On the acquisition and analysis of microscale thermophoresis data

Scheuermann, Thomas H.; Padrick, Shae B.; Gardner, Kevin H.; Brautigam, Chad A.

doi:10.1016/j.ab.2015.12.013

Cited by 147 publications

(195 citation statements)

References 61 publications

(81 reference statements)

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“…On the other hand, addition of BeF 3 − to a sample of EL_REC containing MgCl 2 caused minor changes in the overall spectrum, likely due to the already active-like conformation of the truncated protein (Figure S2D). To further confirm that BeF 3 − binding to EL_PhyR was indeed mimicking the effect of protein phosphorylation, we monitored the binding of the cognate anti-σ factor to fluorescently labeled EL_PhyR (wild-type and D194A mutant) in the presence of 5 mM BeF 3 − using microscale thermophoresis (MST; Seidel et al, 2013; Scheuermann et al, 2016) (Figure 3). Our data show that EL_PhyR strongly binds to its anti-σ factor (K d = 34 ± 17 nM) in an activation-dependent manner, comparable with homologous systems (Campagne et al, 2012; Herrou et al, 2012).…”

Section: Resultsmentioning

confidence: 94%

Basis of Mutual Domain Inhibition in a Bacterial Response Regulator

Corrêa

Gardner

2016

Cell Chemical Biology

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SUMMARY Information transmission in biological signaling networks is commonly considered to be a unidirectional flow of information between protein partners. According to this view, many bacterial response regulator proteins utilize input receiver (REC) domains to “switch” functional outputs, using REC phosphorylation to shift pre-existing equilibria between inactive and active conformations. However, recent data indicate that output domains themselves also shift such equilibria, implying a “mutual inhibition” model. Here we use solution nuclear magnetic resonance to provide a mechanistic basis for such control in a PhyR-type response regulator. Our structure of the isolated, non-phosphorylated REC domain surprisingly reveals a fully active conformation, letting us identify structural and dynamic changes imparted by the output domain to inactivate the full-length protein. Additional data reveal transient structural changes within the full-length protein, facilitating activation. Our data provide a basis for understanding the changes that REC and output domains undergo to set a default “inactive” state.

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Section: Resultsmentioning

confidence: 94%

Basis of Mutual Domain Inhibition in a Bacterial Response Regulator

Corrêa

Gardner

2016

Cell Chemical Biology

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“…The data were analyzed in PALMIST (Scheuermann et al, 2016) and plotted with GUSSI (Brautigam, 2015). …”

Section: Methodsmentioning

confidence: 99%

Nuclear export receptor CRM1 recognizes diverse conformations in nuclear export signals

Fung

Chook

2017

eLife

100

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Nuclear export receptor CRM1 binds highly variable nuclear export signals (NESs) in hundreds of different cargoes. Previously we have shown that CRM1 binds NESs in both polypeptide orientations (Fung et al., 2015). Here, we show crystal structures of CRM1 bound to eight additional NESs which reveal diverse conformations that range from loop-like to all-helix, which occupy different extents of the invariant NES-binding groove. Analysis of all NES structures show 5-6 distinct backbone conformations where the only conserved secondary structural element is one turn of helix that binds the central portion of the CRM1 groove. All NESs also participate in main chain hydrogen bonding with human CRM1 Lys568 side chain, which acts as a specificity filter that prevents binding of non-NES peptides. The large conformational range of NES backbones explains the lack of a fixed pattern for its 3-5 hydrophobic anchor residues, which in turn explains the large array of peptide sequences that can function as NESs.DOI: http://dx.doi.org/10.7554/eLife.23961.001

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“…To examine whether the phosphoinositide and capping protein binding sites overlap the C-terminal tail of twinfilin, we utilized the microscale thermophoresis (MST) method (47)(48)(49) to measure the affinities of wildtype and mutant twinfilins to EGFP-tagged capping protein. Consistent with an earlier study (39), twinfilin lacking the C-terminal tail (TWF1 169-316) exhibited only low affinity towards capping protein (Fig.…”

Section: Resultsmentioning

confidence: 99%

Molecular mechanism for inhibition of twinfilin by phosphoinositides

Hakala

Vattulainen

Lappalainen

2018

Journal of Biological Chemistry

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Membrane phosphoinositides control organization and dynamics of the actin cytoskeleton by regulating the activities of several key actin-binding proteins. Twinfilin is an evolutionarily conserved protein, which contributes to cytoskeletal dynamics by interacting with actin monomers, filaments, and the heterodimeric capping protein. Twinfilin also binds phosphoinositides, which inhibit its interactions with actin, but the underlying mechanism has remained unknown. Here we show that the highaffinity binding site of twinfilin for phosphoinositides is located at the carboxy-terminal tail-region, while the two ADF/cofilin like ADF-H domains of twinfilin bind phosphoinositides only with low affinity. Mutagenesis and biochemical experiments combined with atomistic molecular dynamics simulations reveal that the carboxy-terminal tail of twinfilin interacts with membranes through a multivalent electrostatic interaction with a preference towards PI(3,5)P 2 , PI(4,5)P 2 , and PI(3,4,5)P 3 . This initial interaction places the actin-binding ADF-H domains of twinfilin to a close proximity of the membrane and subsequently promotes their association with the membrane, thus leading to inhibition of the actininteractions. In support to this model, a twinfilin mutant lacking the carboxy-terminal tail inhibits actin filament assembly in a phosphoinositide-insensitive manner. Our mutagenesis data also reveal that the phosphoinositide-and capping proteinbinding sites overlap in the carboxy-terminal tail of twinfilin, suggesting that phosphoinositide-binding additionally inhibits the interactions of twinfilin with the heterodimeric capping protein. The results demonstrate that the conserved carboxy-terminal tail of twinfilin is a multi-functional binding motif, which is crucial for interaction with the heterodimeric capping protein and for tethering twinfilin to phosphoinositiderich membranes.The dynamic interplay between the actin cytoskeleton and plasma membrane is critical for several cellular processes, such as migration, morphogenesis, endocytosis, and phagocytosis. Coordinated polymerization of actin filaments provides a force for generation of membrane invaginations in endocytic processes (1, 2). In migrating cells, polymerization of actin filaments against the plasma membrane at the leading edge pushes the membrane forward to generate plasma membrane protrusions, such as lamellipodia and filopodia (3-6).While actin polymerization controls the geometry of cellular membranes, membrane phospholipids, especially PI(4,5)P 2 , reciprocally regulate the organization and the dynamics of the actin cytoskeleton. Typically an increase in the plasma membrane PI(4,5)P 2 density leads to actin filament assembly beneath the membrane, whereas a decrease in PI(4,5)P 2 concentration results in diminished actin Mechanism of twinfilin-PI(4,5)P 2 interaction filament assembly (7-10). Phosphoinositides regulate actin filament assembly and disassembly by directly interacting with several actin-binding proteins (11). The activities and the pl...

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On the acquisition and analysis of microscale thermophoresis data

Cited by 147 publications

References 61 publications

Basis of Mutual Domain Inhibition in a Bacterial Response Regulator

Basis of Mutual Domain Inhibition in a Bacterial Response Regulator

Nuclear export receptor CRM1 recognizes diverse conformations in nuclear export signals

Molecular mechanism for inhibition of twinfilin by phosphoinositides

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