The structure of the anti-c-myc antibody 9E10 Fab fragment/epitope peptide complex reveals a novel binding mode dominated by the heavy chain hypervariable loops.

The X-ray structure of the Fab fragment from the anti-c-myc antibody 9E10 was determined both as complex with its epitope peptide and for the free Fab. In the complex, two Fab molecules adopt an unusual head to head orientation with the epitope peptide arranged between them. In contrast, the free Fab forms a dimer with different orientation. In the Fab/peptide complex the peptide is bound to one of the two Fabs at the "back" of its extended CDR H3, in a cleft with CDR H1, thus forming a short, three-stranded antiparallel beta-sheet. The N- and C-terminal parts of the peptide are also in contact with the neighboring Fab fragment. Comparison between the CDR H3s of the two Fab molecules in complex with the peptide and those from the free Fab reveals high flexibility of this loop. This structural feature is in line with thermodynamic data from isothermic titration calorimetry.

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. Urea and guanidinium chloride denaturants unfold beta recombinase in a two-step process. The unfolding curves have bends at approximately 5 M and 2.2 M in urea and guanidinium chloride-containing buffers. Assuming a three-state unfolding model (N2-->2I-->2U), the total free energy change from 1 mol of native dimers to 2 mol of unfolded monomers amounts to deltaG(tot) = 17.9 kcal/mol, with deltaG(N2-->2I) = 4.2 kcal/mol for the first transition and deltaG(I-->U) = 6.9 kcal/mol for the second transition. Using sedimentation-equilibrium analytical ultracentrifugation, the presence of beta recombinase monomers was indicated at 5 M urea, and the urea dependence of the circular dichroism at 222 nm strongly suggests that folded monomers represent the unfolding intermediate.

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. Likewise, the binding affinity to DNA is reduced and the association of the two monomeric subunits of the omega2T29A dimer is weakened, as manifested by an increase in the dissociation constant from 3.2 microM for wt omega2 to 6.3 microM for omega2T29A. Denatured dimers are formed upon thermal unfolding of wt omega2 and omega2T29A at ca. 45 microM (D(n)<-->D(u)). Removal of 8 (omega2deltaN8), or even 18 (omega2deltaN18) N-terminal amino acids has no obvious effect either on the core structure or on the activity in comparison to wt omega2. The stability of variants omega2deltaN8 and omega2deltaN18 is similar to that of wt omega2, and their binding to operator DNA is not impaired.

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. This peptide is known to be presented in vivo by both subtypes, and as expected for a self-peptide, TIS-reactive cytotoxic T lymphocytes are absent in the respective individuals. The thermodynamic analyses reveal that both HLA-B27:TIS complexes exhibit comparable, relatively high thermostability (Tm approximately 60 degrees C) and undergo multi-step unfolding reactions, with dissociation of the peptide in the first step. As shown by X-ray crystallography, only subtle structural differences between the subtypes were observed regarding the architecture of their F pockets, including the presence of distinct networks of water molecules. However, no consistent structural differences were found between the peptide presentation modes. In contrast to other peptides displayed by the two HLA-subtypes which show either structural or dynamical differences in their peptide presentation modes, the TIS-complexed HLA-B*2705 and HLA-B*2709 subtypes are an example for thermodynamic and structural equivalence, in agreement with functional data.

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. DNA titration of increasing numbers of heptads with omega2, monitored by circular dichroism measurements, indicates the binding of one omega2 to one heptad (omega2:heptad stoichiometry of 1:1). Spacing of two directly or inversely oriented heptads by 1 to 7 bp reduces the affinity of the protein for its cognate target site. The binding affinity of omega2 for two directly repeated heptads was severely reduced if one of the base pairs of the core 5'-ATCAC-3' sequence of one of the heptads was individually substituted by any other base pair. Hydroxyl radical footprinting shows a protection pattern at the 5'-ATCAC-3' core. These data suggest that each heptad defines an operator half-site and that tight binding of the symmetric omega2 to the central 5'-TCA-3' core of symmetric or asymmetric targets (differently oriented heptads) is probably achieved by structural changes of DNA and/or protein or both.

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. The thermostabilities of a series of HLA-B27 molecules complexed with nonameric and decameric peptides were determined and revealed substantial differences depending on the subtype as well as the residues at the termini of the peptides. In addition we present the crystal structure of the B*2709 subtype complexed with a decameric peptide. This structure provides an explanation for the preference of HLA-B27 for a peptide with an N-terminal arginine as secondary anchor and the lack of preference for tyrosine as peptide C terminus in B*2709. The data show that differences in thermodynamic properties between peptide-complexed HLA-B27 subtypes are correlated with a variety of structural properties.

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. The resulting hybrid inhibitor HEI-TOE I (hybrid inhibitor from E. elaterium and the optimized binding loop of the third domain of turkey ovomucoid inhibitor) shows a specificity and affinity to porcine pancreatic elastase similar to the free inhibitory peptide but with significantly higher proteolytic stability. Isothermal titration calorimetry revealed that elastase binding of HEI-TOE I occurs with a small unfavorable positive enthalpy contribution, a large favorable positive entropy change, and a large negative heat capacity change. In addition, the inhibitory peptide and the hybrid inhibitor HEI-TOE I protected endothelial cells against degradation following treatment with porcine pancreatic elastase.

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 1.4 x 10(5) M (-1) for the weakest binder. For each of the peptides complex formation is enthalpically driven and connected with unfavorable entropic contributions; however, the ratio of enthalpy and entropy contributions to deltaG(0) differs markedly for the individual peptides. A plot of -deltaH(0) vs -TdeltaS(0) shows a linear correlation of the data for a wide variety of experimental conditions as expected for a process with deltaC(p) much larger than deltaS(0). The dissimilarity of deltaC(p) and deltaS(0) also explains why deltaH(0) and TdeltaS(0) show similar temperature dependences resulting in relatively small changes of deltaG(0) with temperature. The heat capacity changes deltaC(p) upon antibody-peptide complex formation determined for three selected peptides vary only in a small range, indicating basic thermodynamic similarity despite different key residues interacting in the complexes. Furthermore, the comparison of van't Hoff and calorimetric enthalpies point to a non-two-state binding mechanism. Protonation effects were excluded by measurements in buffers of different ionization enthalpies. Differences in the solution conformation of the peptides as demonstrated by circular dichroic measurements do not explain different binding affinities of the peptides; specifically a high helix content in solution is not essential for high binding affinity despite the helical epitope conformation in the crystal structure of p24.

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) = 5.1 kcal.mol(-1)), the knot is responsible for a large entropic stabilization of TdeltaS(u,25)(0) = -39.3 kcal mol(-1), which is compensated for by a deltaH(u,25)(0) of -34.2 kcal mol(-1). In the disulfide-deficient mutants, this entropic stabilization disappears, but instead of a decrease, we observe an increase in the thermodynamic stability by about 2 kcal.mol(-1). A detailed crystallographic analysis of the mutant structures suggests a role of the cystine knot motif in protein folding rather than in the stabilization of the folded state. When assuming that the sequential order of the disulfide bridge formation is conserved between VEGF and glycoprotein alpha-subunit, the crystal structure of the mutant C61A-C104A, which deviates by a root mean square deviation of more than 2.2 A from wild-type VEGF, identifies a true folding intermediate of VEGF.

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 epsilon and zeta proteins co-purify as a stable complex that, according to analytical ultracentrifugation and gel filtration, exists as epsilon2zeta2 heterotetramer in solution. Co-crystals of the epsilon2zeta2 complex contain epsilon and zeta in 1:1 molar ratio. Unfolding studies monitoring circular dichroic and fluorescence changes show that the zeta protein has a significantly lower thermodynamic stability than the epsilon protein both in free state and in the complex. Proteolytic studies indicate that zeta protein is more stable in the epsilon2zeta2 complex than in the free state. In vivo studies reveal a short half-life of the epsilon antitoxin (-18 min) and a long lifetime of the zeta toxin (>60 min). When transcription-translation of a plasmid containing the epsilon and zeta genes was inhibited, cell death was observed after a short lag phase that correlates with the disappearance of the epsilon protein from the background.