Label-free methods for optical<i>in vitro</i>characterization of protein–protein interactions

Soltermann, Fabian; Struwe, Weston B.; Kukura, Philipp

doi:10.1039/d1cp01072g

Cited by 25 publications

(26 citation statements)

References 132 publications

(197 reference statements)

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“…in vitro tool used for determining binding kinetics between biological molecules, including antigen-antibody or proteinprotein interactions, among other molecular interactions (37)(38)(39)(40).…”

Section: Discussionmentioning

confidence: 99%

The AIRE G228W mutation disturbs the interaction of AIRE with its partner molecule SIRT1

et al. 2022

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The autoimmune regulator (AIRE) protein functions as a tetramer, interacting with partner proteins to form the “AIRE complex,” which relieves RNA Pol II stalling in the chromatin of medullary thymic epithelial cells (mTECs). AIRE is the primary mTEC transcriptional controller, promoting the expression of a large set of peripheral tissue antigen genes implicated in the negative selection of self-reactive thymocytes. Under normal conditions, the SIRT1 protein temporarily interacts with AIRE and deacetylates K residues of the AIRE SAND domain. Once the AIRE SAND domain is deacetylated, the binding with SIRT1 is undone, allowing the AIRE complex to proceed downstream with the RNA Pol II to the elongation phase of transcription. Considering that the in silico and in vitro binding of the AIRE SAND domain with SIRT1 provides a powerful model system for studying the dominant SAND G228W mutation mechanism, which causes the autoimmune polyglandular syndrome-1, we integrated computational molecular modeling, docking, dynamics between the whole SAND domain with SIRT1, and surface plasmon resonance using a peptide harboring the 211 to 230 residues of the SAND domain, to compare the structure and energetics of binding/release between AIRE G228 (wild-type) and W228 (mutant) SAND domain to SIRT1. We observed that the G228W mutation in the SAND domain negatively influences the AIRE-SIRT1 interaction. The disturbed interaction might cause a disruption in the binding of the AIRE SAND domain with the SIRT1 catalytic site, impairing the AIRE complex to proceed downstream with RNA Pol II.

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Section: Discussionmentioning

confidence: 99%

The AIRE G228W mutation disturbs the interaction of AIRE with its partner molecule SIRT1

et al. 2022

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“…To validate the in silico studies referring to the different interactions of the G228W SAND mutant with the SIRT1 protein, we performed an in vitro study to evaluate this interaction using surface plasmon resonance (SPR). Sensor-based SPR is a label-free, sensitive, precise, and in vitro tool used for determining binding kinetics between biological molecules, including antigen-antibody or protein-protein interactions, among other molecular interactions (Nguyen et al, 2015; Wang et al, 2019; Soltermann et al, 2021; Ribeiro et al, 2021).…”

Section: Discussionmentioning

confidence: 99%

“…This approach confirmed the hypothesis raised in this study, in which we consider that the AIRE G228W mutation changes the interaction with SIRT1, mainly when evaluated using SPR analysis. Sensor-based SPR is a label-free, sensitive, precise, and in vitro tool used for determining binding kinetics between biological molecules, including antigen-antibody or protein-protein interactions, among other molecular interactions (Nguyen et al, 2015; Wang et al, 2019; Soltermann et al, 2021; Ribeiro et al, 2021).…”

Section: Discussionmentioning

confidence: 99%

The AIRE G228W mutation disturbs the interaction of AIRE with its partner molecule SIRT1

Santos

Dametto

Masson

et al. 2021

Preprint

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The in silico and in vitro binding of a peptide covering a part of the autoimmune regulator (AIRE) SAND domain with the SIRT1 protein provides a powerful model system for studying the mechanism of the dominant SAND G228W mutation, which is the causative of APS-1 autoimmune syndrome. It is known that the mutant G228W AIRE protein accumulates more within the nucleus of cells than its wild-type counterpart does. This accumulation is not physiological and is associated with loss of AIRE function. However, the precise molecular mechanism that leads to AIRE accumulation is not yet known. AIRE works as a tetramer and interacts with partner proteins to form the “AIRE complex” that pushes RNA Pol II stalling in the chromatin of medullary thymic epithelial cells. Under normal conditions, the SIRT1 protein temporarily interacts with AIRE and deacetylates Lys residues of the SAND domain. Once AIRE is deacetylated, the binding with SIRT1 is undone, allowing the complex to proceed downstream. Here, we integrate molecular modeling, docking, dynamics, and surface plasmon resonance approaches to compare the structure and energetics of binding/release between AIRE G228 (wild-type) or W228 (mutant) peptides to SIRT1. We find that the proximity of G228W mutation to a K aminoacid residue in the SAND domain promotes a longer-lasting AIRE-SIRT1 interaction. The lasting interaction might cause a delay in the AIRE SAND domain to be released from the SIRT1 catalytic site, which might cause accumulation of the defective AIRE mutant protein in the nuclei of cells.SignificanceThis report reveals the mechanism of the pathogenic and dominant G228W mutation in the AIRE SAND domain. The G228W mutation is found in APS-1 syndrome patients, and it is critical to understand the molecular basis of loss of self-representation, a challenging aspect for immunology. Through modeling, molecular dynamics, and protein binding kinetics, we found that the G228W mutation leads to a stronger physical interaction between the AIRE SAND domain and the SIRT1 protein when compared to the equivalent wild-type segment. The short-term consequence of this stronger interaction is that the release of the AIRE-SIRT1 binding is slower. This might explain the reason that cells carrying the G228W mutation accumulate AIRE protein in their nuclei. This finding reveals with precision the AIRE-SIRT1 binding and the molecular mechanism of the human AIRE G228W mutation.

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“…Functionalizing surfaces and nonmaterial provide a means for targeting through selective binding to receptors at targeted sites [11][12][13][14][15]. Other groups studied the interaction between proteins by monitoring the interferometric contrast modification resulting from an agglomeration of molecules [23]. These interferometric scattering methods proved their sensitivity by detecting single protein and their sensibility by the ability of distinction between different agglomerations [31].…”

Section: -Introductionmentioning

confidence: 99%

Label-free virus-antibody interaction monitoring in real time by common-path interferometry

Alhaddad

Bey

Thouvenin

et al. 2022

Preprint

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Viruses can affect all life forms, raising concerns about virus detection and quantification of small nanoparticles. This paper uses a label-free, full-field, incoherently illuminated common-path interferometric method to detect, track, and quantify biotic nanoparticles. The detection consists of amplifying the light scattered by single nanoparticles in the sample solution. Then, the use of single-particle tracking analysis is used to monitor the change in particle diffusive mobility. With this approach, the recognition signature of T5 phages with purified antibodies targeting the major capsid protein is detected in a few minutes. We also tracked the interaction between SPP1 phages and physiological non-purified serum-containing multiples antibodies molecules. The first interactions occur after around one minute, and the recognition signature is detectable after minutes. In addition, we have been able to differentiate two populations of similar size of empty and full (encapsulating DNA) capsids of T5 in a heterogeneous solution demonstrating the robustness of this label-free detection approach. Furthermore, by combining the diffusion coefficient to the number of tracked particles, we were able to estimate the affinity of the virus-antibodies reaction.

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Label-free methods for opticalin vitrocharacterization of protein–protein interactions

Abstract: Protein–protein interactions are involved in the regulation and function of the majority of cellular processes.

Cited by 25 publications

References 132 publications

The AIRE G228W mutation disturbs the interaction of AIRE with its partner molecule SIRT1

The AIRE G228W mutation disturbs the interaction of AIRE with its partner molecule SIRT1

The AIRE G228W mutation disturbs the interaction of AIRE with its partner molecule SIRT1

Label-free virus-antibody interaction monitoring in real time by common-path interferometry

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