Measuring ligand‐protein binding using NMR diffusion experiments

Dictyostelium Formin C (ForC) is involved in the regulation of local actin cytoskeleton reorganization (e.g. during cellular adhesion or migration). ForC contains formin homology 2 and 3 (FH2 and -3) domains and an N-terminal putative GTPase-binding domain (GBD) but lacks a canonical FH1 region. To better understand the role of the GBD, its structure, dynamics, lipid-binding properties, and cellular functions were analyzed by NMR and CD spectroscopy and by in vivo fluorescence microscopy. Moreover, the program CS-Rosetta was tested for the structure prediction based on chemical shift data only. The ForC GBD adopts an ubiquitinlike ␣/␤-roll fold with an unusually long loop between ␤-strands 1 and 2. Based on the lipid-binding data, the presence of DPC micelles induces the formation of ␣-helical secondary structure and a rearrangement of the tertiary structure. Lipid-binding studies with a mutant protein and a peptide suggest that the ␤1-␤2 loop is not relevant for these conformational changes. Whereas small amounts of negatively charged phosphoinositides (1,2-dioctanoyl-sn-glycero-3-(phosphoinositol 4,5-bisphosphate) and 1,2-dihexanoyl-sn-glycero-3-(phosphoinositol 3,4,5-trisphosphate)) lower the micelle concentration necessary to induce the observed spectral changes, other negatively charged phospholipids (1,2-dihexanoyl-sn-glycero-3-(phospho-L-serine) and 1,2-dihexanoyl-snglycero-3-phospho-(1 -rac-glycerol)) had no such effect. Interestingly, bicelles and micelles composed of diacylphosphocholines had no effect on the GBD structure. Our data suggest a model in which part of the large positively charged surface area of the GBD mediates localization to specific membrane patches, thereby regulating interactions with signaling proteins. Our cellular localization studies show that both the GBD and the FH3 domain are required for ForC targeting to cell-cell contacts and early phagocytic cups and macropinosomes.Formins are widely expressed multidomain proteins that regulate the spatial and temporal organization of the actin and microtubule cytoskeleton, thereby affecting important cellular functions, such as cytokinesis and cell-cell adhesion (1-3). The defining element is the ϳ400-amino acid-encompassing "formin homology 2" (FH2) 2 domain that can tightly associate with the barbed end of elongating actin filaments (4). In nearly all formins, the FH2 domain is preceded by a prolinerich "formin homology 1" (FH1) region that varies in length and the number of binding sites for profilin and that is used to recruit profilin-actin complexes for filament elongation. The influence of the FH2 domain on the rate of filament elongation depends on the length and composition of the FH1 region and the connecting linker (2, 4, 5). Most formins contain additional regulatory domains. The N-terminal "formin homology 3" (FH3) domain mediates interactions with autoinhibitory regulatory elements in the C terminus as well as with other formin regulators. The FH3 domain can be further divided into functional subdomains, such as an armadill...

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“…20. The diffusion properties of free and micellebound protein were determined using a pulsed-field gradient stimulated echo sequence (21).…”

Section: Methodsmentioning

confidence: 99%

Structure, Dynamics, Lipid Binding, and Physiological Relevance of the Putative GTPase-binding Domain of Dictyostelium Formin C

Dames

Junemann

Sass

et al. 2011

Journal of Biological Chemistry

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“…Hence, if a ligand binds to the receptor, its diffusion coefficient will decrease. A variety of NMR pulse sequences have been developed to investigate diffusion coefficients in solution [50]. Diffusion-editing has been successfully used for the characterisation of interactions between small and intermediate sizes proteins.…”

Section: Introductionmentioning

confidence: 99%

NMR Spectroscopy for Studying Interactions of Bioactive Molecules

Novak¹,

Jednačak²

2012

Physico-Chemical Methods in Drug Discovery and Development

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“…Such an interaction can be investigated by means of PFG-NMR by choosing in the NMR spectrum a signal of the ligand. [10] For this application the experiment is particularly quick and reliable, since the D value of the small molecule decreases largely when the species is associated with the protein.…”

Section: Ligand-protein Interactionsmentioning

confidence: 99%

Probing interactions by means of pulsed field gradient nuclear magnetic resonance spectroscopy

2008

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Molecular self-diffusion coefficients (D) of species in solution are related to size and shape and can be used for studying association phenomena. Pulsed field gradient nuclear magnetic resonance (PFG-NMR) spectroscopy has been revealed to be a powerful analytical tool for D measurement in different research fields. The present work briefly illustrates the use of PFG-NMR for assessing the existence of interactions in very different chemical systems: organic and organometallic compounds, colloidal materials and biological aggregates. The application of PFG-NMR is remarkable for understanding the role of anions in homogenous transition metal catalysis and for assessing the aggregation behaviour of biopolymers in material science.

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Measuring ligand‐protein binding using NMR diffusion experiments

Cited by 105 publications

References 84 publications

Structure, Dynamics, Lipid Binding, and Physiological Relevance of the Putative GTPase-binding Domain of Dictyostelium Formin C

Structure, Dynamics, Lipid Binding, and Physiological Relevance of the Putative GTPase-binding Domain of Dictyostelium Formin C

NMR Spectroscopy for Studying Interactions of Bioactive Molecules

Probing interactions by means of pulsed field gradient nuclear magnetic resonance spectroscopy

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