Protein quaternary structures in solution are a mixture of multiple forms

Maricano, Shir; Dey, Debabrata; Listof, Dina; Fleishman, Sarel J.; Sonn-Segev, Adar; Mertens, Haydyn D. T.; Büsch, Florian; Kim, Yong-Seok; Harvey, Sophie R.; Wysocki, Vicki H.; Schreiber, Gideon

doi:10.1039/d2sc02794a

Cited by 28 publications

(15 citation statements)

References 84 publications

(146 reference statements)

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“…7 D ), because having conserved interfaces or positive R 2 do not automatically mean that multimerization is functional. While these fractions are high, they seem to agree with a recent study ( Marciano et al 2022 ), showing that many homomers have several oligomeric forms in solution, and for half of the 17 proteins analyzed by these authors the available structures in the PDB underestimate their real structural diversity; thus, their quaternary structure may not influence function, or their PDB entry may not be the functional or even the dominant form.…”

Section: Discussionsupporting

confidence: 88%

“…In many cases, multimerization is known to be relevant: mutations in protein interfaces are frequently pathogenic ( Yates and Sternberg 2013 ; Sahni et al 2015 ; Livesey and Marsh 2022 ); the interfaces of homomers are frequently conserved across long evolutionary distances ( Levy et al 2008 ); and the importance of quaternary structure for function has been validated for a large number of proteins (see reviews by Marianayagam et al (2004) , Perica et al (2012) , and Marsh and Teichmann (2015) ). Some homomers are known to exist in an equilibrium with their monomeric form ( Nooren and Thornton 2003 ; Dey et al 2010 ; Acuner Ozbabacan et al 2011 ), and recent experimental work indicates that a mixture of different forms is the norm in solvent, rather than the exception ( Marciano et al 2022 ). Such “weak” complexes are nevertheless considered functional, and important in allostery, signaling, or regulation.…”

Section: Introductionmentioning

confidence: 99%

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An Assessment of Quaternary Structure Functionality in Homomer Protein Complexes

Abrusán

Foguet

2023

Molecular Biology and Evolution

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It has been recently suggested that a significant fraction of homomer protein-protein interfaces evolve neutrally, without contributing to function, due to a hydrophobic bias in missense mutations. However, the fraction of such gratuitous complexes is currently unknown. Here we quantified the fraction of homodimers where multimerization is unlikely to contribute to their biochemical function. We show that: 1) Ligand binding-site structure predicts whether a homomer is functional or not; the vast majority of homodimers with multichain binding-sites (MBS) are likely to be functional, while in homodimers with single-chain binding-sites (SBS) and small to medium interfaces, quaternary structure is unlikely to be functional in a significant fraction – 35%, even up to 42% – of complexes. 2) The hydrophobicity of interfaces changes little with the strength of selection, and the amino acid composition of interfaces is shaped by the “hydrophobic ratchet” in both types, but they are not in a strict equilibrium with mutations; particularly cysteines are much more abundant in mutations than in interfaces or surfaces. 3) In MBS homomers the interfaces are conserved, while in a high fraction of SBS homomers the interface is not more conserved than the solvent-accessible surface. 4) MBS homomer interfaces coevolve more strongly with ligand binding sites than the interfaces of SBS homomers, and MBS complexes have higher capacity to transfer information from ligands across the interfaces than SBS homomers, explaining the enrichment of allostery in the former.

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Section: Discussionsupporting

confidence: 88%

Section: Introductionmentioning

confidence: 99%

An Assessment of Quaternary Structure Functionality in Homomer Protein Complexes

Abrusán

Foguet

2023

Molecular Biology and Evolution

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“…The expression and purification of the AIR ligand was conducted following the procedures described ( 71 ). The plasmids pET28-14 His-bdSUMO-mEGFP-5aa-mCherry were introduced into T7 bacteria, which were then cultured in 2YT media supplemented with kanamycin.…”

Section: Methodsmentioning

confidence: 99%

Specificity and promiscuity of JAK recruitment regulates pleiotropy of cytokine-receptor signaling

Zoler,

Meyer,

Bellón

et al. 2023

Preprint

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Promiscuous binding of different Janus kinases (JAKs) to class I/II cytokine receptors has been reported, yet its role in signaling is unclear. To systematically explore JAK pairing in type I interferon (IFN-I) signaling, we generated an artificial IFN-I receptor (AIR) by replacing the extracellular domains of IFNAR1 and IFNAR2 with anti mEGFP and mCherry nanobodies. The heterodimeric AIR restored near-native IFN-I activity, while the homomeric variant of IFNAR2 (AIR-dR2) initiated much weaker signaling despite harboring docking sites for signal transducer and activator of transcription (STAT) proteins. AIR-dR1 was signaling inactive, yet, pulldown uncovered its ICD to bind both TYK2 and JAK1. To further investigate the roles of JAKs on the receptors, knockout (KO) JAK1, JAK2, TYK2, and JAK2/TYK2 were generated. JAK1 KO led to complete loss of IFN-I signaling, which was partially restored by TYK2 overexpression. TYK2 KO cells retained partial activity, which was elevated by JAK1 overexpression, suggesting both JAKs to partially substitute each other. Conversely, JAK2 KO only moderately impacted the biological activity of IFN-Is, even in JAK2/TYK2 KO cells. Live cell micropatterning confirmed promiscuous binding of JAK1, JAK2 and TYK2 to IFNAR1 and IFNAR2, in line with an AlphaFold model that shows JAKs interchangeability on IFNAR ICDs. Similar promiscuity of JAK binding was observed for TPOR and GHR but not EPOR, accompanied by different downstream signaling activity. The competitive binding of JAKs to cytokine receptors together with the highly diverse absolute and relative JAK expression levels can account for cell type-dependent signaling pleiotropy observed for cytokine receptors.One Sentence SummaryPromiscuous and interchangeable binding of JAKs to cytokine receptors enables cell type-specific pleiotropic signaling.

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“…These oligomers can be of homo-or hetero-oligomeric nature, i.e., identical subunits or different subunits, respectively. The oligomeric formation can be obligatory or dynamic (2), and is often crucial for the proper activity of the complex. Examples for oligomeric proteins include the homotetrameric β-galactosidase (3), and the homotrimeric HTRA1 protease (4), where for both the homo-multimer formation is essential for their activity.…”

Section: Introductionmentioning

confidence: 99%

“…This is done by superposing complete complexes and assessing the correct qs by exploiting the available evolutionary information. As for many other structural tasks, AlphaFold has been used in this context as well, where the confidence measures pTM, pLDDT and PAE are used to assess the most probable multimeric state (2,17), or by generating and scoring a variety of complex structures (18). While it is quite natural to use structural information to elucidate the qs of a protein, there are also disadvantages to the above approaches, in particular, the computationally heavy structure prediction when no experimentally solved structure is available.…”

Section: Introductionmentioning

confidence: 99%

Protein language models can capture protein quaternary state

Avraham

Tsaban

Ben-Aharon

et al. 2023

Preprint

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Background: Determining a protein's quaternary state, i.e. how many monomers assemble together to form the functioning unit, is a critical step in protein characterization, and deducing it is not trivial. Many proteins form multimers for their activity, and over 50% are estimated to naturally form homomultimers. Experimental quaternary state determination can be challenging and require extensive work. To complement these efforts, a number of computational tools have been developed for quaternary state prediction, often utilizing experimentally validated structural information. Recently, dramatic advances have been made in the field of deep learning for predicting protein structure and other characteristics. Protein language models that apply computational natural-language models to proteins successfully capture secondary structure, protein cell localization and other characteristics, from a single sequence. Here we hypothesize that information about the protein quaternary state may be contained within protein sequences as well, allowing us to benefit from these novel approaches in the context of quaternary state prediction. Results: We generated embeddings for a large dataset of quaternary state labels, extracted from the curated QSbio dataset. We then trained a model for quaternary state classification and assessed it on a non-overlapping set of distinct folds (ECOD family level). Our model, named QUEEN (QUaternary state prediction using dEEp learNing), performs worse than approaches that include information from solved crystal structures. However, we show that it successfully learned to distinguish multimers from monomers, and that the specific quaternary state is predicted with moderate success, better than a simple model that transfers annotation based on sequence similarity. Our results demonstrate that complex, quaternary state related information is included in these embeddings. Conclusions: QUEEN is the first to investigate the power of embeddings for the prediction of the quaternary state of proteins. As such, it lays out the strength as well as limitations of a sequence-based protein language model approach compared to structure-based approaches. Since it does not require any structural information and is fast, we anticipate that it will be of wide use both for in-depth investigation of specific systems, as well as for studies of large sets of protein sequences. A simple colab implementation is available at: https://colab.research.google.com/github/Orly-A/QUEEN_prediction/blob/main/QUEEN_prediction_notebook.ipynb.

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Protein quaternary structures in solution are a mixture of multiple forms

Abstract: Over half the proteins in the E.coli cytoplasm form homo or hetero-oligomeric structures. Experimentally determined structures are often considered in determining a protein’s oligomeric state, but static structures miss the...

Cited by 28 publications

References 84 publications

An Assessment of Quaternary Structure Functionality in Homomer Protein Complexes

An Assessment of Quaternary Structure Functionality in Homomer Protein Complexes

Specificity and promiscuity of JAK recruitment regulates pleiotropy of cytokine-receptor signaling

Protein language models can capture protein quaternary state

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