Conserved mycobacterial lipoglycoproteins activate TLR2 but also require glycosylation for MHC class II-restricted T cell activation

CD4(+) T cell clones derived from a leprosy lesion and patient blood were used to monitor the isolation and identification of an Ag associated with the self-limited form of the disease. Biochemical purification and genetic analysis identified the T cell Ag as a conserved mycobacterial lipoglycoprotein LprG. LprG-mediated activation of CD4(+) T cells required specific MHC class II restriction molecules and intracellular processing. Although LprG activated TLR2, this alone was not sufficient to stimulate or inhibit T cell activation. A striking finding was that the carbohydrate moieties of LprG were required for optimal T cell activation, because recombinant LprG produced in Escherichia coli, or recombinant LprG produced in Mycobacterium smegmatis and digested by alpha-mannosidase, did not activate T cells. This study demonstrates that the universe of bacterial T cell Ags includes lipoglycoproteins, which act as TLR2 ligands but also require glycosylation for MHC class II-restricted T cell activation in vivo.     

Protein-protein docking with simultaneous optimization of rigid-body displacement and side-chain conformations

Protein-protein docking algorithms provide a means to elucidate structural details for presently unknown complexes. Here, we present and evaluate a new method to predict protein-protein complexes from the coordinates of the unbound monomer components. The method employs a low-resolution, rigid-body, Monte Carlo search followed by simultaneous optimization of backbone displacement and side-chain conformations using Monte Carlo minimization. Up to 10(5) independent simulations are carried out, and the resulting "decoys" are ranked using an energy function dominated by van der Waals interactions, an implicit solvation model, and an orientation-dependent hydrogen bonding potential. Top-ranking decoys are clustered to select the final predictions. Small-perturbation studies reveal the formation of binding funnels in 42 of 54 cases using coordinates derived from the bound complexes and in 32 of 54 cases using independently determined coordinates of one or both monomers. Experimental binding affinities correlate with the calculated score function and explain the predictive success or failure of many targets. Global searches using one or both unbound components predict at least 25% of the native residue-residue contacts in 28 of the 32 cases where binding funnels exist. The results suggest that the method may soon be useful for generating models of biologically important complexes from the structures of the isolated components, but they also highlight the challenges that must be met to achieve consistent and accurate prediction of protein-protein interactions.     

An improved protein decoy set for testing energy functions for protein structure prediction

We have improved the original Rosetta centroid/backbone decoy set by increasing the number of proteins and frequency of near native models and by building on sidechains and minimizing clashes. The new set consists of 1,400 model structures for 78 different and diverse protein targets and provides a challenging set for the testing and evaluation of scoring functions. We evaluated the extent to which a variety of all-atom energy functions could identify the native and close-to-native structures in the new decoy sets. Of various implicit solvent models, we found that a solvent-accessible surface area-based solvation provided the best enrichment and discrimination of close-to-native decoys. The combination of this solvation treatment with Lennard Jones terms and the original Rosetta energy provided better enrichment and discrimination than any of the individual terms. The results also highlight the differences in accuracy of NMR and X-ray crystal structures: a large energy gap was observed between native and non-native conformations for X-ray structures but not for NMR structures.     

Crystal structures and increased stabilization of the protein G variants with switched folding pathways NuG1 and NuG2

We recently described two protein G variants (NuG1 and NuG2) with redesigned first hairpins that were almost twice as stable, folded 100-fold faster, and had a switched folding mechanism relative to the wild-type protein. To test the structural accuracy of our design algorithm and to provide insights to the dramatic changes in the kinetics and thermodynamics of folding, we have now determined the crystal structures of NuG1 and NuG2 to 1.8 A and 1.85 A, respectively. We find that they adopt hairpin structures that are closer to the computational models than to wild-type protein G; the RMSD of the NuG1 hairpin to the design model and the wild-type structure are 1.7 A and 5.1 A, respectively. The crystallographic B factor in the redesigned first hairpin of NuG1 is systematically higher than the second hairpin, suggesting that the redesigned region is somewhat less rigid. A second round of structure-based design yielded new variants of NuG1 and NuG2, which are further stabilized by 0.5 kcal/mole and 0.9 kcal/mole.     

Rates of DNA sequence evolution are not sex-biased in Drosophila melanogaster and D. simulans

To determine whether male- or female-biased mutation rates have affected the molecular evolution of Drosophila melanogaster and D. simulans, we calculated the male-to-female ratio of germline cell divisions ([symbol: see text]) from germline generation data and the male-to-female ratio of mutation rate ([symbol: see text]) by comparing chromosomal levels of nucleotide divergence. We found that the ratio of germline cell divisions changes from indicating a weak female bias to indicating a weak male bias as the age of reproduction increases. The range of [symbol: see text] values that we observed, however, does not lead us to expect much, if any, difference in mutation rate between the sexes. Silent and intron nucleotide divergence were compared between nine loci on the X chromosome and nine loci on the second and third chromosomes. The average levels of nucleotide divergence were not significantly different across the chromosomes, although both silent and intron sites show a trend toward slightly more divergence on the X. These results indicate a lack of sex- or chromosome-biased molecular evolution in D. melanogaster and D. simulans.      

Spermatogenic cells of the prepuberal mouse: isolation and morphological characterization

A procedure is described which permits the isolation from the prepuberal mouse testis of highly purified populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, leptotene and zygotene primary spermatocytes, pachytene primary spermatocytes and Sertoli cells. The successful isolation of these prepuberal cell types was accomplished by: (a) defining distinctive morphological characteristics of the cells, (b) determining the temporal appearance of spermatogenic cells during prepuberal development, (c) isolating purified seminiferous cords, after dissociation of the testis with collagenase, (d) separating the trypsin-dispersed seminiferous cells by sedimentation velocity at unit gravity, and (e) assessing the identity and purity of the isolated cell types by microscopy. The seminiferous epithelium from day 6 animals contains only primitive type A spermatogonia and Sertoli cells. Type A and type B spermatogonia are present by day 8. At day 10, meiotic prophase is initiated, with the germ cells reaching the early and late pachytene stages by 14 and 18, respectively. Secondary spermatocytes and haploid spermatids appear throughout this developmental period. The purity and optimum day for the recovery of specific cell types are as follows: day 6, Sertoli cells (purity>99 percent) and primitive type A spermatogonia (90 percent); day 8, type A spermatogonia (91 percent) and type B spermatogonia (76 percent); day 18, preleptotene spermatocytes (93 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent).