Implications of structural and thermodynamic studies of HLA-B27 subtypes exhibiting differential association with ankylosing spondylitis.

Structural and thermodynamic properties of HLA-B27 molecules provide the basis for their function within the immune system and are probably also central for the understanding of the pathology of HLA-B27-associated diseases such as ankolysing spondylitis (AS). Several HLA-B27 alleles are AS-associated, whereas some are not, although the protein encoded by the former may differ in only a single amino acid exchange from those specified by the latter. This indicates that subtype-specific polymorphic residues play a key role in determining whether an HLA-B27 subtype is AS-associated or not and open the possibility to correlate structural, thermodynamic and functional characteristics ofa given subtype with the disease association.

Early stages of misfolding and association of beta2-microglobulin: insights from infrared spectroscopy and dynamic light scattering.

Conformational changes associated with the assembly of recombinant beta 2-microglobulin in vitro under acidic conditions were investigated using infrared spectroscopy and static and dynamic light scattering. In parallel, the morphology of the different aggregated species obtained under defined conditions was characterized by electron microscopy. The initial salt-induced aggregate form of beta 2-microglobulin, composed of small oligomers (dimers to tetramers), revealed the presence of beta-strands organized in an intramolecular-like fashion.

Molecular determinants of major histocompatibility complex class I complex stability: shaping antigenic features through short and long range electrostatic interactions.

A single amino acid exchange between the major histocompatibility complex molecules HLA-B(*)2705 and HLA-B(*)2709 (Asp-116/His) is responsible for the emergence of distinct HLA-B27-restricted T cell repertoires in individuals harboring either of these two subtypes and could correlate with their differential association with the autoimmune disease ankylosing spondylitis. By using fluorescence depolarization and pK(a) calculations, we investigated to what extent electrostatic interactions contribute to shape antigenic differences between these HLA molecules complexed with viral, self, and non-natural peptide ligands. In addition to the established main anchor of peptides binding to HLA-B27, arginine at position 2 (pArg-2), and the secondary anchors at the peptide termini, at least two further determinants contribute to stable peptide accommodation.

HLA-B27 subtypes differentially associated with disease exhibit conformational differences in solution.

Human leukocyte antigen (HLA) class I molecules consist of a heavy chain, beta(2)-microglobulin, and a peptide that are noncovalently bound. Certain HLA-B27 subtypes are associated with ankylosing spondylitis (such as HLA-B*2705), whereas others (such as HLA-B*2709) are not. Both differ in only one residue (Asp116 and His116, respectively) in the F pocket that accommodates the peptide C-terminus.

Helicobacter exploits integrin for type IV secretion and kinase activation.

Integrins are important mammalian receptors involved in normal cellular functions as well as pathogenesis of chronic inflammation and cancer. We propose that integrins are exploited by the gastric pathogen and type-1 carcinogen Helicobacter pylori for injection of the bacterial oncoprotein cytotoxin-associated gene A (CagA) into gastric epithelial cells. Virulent H.

Conformation and stability of the Streptococcus pyogenes pSM19035-encoded site-specific beta recombinase, and identification of a folding intermediate.

Solution properties of beta recombinase were studied by circular dichroism and fluorescence spectroscopy, size exclusion chromatography, analytical ultracentrifugation, denaturant-induced unfolding and thermal unfolding experiments. In high ionic strength buffer (1 M NaCl) beta recombinase forms mainly dimers, and strongly tends to aggregate at ionic strength lower than 0.3 M NaCl.

Role of the N-terminal region and of beta-sheet residue Thr29 on the activity of the omega2 global regulator from the broad-host range Streptococcus pyogenes plasmid pSM19035.

The dimeric regulatory protein wild-type omega (wt omega2) binds to arrays of 7-bp sequences (heptads) present in the operator DNA region of copy control and partition functions of plasmid pSM19035. Each omega2 protein probably binds with an antiparallel beta-sheet structure in the major groove of the 7-bp subsite of the operator DNA. Exchange of threonine at position 29 to alanine (T29A) drastically affects the activity of variant protein omega2T29A both in vivo and in vitro, and reduces the thermodynamic stability deltaG(o)u, but does not change the conformation.

The structure of the TRAPP subunit TPC6 suggests a model for a TRAPP subcomplex.

The TRAPP (transport protein particle) complexes are tethering complexes that have an important role at the different steps of vesicle transport. Recently, the crystal structures of the TRAPP subunits SEDL and BET3 have been determined, and we present here the 1.7 Angstroms crystal structure of human TPC6, a third TRAPP subunit.

Arc repressor-operator DNA interactions and contribution of Phe10 to binding specificity.

Solution properties of Arc repressors (wild-type and F10H variant) from Salmonella bacteriophage P22 and their complexes with operator DNA (Arc-wt-DNA and Arc-F10H-DNA) were characterized by circular dichroism, fluorescence, and Raman difference spectroscopy and compared with the crystal structures of free and DNA-bound Arc repressors (wild-type and F10V variant). From the crystal structure of Arc-wt-operator DNA complex, it is known that amino acids Phe10/10' flip out of the hydrophobic protein core, and in the Arc-F10V-DNA complex, the methyl groups of Val10/10' rotate toward the DNA. Arc-wt and Arc-F10H significantly perturb the Raman signatures of the operator DNA upon complex formation.

Thermodynamic and structural equivalence of two HLA-B27 subtypes complexed with a self-peptide.

The F pocket of major histocompatibility complex (in humans HLA) class I molecules accommodates the C terminus of the bound peptide. Residues forming this pocket exhibit considerable polymorphism, and a single difference (Asp116 in HLA-B*2705 and His116 in HLA-B*2709 heavy chains) confers differential association of these two HLA-B27 subtypes to the autoimmune disease ankylosing spondylitis. As peptide presentation by HLA molecules is of central importance for immune responses, we performed thermodynamic (circular dichroism, differential scanning calorimetry, fluorescence polarization) and X-ray crystallographic analyses of both HLA-B27 subtypes complexed with the epidermal growth factor response factor 1-derived self-peptide TIS (RRLPIFSRL) to understand the impact of the Asp116His exchange on peptide display.

The microglia-activating potential of thrombin: the protease is not involved in the induction of proinflammatory cytokines and chemokines.

The serine protease thrombin is known as a blood coagulation factor. Through limited cleavage of proteinase-activated receptors it can also control growth and functions in various cell types, including neurons, astrocytes, and microglia (brain macrophages). A number of previous studies indicated that thrombin induces the release of proinflammatory cytokines and chemokines from microglial cells, suggesting another important role for the protease beyond hemostasis.

Recognition of DNA by omega protein from the broad-host range Streptococcus pyogenes plasmid pSM19035: analysis of binding to operator DNA with one to four heptad repeats.

pSM19035-encoded omega protein forms a dimer (omega2) that binds to a set of 7-bp repeats with sequence 5'-NATCACN-3'. Upon binding to its cognate sites, omega2 regulates transcription of genes required for copy number control and stable inheritance of plasmids, and promotes accurate plasmid segregation. Protein omega2 binds poorly to one heptad but the affinity to DNA increases with two and more unspaced heptads in direct or inverted orientation.

Thermodynamic and structural analysis of peptide- and allele-dependent properties of two HLA-B27 subtypes exhibiting differential disease association.

Selected HLA-B27 subtypes are associated with spondyloarthropathies, but the underlying mechanism is not understood. To explain this association in molecular terms, a comparison of peptide-dependent dynamic and structural properties of the differentially disease-associated subtypes HLA-B*2705 and HLA-B*2709 was carried out. These molecules differ only by a single amino acid at the floor of the peptide binding groove.

Design and characterization of a hybrid miniprotein that specifically inhibits porcine pancreatic elastase.

Studying protease/peptide inhibitor interactions is a useful tool for understanding molecular recognition in general and is particularly relevant for the rational design of inhibitors with therapeutic potential. An inhibitory peptide (PMTLEYR) derived from the third domain of turkey ovomucoid inhibitor and optimized for specific porcine pancreatic elastase inhibition was introduced into an inhibitor scaffold to increase the proteolytic stability of the peptide. The trypsin-specific squash inhibitor EETI II from Ecballium elaterium was chosen as the scaffold.

Interaction of epitope-related and -unrelated peptides with anti-p24 (HIV-1) monoclonal antibody CB4-1 and its Fab fragment.

The binding of four epitope-related peptides and three library-derived, epitope-unrelated peptides of different lengths (10-14 amino acids) and sequence by anti-p24 (HIV-1) monoclonal antibody CB4-1 and its Fab fragment was studied by isothermal titration calorimetry. The binding constants K(A) at 25 degrees C vary between 5.1 x 10(7) M (-1) for the strongest and 1.

In vitro and in vivo stability of the epsilon2zeta2 protein complex of the broad host-range Streptococcus pyogenes pSM19035 addiction system.

Streptococcus pyogenes pSM19035-encoded epsilon (10.7 kDa) and zeta (32.4 kDa) proteins are necessary to secure stable plasmid inheritance in bacteria, with zeta acting as toxin that kills plasmid-deprived cells and epsilon as an antitoxin that neutralises the activity of zeta.

The cystine knot promotes folding and not thermodynamic stability in vascular endothelial growth factor.

Cystine knots consist of three intertwined disulfide bridges and are considered major determinants of protein stability in proteins in which they occur. We questioned this function and observed that removal of individual disulfide bridges in human vascular endothelial growth factor (VEGF) does not reduce its thermodynamic stability but reduces its unexpected high thermal stability of 108 degrees C by up to 40 degrees C. In wild-type VEGF (deltaG(u,25)(0) = 5.

NblA from Anabaena sp. PCC 7120 is a mostly alpha-helical protein undergoing reversible trimerization in solution.

The nblA family of genes encodes for small proteins necessary for the ordered degradation of phycobilisomes under certain stress conditions, a process known as chlorosis. Genes homologous to nblA seem to occur in all phycobilisome-containing organisms. However, to date, no molecular mechanism is known for the action of NblA, nor have the gene products been characterized to understand the physical properties of the molecule and thus help elucidate the mechanism on a structural basis.

Efficient eukaryotic expression system for authentic human sex hormone-binding globulin.

Sex hormone-binding globulin (SHBG) is the main carrier for androgens and oestrogens in humans. It mediates the transport of steroid hormones in the circulation and testicular fluid, and regulates their bioavailability to steroid-responsive tissues. In addition, the protein interacts with membrane receptors expressed in target tissues.

Cross-reactive binding of cyclic peptides to an anti-TGFalpha antibody Fab fragment: an X-ray structural and thermodynamic analysis.

The monoclonal antibody tAb2 binds the N-terminal sequence of transforming growth factor alpha, VVSHFND. With the help of combinatorial peptide libraries it is possible to find homologous peptides that bind tAb2 with an affinity similar to that of the epitope. The conformational flexibility of short peptides can be constrained by cyclization in order to improve their affinity to the antibody and their stability towards proteolysis.

Stability and DNA-binding properties of the omega regulator protein from the broad-host range Streptococcus pyogenes plasmid pSM19035.

At the transcriptional level, the pSM19035-encoded omega protein coordinates the expression of proteins required for control of copy number and maintenance of plasmids. Using circular dichroism, fluorescence spectroscopy, ultracentrifugation and an electrophoretic mobility shift assay, the wild-type omega protein and a variant with a C-terminal hexa-histidine tag (omega-H(6)) were characterized. The omega protein is mainly alpha-helical (42%), occurs as homodimer in solution, unfolds thermally with half transition temperatures, T(m), between approximately 43 and approximately 78 degrees C depending on the ionic strength of the buffer, and binds PcopS-DNA with high affinity.

Interaction of a designed interleukin-10 epitope mimic with an antibody studied by isothermal titration microcalorimetry.

The mechanism of recognition of proteins and peptides by antibodies and the factors determining binding affinity and specificity are mediated by essentially the same features. However, additional effects of the usually unfolded and flexible solution structure of peptide ligands have to be considered. In an earlier study we designed and optimized six peptides (pepI to pepVI) mimicking the discontinuous binding site of interleukin-10 for the anti-interleukin-10 monoclonal antibody (mab) CB/RS/1.

Expression and purification of dynamin II domains and initial studies on structure and function.

Dynamin II, a large GTP-binding protein, is involved in endocytosis and in vesicle formation at the trans-Golgi network. To further elucidate functions of dynamin II, the pleckstrin homology domain (PHD), the proline-rich domain (PRD), and the C-terminal part of dynamin II (dynamin(500-870)) were expressed in Escherichia coli. The PHD, tagged C-terminally by a (His)(6) peptide, was expressed to 15% of cellular proteins and could be purified on nickel-chelating agarose.

The C-terminal subdomain (IF2 C-2) contains the entire fMet-tRNA binding site of initiation factor IF2.

Previous protein unfolding studies had suggested that IF2 C, the 24. 5-kDa fMet-tRNA binding domain of Bacillus stearothermophilus translation initiation factor IF2, may consist of two subdomains. In the present work, the four Phe residues of IF2 C (positions 531, 599, 657, and 721) were replaced with Trp, yielding four variant proteins having intrinsic fluorescence markers in different positions of the molecule.