Making ends meet: the importance of the N- and C-termini for the structure, stability, and function of the third SH3 domain of CIN85.

SH3 domains are common structure, interaction, and regulation modules found in more than 200 human proteins. In this report, we studied the third SH3 domain from the human CIN85 adaptor protein, which plays an important role in both receptor tyrosine kinase downregulation and phosphatidylinositol 3 kinase inhibition. The structure of this domain includes an additional 90° kink after the last canonical ß-strand and features unusual interactions between the termini well outside the boundaries of the standard SH3 domain definition. The extended portions of the domain are well-structured and held together entirely by side chain-side chain interactions. Extensive expression screening showed that these additional contacts provide significantly increased stability to the domain. A similar 90° kink is found in only one other SH3 domain structure, while side chain contacts linking the termini have never been described before. As a result of the increased size of CIN85 SH3 domain C, the proximal proline rich region is positioned such that a possible intramolecular interaction is structurally inhibited. Using the key interactions of the termini as the basis for sequence analysis allowed the identification of several SH3 domains with flanking sequences that could adopt similar structures. This work illustrates the importance of careful experimental analysis of domain boundaries even for a well-characterized fold such as the SH3 domain.

Structural characterization of the N-terminal oligomerization domain of the bacterial chromatin-structuring protein, H-NS.

The H-NS protein plays a key role in condensing DNA and modulating gene expression in bacterial nucleoids. The mechanism by which this is achieved is dependent, at least in part, on the oligomerization of the protein. H-NS consists of two distinct domains; the N-terminal domain responsible for protein oligomerization, and the C-terminal DNA binding domain, which are separated by a flexible linker region. We present a multidimensional NMR study of the amino-terminal 64 residues of H-NS (denoted H-NS1-64) from Salmonella typhimurium, which constitute the oligomerization domain. This domain exists as a homotrimer, which is predicted to be self-associated through a coiled-coil configuration. NMR spectra show an equivalent magnetic environment for each monomer indicating that the polypeptide chains are arranged in parallel with complete 3-fold symmetry. Despite the limited resonance dispersion, an almost complete backbone assignment for 1H(N), 1H(alpha), 15N, 13CO and 13C(alpha) NMR resonances was obtained using a suite of triple resonance experiments applied to uniformly 15N-, 13C/15N- and 2H/13C/15N-labelled H-NS1-64 samples. The secondary structure of H-NS1-64 has been identified on the basis of the analysis of 1H(alpha), 13C(alpha), 13Cbeta and 13CO chemical shifts, NH/solvent exchange rates, intra-chain H(N)-H(N) and medium-range nuclear Overhauser enhancements (NOEs). Within the context of the homotrimer, each H-NS1-64 protomer consists of three alpha-helices spanning residues 2-8, 12-20 and 22-53, respectively. A topological model is presented for the symmetric H-NS1-64 trimer based upon the combined analysis of the helical elements and the pattern of backbone amide group 15N nuclear relaxation rates within the context of axially asymmetric diffusion tensor. In this model, the longest of the three helices (helix 3, residues 22-53) forms a coiled-coil interface with the other chains in the homotrimer. The two shorter N-terminal helices fold back onto the outer surface of the coiled-coil core and potentially act to stabilise this configuration.

NMR analysis of the N-terminal SRCR domain of human CD5: engineering of a glycoprotein for superior characteristics in NMR experiments.

CD5 is a type-I transmembrane glycoprotein found on thymocytes, T-cells and a subset of B-cells. The extracellular region consists of three domains belonging to the scavenger receptor cysteine-rich (SRCR) superfamily for which the three-dimensional polypeptide fold is as yet unknown. Glycosylated CD5 domain 1 (CD5d1) has been obtained by expression by secretion from both Chinese hamster ovary (CHO) cells and Pichia pastoris. Recombinant CD5d1 expressed in this manner was shown to be correctly folded by binding to anti-CD5 L17F12/Leu1 monoclonal antibody. Preliminary nuclear magnetic resonance (NMR) spectra obtained for CD5d1 (residues 1-118) had spectral dispersion typical of a folded protein, but otherwise of such poor quality that NMR structural studies were not feasible. The analysis of glycoproteins by NMR is frustrated by sample heterogeneity and poor spectral quality associated with glycan resonance overlap and the potential for increased line-widths due to the large hydrodynamic volume. In order to pursue NMR structural studies of CD5d1 it was necessary to optimize the quality of NMR spectra of CD5d1. A range of constructs of varying length and carbohydrate content were expressed in CHO cells and in P. pastoris. In addition the P. pastoris CD5d1 proved susceptible to N-glycan cleavage with endoglycosidase H. The protein products were characterised using size exclusion chromatography, NMR measurement of translational self-diffusion coefficients and two-dimensional 1H nuclear Overhauser effect spectroscopy experiments. Removal of an eight residue O-glycosylated C-terminal peptide, in particular, resulted in significant improvements in the quality of the CD5d1 NMR data, while retaining native protein structure. Two-dimensional heteronuclear NMR spectroscopy of nitrogen-15 isotope labelled deglycosylated CD5d1 (residues 1-110) prepared from P. pastoris suggests that this protein product is now amenable to solution structure determination.

Correlation between conformational and binding properties of nebulin repeats.

Nebulin, a large protein (600 to 800 kDa) located in the thin filament of striated vertebrate muscle, is assumed to bind and stabilise F-actin. Complete sequence determination of human nebulin has only recently been accomplished showing a uniform modular structure along the whole length of the molecule. Up to 97% of the sequence is assembled from repeats of a sequence motif 35 amino acid residues long. This architecture suggests that a structural and functional understanding of such a large molecule may be possible by characterising single repeats and reconstructing from them the behaviour of the whole molecule. In the present study, we extend and generalise to the whole molecule previous work carried out on single repeats from a limited region of nebulin. Knowledge of the complete sequence allowed extensive analysis of the single repeats revealing a progressive N to C-terminal divergence that is mirrored by an increase of the alpha-helix propensity. A number of synthetic peptides spanning the sequences of selected repeats were obtained and their conformational and binding properties studied in detail. All the peptides showed a tendency to fold as transient helices in aqueous solution with helix content as observed by CD and NMR studies in excellent agreement with predictions. A higher helical tendency of repeats near the C terminus was observed. Analysis of the influence of charged media as well as trifluoroethanol on the folding of single repeats strongly suggested that the mechanism by which the nebulin alpha-helix is stabilised is mostly electrostatic. Peptides with higher helical content also showed a higher binding affinity to F-actin. Considerably varying effects were observed for the peptides on F-actin viscosity and polymerisation. We discuss the divergence in sequence and helical tendency and its correlation to the functional data with regard to their significance for the assembly of the thin filament during myogenesis.

Secondary structure determination by NMR spectroscopy of an immunoglobulin-like domain from the giant muscle protein titin.

We present the complete 15N and 1H NMR assignment and the secondary structure of an immunoglobulin-like domain from the giant muscle protein titin. The assignment was obtained using homonuclear and 15N heteronuclear 2D and 3D experiments. The complementarity of 3D TOCSY-NOESY and 3D 15N NOESY-HSQC experiments, using WATERGATE for water suppression, allowed an efficient assignment of otherwise ambiguous cross peaks and was helpful in overcoming poor TOCSY transfer for some amino acids. The secondary structure is derived from specific NOEs between backbone alpha- and amide protons, secondary chemical shifts of alpha-protons and chemical exchange for the backbone amide protons. It consists of eight beta-strands, forming two beta-sheets with four strands each, similar to the classical beta-sandwich of the immunoglobulin superfamily, as previously predicted by sequence analysis. Two of the beta-strands are connected by type II beta-turns; the first beta-strand forms a beta-bulge. The whole topology is very similar to the only intracellular immunoglobulin-like domain for which a structure has been determined so far, i.e., telokin.

Tertiary structure of an immunoglobulin-like domain from the giant muscle protein titin: a new member of the I set.

BACKGROUND: Titin is a gigantic protein located in the thick filament of vertebrate muscles. The putative functions of titin range from interactions with myosin and other muscle proteins to a role in muscle recoil. Analysis of its complete sequence has shown that titin is a multi-domain protein containing several copies of modules of 100 amino acids each. These are thought to belong to the fibronectin type-III and immunoglobulin superfamilies. So far, a complete structural determination has not been carried out on any of the titin modules. RESULTS: The three-dimensional structure of an immunoglobulin module, located in the M-line of the sarcomere close to the titin C terminus and called 'M5', was determined by multi-dimensional NMR spectroscopy. The structure has the predicted immunoglobulin fold with two beta-sheets packed against each other. Each sheet contains four strands. The structure of M5 belongs to the I (intermediate) set of the immunoglobulin superfamily and is very similar to telokin, which is also found in muscles. Although M5 and telokin have relatively little sequence similarity, the two proteins clearly share the same hydrophobic core. The major difference between telokin and the titin M5 module is the absence of the C' strand in the latter. CONCLUSIONS: The titin domains and several of the immunoglobulin-like domains from other modular muscle proteins are highly conserved at the positions corresponding to the hydrophobic core of M5. Our results indicate that it may be possible to use the structure of M5 as a molecular template to model most of the other immunoglobulin-like domains in muscle titin.