Monomeric and dimeric conformation of the vinculin tail five-helix bundle in solution studied by EPR spectroscopy.

The cytoskeletal adaptor protein vinculin plays an important role in the control of cell adhesion and migration, linking the actin cytoskeleton to adhesion receptor complexes in cell adhesion sites. The conformation of the vinculin tail dimer, which is crucial for protein function, was analyzed using site-directed spin labeling in electron paramagnetic resonance spectroscopy. Interspin distances for a set of six singly and four doubly spin-labeled mutants of the tail domain of vinculin were determined and used as constraints for modeling of the vinculin tail dimer. A comparison of the results obtained by molecular dynamic simulations and a rotamer library approach reveals that the crystal structure of the vinculin tail monomer is essentially preserved in aqueous solution. The orientation of monomers within the dimer observed previously by x-ray crystallography agrees with the solution electron paramagnetic resonance data. Furthermore, the distance between positions 1033 is shown to increase by >3 nm upon interaction of the vinculin tail domain with F-actin.

Structural and dynamic characterization of a vinculin binding site in the talin rod.

Talin is a key protein involved in linking integrins to the actin cytoskeleton. The long flexible talin rod domain contains a number of binding sites for vinculin, a cytoskeletal protein important in stabilizing integrin-mediated cell-matrix junctions. Here we report the solution structure of a talin rod polypeptide (residues 1843-1973) which contains a single vinculin binding site (VBS; residues 1944-1969). Like other talin rod polypeptides, it consists of a helical bundle, in this case a four-helix bundle with a right-handed topology. The residues in the VBS important for vinculin binding were identified by studying the binding of a series of VBS-related peptides to the vinculin Vd1 domain. The key binding determinants are buried in the interior of the helical bundle, suggesting that a substantial structural change in the talin polypeptide is required for vinculin binding. Direct evidence for this was obtained by NMR and EPR spectroscopy. [1H,15N]-HSQC spectra of the talin fragment indicate that vinculin binding caused approximately two-thirds of the protein to adopt a flexible random coil. For EPR spectroscopy, nitroxide spin labels were attached to the talin polypeptide via appropriately located cysteine residues. Measurements of inter-nitroxide distances in doubly spin-labeled protein showed clearly that the helical bundle is disrupted and the mobility of the helices, except for the VBS helix, is markedly increased. Binding of vinculin to talin is thus a clear example of the unusual phenomenon of protein unfolding being required for protein/protein interaction.

Assessing oligomerization of membrane proteins by four-pulse DEER: pH-dependent dimerization of NhaA Na+/H+ antiporter of E. coli.

The pH dependence of the structure of the main Na(+)/H(+) antiporter NhaA of Escherichia coli is studied by continuous-wave (CW) and pulse electron paramagnetic resonance (EPR) techniques on singly spin-labeled mutants. Residues 225 and 254 were selected for site-directed spin labeling, as previous work suggested that they are situated in domains undergoing pH-dependent structural changes. A well-defined distance of 4.4 nm between residues H225R1 in neighboring molecules is detected by a modulation in double electron-electron resonance data. This indicates that NhaA exists as a dimer, as previously suggested by a low-resolution electron density map and cross-linking experiments. The modulation depth decreases reversibly when pH is decreased from 8 to 5.8. A quantitative analysis suggests a dimerization equilibrium, which depends moderately on pH. Furthermore, the mobility and polarity of the environment of a spin label attached to residue 225 change only slightly with changing pH, while no other changes are detected by CW EPR. As antiporter activity of NhaA changes drastically in the studied pH range, residues 225 and 254 are probably located not in the sensor or ion translocation sites themselves but in domains that convey the signal from the pH sensor to the translocation site.

Structural dynamics of the DnaK-peptide complex.

The molecular chaperone DnaK recognizes and binds substrate proteins via a stretch of seven amino acid residues that is usually only exposed in unfolded proteins. The binding kinetics are regulated by the nucleotide state of DnaK, which alternates between DnaK.ATP (fast exchange) and DnaK.ADP (slow exchange). These two forms cycle with a rate mainly determined by the ATPase activity of DnaK and nucleotide exchange. The different substrate binding properties of DnaK are mainly attributed to changes of the position and mobility of a helical region in the C-terminal peptide-binding domain, the so-called LID. It closes the peptide-binding pocket and thus makes peptide binding less dynamic in the ADP-bound state, but does not (strongly) interact with peptides directly. Here, we address the question if nucleotide-dependent structural changes may be observed in the peptide-binding region that could also be connected to peptide binding kinetics and more importantly could induce structural changes in peptide stretches using the energy available from ATP hydrolysis. Model peptides containing two cysteine residues at varying positions were derived from the structurally well-documented peptide NRLLLTG and labelled with electron spin sensitive probes. Measurements of distances and mobilities of these spin labels by electron paramagnetic resonance spectroscopy (EPR) of free peptides or peptides bound to the ATP and ADP-state of DnaK, respectively, showed no significant changes of mobility nor distance of the two labels. This indicates that no structural changes that could be sensed by the probes at the position of central leucine residues located in the center of the binding region occur due to different nucleotide states. We conclude from these studies that the ATPase activity of DnaK is not connected to structural changes of the peptide-binding pocket but rather only has an effect on the LID domain or other further remote residues.