NMR study of the differential contributions of residues of transforming growth factor alpha to association with its receptor

A heteronuclear NMR study of human transforming growth factor alpha (TGFalpha) in complex with the epidermal growth factor receptor extracellular domain (EGFR-ED) provided an effective method for delineating the relative contributions of the residues of the ligand to its affinity for the receptor. In conjunction with previously obtained mutagenesis data, these results indicate that while a large number of residues are involved in complex formation and make up the binding interface, a small subset contribute most of the binding energy. They also show that while the residues which contribute to receptor binding are localized on one face of the molecule, the specific residues that play the major role in the affinity of TGFalpha in the complex are in two distinct regions of TGFalpha. This suggests that two small functional epitopes each composed of two residues exist within a larger structural epitope presented on the binding face. These results give the most detailed picture to date of the receptor binding determinants and yield further insight into the formation of the ligand-receptor complex.     

Mac-1-negative B-1b phenotype of natural antibody-producing cells, including those responding to Gal alpha 1,3Gal epitopes in alpha 1,3-galactosyltransferase-deficient mice

Human natural Abs against Galalpha1-3Galbeta1-4GlcNAc (Gal) epitopes are a major barrier to xenotransplantation. Studies in this report, which use combined multiparameter flow cytometric sorting and enzyme-linked immunospot assay, demonstrate that anti-Gal IgM-producing cells are found exclusively in a small B cell subpopulation (i.e., CD21(-/low) IgM(high) B220(low) CD5(-) Mac-1(-) 493(-) cells) in the spleens of alpha1, 3-galactosyltransferase-deficient mice. All IgM-producing cells were detected in a similar splenic subpopulation of alpha1, 3-galactosyltransferase-deficient and wild-type mice. A higher frequency of B cells with anti-Gal surface IgM receptors was observed in the peritoneal cavity than in the spleen, but these did not actively secrete Abs, and showed phenotypic properties of B-1b cells (CD21(-/low) IgM(high) CD5(-) CD43(+) Mac-1(+)). However, these became Mac-1(-) and developed anti-Gal Ab-producing activity after in vitro culture with LPS. The splenic B cells with anti-Gal receptors consisted of both Mac-1(+) B-1b cells and Mac-1(-) B-1b-like cells. The latter comprised most anti-Gal IgM-producing cells. Our studies indicate that anti-Gal natural IgM Abs are produced by a B1b-like, Mac-1(-) splenic B cell population and not by plasma cells or B-1a cells. They are consistent with a model whereby B-1b cells lose Mac-1 expression upon Ag exposure and that these, rather than plasma cells, become the major IgM Ab-producing cell population.     

Human CC chemokine I-309, structural consequences of the additional disulfide bond

I-309 is a member of the CC subclass of chemokines and is one of only three human chemokines known to contain an additional, third disulfide bond. The three-dimensional solution structure of I-309 was determined by (1)H nuclear magnetic resonance spectroscopy and dynamic simulated annealing. The structure of I-309, which remains monomeric at high concentrations, was determined on the basis of 978 experimental restraints. The N-terminal region of I-309 was disordered, as has been previously observed for the CC chemokine eotaxin but not others such as MCP-1 and RANTES. This was followed in I-309 by a well-ordered region between residues 13 and 69 that consisted of a 3(10)-helix, a triple-stranded antiparallel beta-sheet, and finally a C-terminal alpha-helix. Root-mean-square deviations of 0.61 and 1.16 were observed for the backbone and heavy atoms, respectively. A comparison of I-309 to eotaxin and HCC-2 revealed a significant structural change in the C-terminal region of the protein. The alpha-helix normally present in chemokines was terminated early and was followed by a short section of extended strand. These changes were a direct result of the additional disulfide bond present in this protein. An examination of the I-309 structure will aid in an understanding of the specificity of this protein with its receptor, CCR8.     

Structure-based thermodynamic analysis of the dissociation of protein phosphatase-1 catalytic subunit and microcystin-LR docked complexes

The relationship between the structure of a free ligand in solution and the structure of its bound form in a complex is of great importance to the understanding of the energetics and mechanism of molecular recognition and complex formation. In this study, we use a structure-based thermodynamic approach to study the dissociation of the complex between the toxin microcystin-LR (MLR) and the catalytic domain of protein phosphatase-1 (PP-1c) for which the crystal structure of the complex is known. We have calculated the thermodynamic parameters (enthalpy, entropy, heat capacity, and free energy) for the dissociation of the complex from its X-ray structure and found the calculated dissociation constant (4.0 x 10(-11)) to be in excellent agreement with the reported inhibitory constant (3.9 x 10(-11)). We have also calculated the thermodynamic parameters for the dissociation of 47 PP-1c:MLR complexes generated by docking an ensemble of NMR solution structures of MLR onto the crystal structure of PP-1c. In general, we observe that the lower the root-mean-square deviation (RMSD) of the docked complex (compared to the X-ray complex) the closer its free energy of dissociation (deltaGd(o)) is to that calculated from the X-ray complex. On the other hand, we note a significant scatter between the deltaGd(o) and the RMSD of the docked complexes. We have identified a group of seven docked complexes with deltaGd(o) values very close to the one calculated from the X-ray complex but with significantly dissimilar structures. The analysis of the corresponding enthalpy and entropy of dissociation shows a compensation effect suggesting that MLR molecules with significant structural variability can bind PP-1c and that substantial conformational flexibility in the PP-1c:MLR complex may exist in solution.     

Allogeneic bone marrow transplantation with co-stimulatory blockade induces macrochimerism and tolerance without cytoreductive host treatment

Allogeneic bone marrow transplantation (in immunocompetent adults) has always required cytoreductive treatment of recipients with irradiation or cytotoxic drugs to achieve lasting engraftment at levels detectable by non-PCR-based techniques ('macrochimerism' or 'mixed chimerism'). Only syngeneic marrow engraftment at such levels has been achieved in unconditioned hosts. This requirement for potentially toxic myelosuppressive host pre-conditioning has precluded the clinical use of allogeneic bone marrow transplantation for many indications other than malignancies, including tolerance induction. We demonstrate here that treatment of naive mice with a high dose of fully major histocompatibility complex-mismatched allogeneic bone marrow, followed by one injection each of monoclonal antibody against CD154 and cytotoxic T-lymphocyte antigen 4 immunoglobulin, resulted in multi-lineage hematopoietic macrochimerism (of about 15%) that persisted for up to 34 weeks. Long-term chimeras developed donor-specific tolerance (donor skin graft survival of more than 145 days) and demonstrated ongoing intrathymic deletion of donor-reactive T cells. A protocol of high-dose bone marrow transplantation and co-stimulatory blockade can thus achieve allogeneic bone marrow engraftment without cytoreduction or T-cell depletion of the host, and eliminates a principal barrier to the more widespread use of allogeneic bone marrow transplantation. Although efforts have been made to minimize host pre-treatment for allogeneic bone marrow transplantation for tolerance induction, so far none have succeeded in eliminating pre-treatment completely. Our demonstration that this can be achieved provides the rationale for a safe approach for inducing robust transplantation tolerance in large animals and humans.     

Growing an actin gel on spherical surfaces

Inspired by the motility of the bacteria Listeria monocytogenes, we have experimentally studied the growth of an actin gel around spherical beads grafted with ActA, a protein known to be the promoter of bacteria movement. On ActA-grafted beads F-actin is formed in a spherical manner, whereas on the bacteria a "comet-like" tail of F-actin is produced. We show experimentally that the stationary thickness of the gel depends on the radius of the beads. Moreover, the actin gel is not formed if the ActA surface density is too low. To interpret our results, we propose a theoretical model to explain how the mechanical stress (due to spherical geometry) limits the growth of the actin gel. Our model also takes into account treadmilling of actin. We deduce from our work that the force exerted by the actin gel on the bacteria is of the order of 10 pN. Finally, we estimate from our theoretical model possible conditions for developing actin comet tails.     

Interaction of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pili strain PAK with a cross-reactive antibody: conformation of the bound peptide

The C-terminal receptor binding region of Pseudomonas aeruginosa pilin protein strain PAK (residues 128-144) has been the target for the design of a vaccine effective against P. aeruginosa infections. We have recently cloned and expressed a (15)N-labeled PAK pilin peptide spanning residues 128-144 of the PAK pilin protein. The peptide exists as a major (trans) and minor (cis) species in solution, arising from isomerization around a central Ile(138)-Pro(139) peptide bond. The trans isomer adopts two well-defined turns in solution, a type I beta-turn spanning Asp(134)-Glu-Gln-Phe(137) and a type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142). The cis isomer adopts only one well-defined type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142) but displays evidence of a less ordered turn spanning Asp(132)-Gln-Asp-Glu(135). These turns have been implicated in cross-reactive antibody recognition. (15)N-edited NMR spectroscopy was used to study the binding of the (15)N-labeled PAK pilin peptide to an Fab fragment of a cross-reactive monoclonal antibody, PAK-13, raised against the intact PAK pilus. The results of these studies are as follows: the trans and cis isomers bind with similar affinity to the Fab, despite their different topologies; both isomers maintain the conformational integrity of their beta-turns when bound; binding leads to the preferential stabilization of the first turn over the second turn in each isomer; and binding leads to the perturbation of resonances within regions of the trans and cis backbone that undergo microsecond to millisecond motions. These slow motions may play a role in induced fit binding of the first turn to Fab PAK-13, which would allow the same antibody combining site to accommodate either trans or cis topology. More importantly for vaccine design, these motions may also play a role in the development of a broad-spectrum vaccine capable of generating an antibody therapeutic effective against the multiple strains of P. aeruginosa.     

New mechanistic insights into the reactivity of the R2 protein of E. coli ribonucleotide reductase (RNR)

Further to a linear free-energy correlation of cross-reaction rate constants k12 for the reaction of eight organic radicals (OR), e.g. MV*+, from methyl viologen, with cytochrome c(III), we consider here similar studies for the reduction of the R2 protein of Escherichia coli ribonucleotide reductase, which has FeIII2 and Tyr* redox components. The same two techniques of pulse radiolysis and stopped-flow were used. Cross-reaction rate constants (22 degrees C) at pH 7.0, I=0.100 M (NaCl), were determined for the reduction of active-R2 with the eight ORs, reduction potentials E0(1) from -0.446 to +0.194 V. Samples of active-R2 have an FeIII2 met-R2 component, which in the present studies was close to 40%. Concurrent reactions have to be taken into account for the five most reactive ORs, corresponding to reduction of the FeIII2 of met-R2 and then of active-R2. Separate experiments on met-R2 reproduced the first of these rate constants, which on average is approximately 66% larger than the second rate constant. A single Marcus free-energy plot of log k12-0.5 log10f versus -E0(1)/0.059 describes all the data and the slope of 0.54 is in satisfactory agreement with the theoretical value of 0.50. Such behaviour is unexpected since the Tyr* is a much stronger oxidant (E0 approximately 1.0 V versus NHE) as compared to FeIII2 (E0 close to zero). X-ray structures of the met- and red-R2 states have indicated that electroneutrality of the approximately 10 A buried active site is maintained. Proton transfer is therefore proposed as a rapid sequel to electron transfer. Other reactions considered are the much slower conventional time-range reductions of active-R2 with hydrazine and dithionite. For these reactions one and/or two-equivalent changes are possible. With both reductants, met-R2 reacts about four-fold faster than active-R2, and as with the ORs the less strongly oxidising FeIII2 component is reduced before the Tyr*.