Natural Conformational Sampling of Human TNFα Visualized by Double Electron-Electron Resonance

Carrington, Bruce; Myers, William K.; Horanyi, P.S.; Calmiano, Mark; Lawson, Alastair D. G.

doi:10.1016/j.bpj.2017.06.007

Cited by 12 publications

(12 citation statements)

References 61 publications

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“…These data suggest that the antibody may recognise a similarly perturbed open form of the trimer that exists naturally but is only represented by a small fraction of the TNF population and is potentially only sampled transiently. This supports the conclusions made in previous studies using molecular dynamic simulations 5 and DEER analysis of spin-labelled trimer 10 .…”

Section: Resultssupporting

confidence: 92%

A conformation-selective monoclonal antibody against a small molecule-stabilised signalling-deficient form of TNF

et al. 2021

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We have recently described the development of a series of small-molecule inhibitors of human tumour necrosis factor (TNF) that stabilise an open, asymmetric, signalling-deficient form of the soluble TNF trimer. Here, we describe the generation, characterisation, and utility of a monoclonal antibody that selectively binds with high affinity to the asymmetric TNF trimer–small molecule complex. The antibody helps to define the molecular dynamics of the apo TNF trimer, reveals the mode of action and specificity of the small molecule inhibitors, acts as a chaperone in solving the human TNF–TNFR1 complex crystal structure, and facilitates the measurement of small molecule target occupancy in complex biological samples. We believe this work defines a role for monoclonal antibodies as tools to facilitate the discovery and development of small-molecule inhibitors of protein–protein interactions.

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Section: Resultssupporting

confidence: 92%

A conformation-selective monoclonal antibody against a small molecule-stabilised signalling-deficient form of TNF

et al. 2021

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“…The shape of our FES suggests that the asymmetric open state would be stable without a bound inhibitor, as there is a significant free-energy barrier to returning to the symmetric closed conformation, which is calculated to be about 5 kcal mol −1 more stable. Hence, we expect that this new conformation of TNF is formed by conformational selection as suggested by Double Electron-Electron Resonance (DEER) analysis 20 and stabilised by UCB- 6876 . This model supports our hypothesis that the compounds can bind to and stabilise a naturally sampled transient TNF trimer conformation.…”

Section: Resultsmentioning

confidence: 99%

Small molecules that inhibit TNF signalling by stabilising an asymmetric form of the trimer

et al. 2019

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Tumour necrosis factor (TNF) is a cytokine belonging to a family of trimeric proteins; it has been shown to be a key mediator in autoimmune diseases such as rheumatoid arthritis and Crohn’s disease. While TNF is the target of several successful biologic drugs, attempts to design small molecule therapies directed to this cytokine have not led to approved products. Here we report the discovery of potent small molecule inhibitors of TNF that stabilise an asymmetrical form of the soluble TNF trimer, compromising signalling and inhibiting the functions of TNF in vitro and in vivo. This discovery paves the way for a class of small molecule drugs capable of modulating TNF function by stabilising a naturally sampled, receptor-incompetent conformation of TNF. Furthermore, this approach may prove to be a more general mechanism for inhibiting protein–protein interactions.

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“…While spin labels with attachment sites in α-helices have been investigated quite extensively, relatively little has been done on spin labels with attachment sites in β-sheets. The currently available crystallographic evidence includes 37 realizations of solvent-facing R1 tags in the β-sheet regions, including our own previous work on GB1 [77][78][79][80][81] . The sample is dominated by χ χ = t m ( , ) (…”

Section: Simple Determinants Of the Esr Spectral Shapes The Results Inmentioning

confidence: 99%

Structural and dynamic origins of ESR lineshapes in spin-labeled GB1 domain: the insights from spin dynamics simulations based on long MD trajectories

Izmailov

Rabdano

Hasanbasri

et al. 2020

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Site-directed spin labeling (SDSL) eSR is a valuable tool to probe protein systems that are not amenable to characterization by x-ray crystallography, nMR or eM. While general principles that govern the shape of SDSL eSR spectra are known, its precise relationship with protein structure and dynamics is still not fully understood. To address this problem, we designed seven variants of GB1 domain bearing R1 spin label and recorded the corresponding MD trajectories (combined length 180 μs). the MD data were subsequently used to calculate time evolution of the relevant spin density matrix and thus predict the eSR spectra. the simulated spectra proved to be in good agreement with the experiment. further analysis confirmed that the spectral shape primarily reflects the degree of steric confinement of the R1 tag and, for the well-folded protein such as GB1, offers little information on local backbone dynamics. The rotameric preferences of R1 side chain are determined by the type of the secondary structure at the attachment site. the rotameric jumps involving dihedral angles χ 1 and χ 2 are sufficiently fast to directly influence the ESR lineshapes. However, the jumps involving multiple dihedral angles tend to occur in (anti)correlated manner, causing smaller-than-expected movements of the R1 proxyl ring. Of interest, ESR spectra of GB1 domain with solvent-exposed spin label can be accurately reproduced by means of Redfield theory. In particular, the asymmetric character of the spectra is attributable to Redfield-type cross-correlations. We envisage that the current MD-based, experimentally validated approach should lead to a more definitive, accurate picture of SDSL ESR experiments. Over the last three decades, the field of structural biology has made a tremendous progress. This progress should be mainly credited to high-resolution X-ray crystallography and, to a lesser extent, solution-state NMR. More recently, cryo-EM microscopy became a major source of medium-resolution data. Furthermore, solid-state NMR emerged as a valuable addition to the repertoire of structure-solving techniques. However, there are many important protein systems, which defy conventional structure-solving strategies. X-ray diffractometry is contingent on one's ability to obtain a crystalline sample. NMR spectroscopy is limited by the size of the system. Generally, all techniques, including cryo-EM microscopy, tend to have difficulties with those systems that are highly inhomogeneous and/or highly dynamic. Many of the most important cellular systems fall in this category, e.g. chromatin, nuclear pore complex, cilia, etc. Some of the aberrant protein assemblies also have these characteristics, e.g. the so-called protofibrils, neurofibrillary tangles, etc. For those more challenging samples, valuable structural information can often be obtained by means of ESR spectroscopy. Applications of ESR spectroscopy to protein samples usually rely on the popular spin-labeling reagent, (1-oxyl-2,2,5,5-tetramethylpyrrolinyl-3-methyl)-methanethiosulfonate (abbre...

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Natural Conformational Sampling of Human TNFα Visualized by Double Electron-Electron Resonance

Cited by 12 publications

References 61 publications

A conformation-selective monoclonal antibody against a small molecule-stabilised signalling-deficient form of TNF

A conformation-selective monoclonal antibody against a small molecule-stabilised signalling-deficient form of TNF

Small molecules that inhibit TNF signalling by stabilising an asymmetric form of the trimer

Structural and dynamic origins of ESR lineshapes in spin-labeled GB1 domain: the insights from spin dynamics simulations based on long MD trajectories

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