Comparison of the backbone dynamics of wild-type Hydrogenobacter thermophilus cytochrome c(552) and its b-type variant.

Cytochrome c552 from the thermophilic bacterium Hydrogenobacter thermophilus is a typical c-type cytochrome which binds heme covalently via two thioether bonds between the two heme vinyl groups and two cysteine thiol groups in a CXXCH sequence motif. This protein was converted to a b-type cytochrome by substitution of the two cysteine residues by alanines (Tomlinson and Ferguson in Proc Natl Acad Sci USA 97:5156-5160, 2000a). To probe the significance of the covalent attachment of the heme in the c-type protein, (15)N relaxation and hydrogen exchange studies have been performed for the wild-type and b-type proteins. The two variants share very similar backbone dynamic properties, both proteins showing high (15)N order parameters in the four main helices, with reduced values in an exposed loop region (residues 18-21), and at the C-terminal residue Lys80. Some subtle changes in chemical shift and hydrogen exchange protection are seen between the wild-type and b-type variant proteins, not only for residues at and neighbouring the mutation sites, but also for some residues in the heme binding pocket. Overall, the results suggest that the main role of the covalent linkages between the heme group and the protein chain must be to increase the stability of the protein.

Asymmetric mode of Ca²âº-S100A4 interaction with nonmuscle myosin IIA generates nanomolar affinity required for filament remodeling.

Filament assembly of nonmuscle myosin IIA (NMIIA) is selectively regulated by the small Ca²âº-binding protein, S100A4, which causes enhanced cell migration and metastasis in certain cancers. Our NMR structure shows that an S100A4 dimer binds to a single myosin heavy chain in an asymmetrical configuration. NMIIA in the complex forms a continuous helix that stretches across the surface of S100A4 and engages the Ca²âº-dependent binding sites of each subunit in the dimer. Synergy between these sites leads to a very tight association (K(D) ∼1 nM) that is unique in the S100 family. Single-residue mutations that remove this synergy weaken binding and ameliorate the effects of S100A4 on NMIIA filament assembly and cell spreading in A431 human epithelial carcinoma cells. We propose a model for NMIIA filament disassembly by S100A4 in which initial binding to the unstructured NMIIA tail initiates unzipping of the coiled coil and disruption of filament packing.

Self-association of calcium-binding protein S100A4 and metastasis.

Elevated levels of the calcium-binding protein S100A4 promote metastasis and in carcinoma cells are associated with reduced survival of cancer patients. S100A4 interacts with target proteins that affect a number of activities associated with metastatic cells. However, it is not known how many of these interactions are required for S100A4-promoted metastasis, thus hampering the design of specific inhibitors of S100A4-induced metastasis. Intracellular S100A4 exists as a homodimer through previously identified, well conserved, predominantly hydrophobic key contacts between the subunits. Here it is shown that mutating just one key residue, phenylalanine 72, to alanine is sufficient to reduce the metastasis-promoting activity of S100A4 to 50% that of the wild type protein, and just 2 or 3 specific mutations reduces the metastasis-promoting activity of S100A4 to less than 20% that of the wild type protein. These mutations inhibit the self-association of S100A4 in vivo and reduce markedly the affinity of S100A4 for at least two of its protein targets, a recombinant fragment of non-muscle myosin heavy chain isoform A, and p53. Inhibition of the self-association of S100 proteins might be a novel means of inhibiting their metastasis-promoting activities.

Clusters in an intrinsically disordered protein create a protein-binding site: the TolB-binding region of colicin E9.

The 61-kDa colicin E9 protein toxin enters the cytoplasm of susceptible cells by interacting with outer membrane and periplasmic helper proteins and kills them by hydrolyzing their DNA. The membrane translocation function is located in the N-terminal domain of the colicin, with a key signal sequence being a pentapeptide region that governs the interaction with the helper protein TolB (the TolB box). Previous NMR studies [Collins et al. (2002) J. Mol. Biol. 318, 787-904; MacDonald et al. (2004), J. Biomol. NMR 30, 81-96] have shown that the N-terminal 83 residues of colicin E9, which includes the TolB box, is intrinsically disordered and contains clusters of interacting side chains. To further define the properties of this region of colicin E9, we have investigated the effects on the dynamical and TolB-binding properties of three mutations of colicin E9 that inactivate it as a toxin. The mutations were contained in a fusion protein consisting of residues 1-61 of colicin E9 connected to the N terminus of the E9 DNase by an eight-residue linking sequence. The NMR data reveals that the mutations cause major alterations to the properties of some of the clusters, consistent with some form of association between them and other more distant parts of the amino acid sequence, particularly toward the N terminus of the protein. However, (15)N T(2) measurements indicates that residues 5-13 of the fusion protein bound to the 43-kDa TolB remain as flexible as they are in the free protein. The NMR data point to considerable dynamic ordering within the intrinsically disordered translocation domain of the colicin that is important for creating the TolB-binding site. Furthermore, amino acid sequence considerations suggest that the clusters of amino acids occur because of the size and polarities of the side chains forming them influenced by the propensities of the residues within the clusters and those immediately surrounding them in sequence space to form beta turns.

Photo-CIDNP NMR spectroscopy of a heme-containing protein.

There are relatively few examples of the application of photo-CIDNP NMR spectroscopy to chromophore-containing proteins. The most likely reason for this is that simultaneous absorption of light by the photosensitiser molecule and the protein chromophore reduces the effectiveness of the photochemical reaction that produces the observed nuclear polarisation. We present details of experiments performed on the air-oxidised form of a small cytochrome, from the thermophilic bacterium Hydrogenobacter thermophilus, using both the wild-type protein and apo and holo forms of a double alanine b-type mutant. We show that, along with the apo state, it is possible to generate CIDNP in the air-oxidised form of the b-type mutant, but not in the corresponding c-type cytochrome. This finding is supported by control experiments using horse-heart cytochrome c.

Characterisation of a mobile protein-binding epitope in the translocation domain of colicin E9.

The 61 kDa colicin E9 protein toxin enters the cytoplasm of susceptible cells by interacting with outer membrane and periplasmic helper proteins, and kills them by hydrolysing their DNA. The membrane translocation function is located in the N-terminal domain of the colicin, with a key signal sequence being a pentapeptide region that governs the interaction with the helper protein TolB (the TolB box). Previous NMR studies (Collins et al., 2002 J. Mol. Biol. 318, 787-804) have shown that the N-terminal 83 residues of colicin E9, which includes the TolB box, is largely unstructured and highly flexible. In order to further define the properties of this region we have studied a fusion protein containing residues 1-61 of colicin E9 connected to the N-terminus of the E9 DNase by an eight-residue linking sequence. 53 of the expected 58 backbone NH resonances for the first 61 residues and all of the expected 7 backbone NH resonances of the linking sequence were assigned with 3D (1)H-(13)C-(15)N NMR experiments, and the backbone dynamics of these regions investigated through measurement of (1)H-(15)N relaxation properties. Reduced spectral density mapping, extended Lipari-Szabo modelling, and fitting backbone R(2) relaxation rates to a polymer dynamics model identifies three clusters of interacting residues, each containing a tryptophan. Each of these clusters is perturbed by TolB binding to the intact colicin, showing that the significant region for TolB binding extends beyond the recognized five amino acids of the TolB box and demonstrating that the binding epitope for TolB involves a considerable degree of order within an otherwise disordered and flexible domain. Abbreviations : Im9, the immunity protein for colicin E9; E9 DNase, the endonuclease domain of colicin E9; HSQC, heteronuclear single quantum coherence; ppm, parts per million; DSS, 2,2-(dimethylsilyl)propanesulfonic acid; TSP, sodium 3-trimethylsilypropionate; T(1 - 61)-DNase fusion protein, residues 1-61 of colicin E9 connected to the N-terminus of the E9 DNase by an eight residue thrombin cleavage sequence.

Structural dynamics of the membrane translocation domain of colicin E9 and its interaction with TolB.

In order for the 61 kDa colicin E9 protein toxin to enter the cytoplasm of susceptible cells and kill them by hydrolysing their DNA, the colicin must interact with the outer membrane BtuB receptor and Tol translocation pathway of target cells. The translocation function is located in the N-terminal domain of the colicin molecule. (1)H, (1)H-(1)H-(15)N and (1)H-(13)C-(15)N NMR studies of intact colicin E9, its DNase domain, minimal receptor-binding domain and two N-terminal constructs containing the translocation domain showed that the region of the translocation domain that governs the interaction of colicin E9 with TolB is largely unstructured and highly flexible. Of the expected 80 backbone NH resonances of the first 83 residues of intact colicin E9, 61 were identified, with 43 of them being assigned specifically. The absence of secondary structure for these was shown through chemical shift analyses and the lack of long-range NOEs in (1)H-(1)H-(15)N NOESY spectra (tau(m)=200 ms). The enhanced flexibility of the region of the translocation domain containing the TolB box compared to the overall tumbling rate of the protein was identified from the relatively large values of backbone and tryptophan indole (15)N spin-spin relaxation times, and from the negative (1)H-(15)N NOEs of the backbone NH resonances. Variable flexibility of the N-terminal region was revealed by the (15)N T(1)/T(2) ratios, which showed that the C-terminal end of the TolB box and the region immediately following it was motionally constrained compared to other parts of the N terminus. This, together with the observation of inter-residue NOEs involving Ile54, indicated that there was some structural ordering, resulting most probably from the interactions of side-chains. Conformational heterogeneity of parts of the translocation domain was evident from a multiplicity of signals for some of the residues. Im9 binding to colicin E9 had no effect on the chemical shifts or other NMR characteristics of the region of colicin E9 containing the TolB recognition sequence, though the interaction of TolB with intact colicin E9 bound to Im9 did affect resonances from this region. The flexibility of the translocation domain of colicin E9 may be connected with its need to recognise protein partners that assist it in crossing the outer membrane and in the translocation event itself.