Structure of PBP-A from Thermosynechococcus elongatus, a Penicillin-Binding Protein Closely Related to Class A β-Lactamases

Molecular evolution has always been a subject of discussions, and researchers are interested in understanding how proteins with similar scaffolds can catalyze different reactions. In the superfamily of serine penicillin-recognizing enzymes, d-alanyl-d-alanine peptidases and β-lactamases are phylogenetically linked but feature large differences of reactivity towards their respective substrates. In particular, while β-lactamases hydrolyze penicillins very fast, leading to their inactivation, these molecules inhibit d-alanyl-d-alanine peptidases by forming stable covalent penicilloyl enzymes. In cyanobacteria, we have discovered a new family of penicillin-binding proteins (PBPs) presenting all the sequence features of class A β-lactamases but having a six-amino-acid deletion in the conserved Ω-loop and lacking the essential Glu166 known to be involved in the penicillin hydrolysis mechanism. With the aim of evolving a member of this family into a β-lactamase, PBP-A from Thermosynechococcus elongatus has been chosen because of its thermostability. Based on sequence alignments, introduction of a glutamate in position 158 of the shorter Ω-loop afforded an enzyme with a 50-fold increase in the rate of penicillin hydrolysis. The crystal structures of PBP-A in the free and penicilloylated forms at 1.9 Å resolution and of L158E mutant at 1.5 Å resolution were also solved, giving insights in the catalytic mechanism of the proteins. Since all the active-site elements of PBP-A-L158E, including an essential water molecule, are almost perfectly superimposed with those of a class A β-lactamase such as TEM-1, the question why our mutant is still 5 orders of magnitude less active as a penicillinase remains and our results emphasize how far we are from understanding the secrets of enzymes. Based on the few minor differences between the active sites of PBP-A and TEM-1, mutations were introduced in the L158E enzyme, but while activities on d-Ala-d-Ala mimicking substrates were severely impaired, further improvement in penicillinase activity was unsuccessful.     

Genome-wide association study identifies candidate genes for male fertility traits in humans

Despite the fact that hundreds of genes are known to affect fertility in animal models, relatively little is known about genes that influence natural fertility in humans. To broadly survey genes contributing to variation in male fertility, we conducted a genome-wide association study (GWAS) of two fertility traits (family size and birth rate) in 269 married men who are members of a founder population of European descent that proscribes contraception and has large family sizes. Associations between ～250,000 autosomal SNPs and the fertility traits were examined. A total of 41 SNPs with p ≤ 1 × 10(-4) for either trait were taken forward to a validation study of 123 ethnically diverse men from Chicago who had previously undergone semen analyses. Nine (22%) of the SNPs associated with reduced fertility in the GWAS were also associated with one or more of the ten measures of reduced sperm quantity and/or function, yielding 27 associations with p values < 0.05 and seven with p values < 0.01 in the validation study. On the basis of 5,000 permutations of our data, the probabilities of observing this many or more small p values were 0.0014 and 5.6 × 10(-4), respectively. Among the nine associated loci, outstanding candidates for male fertility genes include USP8, an essential deubiquitinating enzyme that has a role in acrosome assembly; UBD and EPSTI1, which have potential roles in innate immunity; and LRRC32, which encodes a latent transforming growth factor β (TGF-β) receptor on regulatory T cells. We suggest that mutations in these genes that are more severe may account for some of the unexplained infertility (or subfertility) in the general population.     

Resequencing candidate genes implicates rare variants in asthma susceptibility

Common variation in over 100 genes has been implicated in the risk of developing asthma, but the contribution of rare variants to asthma susceptibility remains largely unexplored. We selected nine genes that showed the strongest signatures of weak purifying selection from among 53 candidate asthma-associated genes, and we sequenced the coding exons and flanking noncoding regions in 450 asthmatic cases and 515 nonasthmatic controls. We observed an overall excess of p values <0.05 (p = 0.02), and rare variants in four genes (AGT, DPP10, IKBKAP, and IL12RB1) contributed to asthma susceptibility among African Americans. Rare variants in IL12RB1 were also associated with asthma susceptibility among European Americans, despite the fact that the majority of rare variants in IL12RB1 were specific to either one of the populations. The combined evidence of association with rare noncoding variants in IL12RB1 remained significant (p = 3.7 × 10(-4)) after correcting for multiple testing. Overall, the contribution of rare variants to asthma susceptibility was predominantly due to noncoding variants in sequences flanking the exons, although nonsynonymous rare variants in DPP10 and in IL12RB1 were associated with asthma in African Americans and European Americans, respectively. This study provides evidence that rare variants contribute to asthma susceptibility. Additional studies are required for testing whether prioritizing genes for resequencing on the basis of signatures of purifying selection is an efficient means of identifying novel rare variants that contribute to complex disease.     

Complete genome sequence of the highly hemolytic strain Bacillus cereus F837/76

Highly hemolytic strain Bacillus cereus F837/76 was isolated in 1976 from a contaminated prostate wound. The complete nucleotide sequence of this strain reported here counts nearly 36,500 single-nucleotide differences from the closest sequenced strain, Bacillus thuringiensis Al Hakam. F827/76 also contains a 10-kb plasmid that was not detected in the Al Hakam strain.     

Structural basis of HIV-1 neutralization by affinity matured Fabs directed against the internal trimeric coiled-coil of gp41

The conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 is transiently exposed during the fusion process by forming a pre-hairpin intermediate, thus representing an attractive target for the design of fusion inhibitors and neutralizing antibodies. In previous studies we reported a series of broadly neutralizing mini-antibodies derived from a synthetic naïve human combinatorial antibody library by panning against a mimetic of the trimeric N-HR coiled coil, followed by affinity maturation using targeted diversification of the CDR-H2 loop. Here we report crystal structures of the N-HR mimetic 5-Helix with two Fabs that represent the extremes of this series: Fab 8066 is broadly neutralizing across a wide panel of B and C type HIV-1 viruses, whereas Fab 8062 is non-neutralizing. The crystal structures reveal important differences in the conformations of the CDR-H2 loops in the complexes that propagate into other regions of the antigen-antibody interface, and suggest that both neutralization properties and affinity for the target can be attributed, at least in part, to the differences in the interactions of the CDR-H2 loops with the antigen. Furthermore, modeling of the complex of an N-HR trimer with three Fabs suggests that the CDR-H2 loop may be involved in close intermolecular contacts between neighboring antibody molecules, and that such contacts may hinder the formation of complexes between the N-HR trimer and more than one antibody molecule depending on the conformation of the bound CDR-H2 loop which is defined by its interactions with antigen. Comparison with the crystal structure of the complex of 5-Helix with another neutralizing monoclonal antibody known as D5, derived using an entirely different antibody library and panning procedure, reveals remarkable convergence in the optimal sequence and conformation of the CDR-H2 loop.     

The CFTR Met 470 Allele Is Associated with Lower Birth Rates in Fertile Men from a Population Isolate

Although little is known about the role of the cystic fibrosis transmembrane regulator (CFTR) gene in reproductive physiology, numerous variants in this gene have been implicated in etiology of male infertility due to congenital bilateral absence of the vas deferens (CBAVD). Here, we studied the fertility effects of three CBAVD–associated CFTR polymorphisms, the (TG)m and polyT repeat polymorphisms in intron 8 and Met470Val in exon 10, in healthy men of European descent. Homozygosity for the Met470 allele was associated with lower birth rates, defined as the number of births per year of marriage (P = 0.0029). The Met470Val locus explained 4.36% of the phenotypic variance in birth rate, and men homozygous for the Met470 allele had 0.56 fewer children on average compared to Val470 carrier men. The derived Val470 allele occurs at high frequencies in non-African populations (allele frequency  = 0.51 in HapMap CEU), whereas it is very rare in African population (Fst  = 0.43 between HapMap CEU and YRI). In addition, haplotypes bearing Val470 show a lack of genetic diversity and are thus longer than haplotypes bearing Met470 (measured by an integrated haplotype score [iHS] of −1.93 in HapMap CEU). The fraction of SNPs in the HapMap Phase2 data set with more extreme Fst and iHS measures is 0.003, consistent with a selective sweep outside of Africa. The fertility advantage conferred by Val470 relative to Met470 may provide a selective mechanism for these population genetic observations.     

Initiator elements function to determine the activity state of BX-C enhancers

A >300 kb cis-regulatory region is required for the proper expression of the three bithorax complex (BX-C) homeotic genes. Based on genetic and transgenic analysis, a model has been proposed in which the numerous BX-C cis-regulatory elements are spatially restricted through the activation or repression of parasegment-specific chromatin domains. Particular early embryonic enhancers, called initiators, have been proposed to control this complex process. Here, in order to better understand the process of domain activation, we have undertaken a systematic in situ dissection of the iab-6 cis-regulatory domain using a new method, called InSIRT. Using this method, we create and genetically characterize mutations affecting iab-6 function, including mutations specifically modifying the iab-6 initiator. Through our mutagenesis of the iab-6 initiator, we provide strong evidence that initiators function not to directly control homeotic gene expression but rather as domain control centers to determine the activity state of the enhancers and silencers within a cis-regulatory domain.