potato exhibited similar properties. The structural features of the modeled protein complexes were consistent with the experimental data.

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Cross-linking of the Endolysosomal System Reveals Flotillin Structures and Putative Cargo

10.1101/2022.01.12.475930 ◽

2022 ◽

Author(s):

Jasjot Singh ◽

Hadeer Elhabashy ◽

Pathma Muthukottiappan ◽

Markus Stepath ◽

Martin Eisenacher ◽

...

Keyword(s):

Protein Interactions ◽

Protein Complexes ◽

Decisive Role ◽

Cross Linking ◽

Protein Protein Interactions ◽

Lysosome Biogenesis ◽

Early Endosomes ◽

Cross Links ◽

Related Proteins ◽

G Protein Coupled

Lysosomes are well-established as the main cellular organelles for the degradation of macromolecules and emerging as regulatory centers of metabolism. They are of crucial importance for cellular homeostasis, which is exemplified by a plethora of disorders related to alterations in lysosomal function. In this context, protein complexes play a decisive role, regulating not only metabolic lysosomal processes, but also lysosome biogenesis, transport, and interaction with other organelles. Using cross-linking mass spectrometry, we analyzed lysosomes and early endosomes. Based on the identification of 5,376 cross-links, we investigated protein-protein interactions and structures of lysosome- and endosome-related proteins. In particular, we present evidence for a tetrameric assembly of the lysosomal hydrolase PPT1 and heterodimeric/-multimeric structures of FLOT1/FLOT2 at lysosomes and early endosomes. For FLOT1-/FLOT2-positive early endosomes, we identified >300 proteins presenting putative cargo, and confirm the latrophilin family of adhesion G protein-coupled receptors as substrates for flotillin-dependent endocytosis.

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