Subfamily-Specific Adaptations in the Structures of Two Penicillin-Binding Proteins from Mycobacterium tuberculosis

Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows that Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis

TASOR is a pseudo-PARP that directs HUSH complex assembly and epigenetic transposon control

Abstract: The HUSH complex represses retroviruses, transposons and genes to maintain the integrity of vertebrate genomes. HUSH regulates deposition of the epigenetic mark H3K9me3, but how its three core subunits — TASOR, MPP8 and Periphilin — contribute to assembly and targeting of the complex remains unknown. Here, we define the biochemical basis of HUSH assembly and find that its modular architecture resembles the yeast RNA-induced transcriptional silencing complex. TASOR, the central HUSH subunit, associates with RNA processing components. TASOR is required for H3K9me3 deposition over LINE-1 repeats and repetitive exons in transcribed genes. In the context of previous studies, this suggests that an RNA intermediate is important for HUSH activity. We dissect the TASOR and MPP8 domains necessary for transgene repression. Structure-function analyses reveal TASOR bears a catalytically-inactive PARP domain necessary for targeted H3K9me3 deposition. We conclude that TASOR is a multifunctional pseudo-PARP that directs HUSH assembly and epigenetic regulation of repetitive genomic targets

A type III complement factor D deficiency: Structural insights for inhibition of the alternative pathway.

Abstract Background: Complement factor D (FD) is the rate-limiting enzyme of the alternative complement pathway. Previous reports of FD deficiency featured absent plasma FD (type I deficiency) and susceptibility to meningococcal infection. A new FD mutant, which is non-functional but fully expressed, was identified in a patient with invasive meningococcal disease. Objectives: We sought to investigate the molecular features of this novel FD mutant. Methods: We performed complement haemolytic assays, western blot analysis of serum FD and Sanger sequencing of the CFD gene. Recombinant mutant FD was assessed by in vitro catalytic assays, circular dichroism, thermal shift assays, esterolytic assays and surface plasmon resonance. Molecular dynamics simulation was used to visualise the structural changes in mutant FD. Results: A homozygous single-nucleotide variation of the CFD gene in the patient and their sibling resulted in an arginine to proline (R176P) substitution in FD. While R176P FD was stable and fully expressed in blood, it had minimal catalytic activity. Mutation R176P caused key FD-C3bB binding exosite loop 156-162 to lose its binding-competent conformation and stabilised the inactive conformation of FD. Consequently, R176P FD was unable to bind its natural substrate, C3bB. Neither patient nor sibling demonstrated the glucose homeostasis impairment that occurs in FD-null mice. Conclusions: Here, we report the first genetically confirmed functional, or type III, deficiency of an activating complement serine protease. This novel mechanism of FD inhibition can inform further development of alternative pathway inhibitors to treat common inflammatory diseases such as age-related macular degeneration

Peptidoglycan Degrading and Sensing Systems of Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) cell wall, built on a cross-linked sugar-peptide polymer called peptidoglycan, protects the bacterial cell from adverse environments. Peptidoglycan homeostasis is maintained by extracellular peptidoglycan synthases and hydrolases. Intricate coordination of their activities is required to maintain structural integrity of the cell wall during growth, division, and response to stress. The sensor protein kinase B (PknB) is likely to play a critical role in monitoring the state of the peptidoglycan outside the cell and inducing subsequent metabolic changes inside. In this study, computational, biochemical, and structural approaches were used to characterize the peptidoglycan hydrolases of Mtb and to investigate the molecular mechanism of peptidoglycan signaling through PknB.Peptidoglycan hydrolases are critical players in bacterial growth, division, cell shape determination, and peptidoglycan fragment-mediated communication. Computational analysis identified 22 mycobacterial peptidoglycan hydrolases based on homology to known enzymes from model organisms. The peptidoglycan degradation machinery of Mtb includes 4 N-acetylmuramoyl-L-alanine amidases, 8 lytic transglycosidases, and 10 peptidases of various specificities. Ten of these enzymes form a core set of mycobacterial peptidoglycan hydrolases, while four of them are essential for growth in Mtb. Comprehensive biochemical and structural investigation of the Mtb peptidoglycan hydrolases was initiated by cloning and heterologously expressing constructs representing all 22 Mtb peptidoglycan hydrolases in Escherichia coli. Robust expression was observed for all but one target protein. Twelve were successfully purified on large scale.The peptidoglycan amidases Rv3717 and Rv3915 share similar catalytic cores yet have non-redundant functions in peptidoglycan turnover. Hydrolase activity assays using polymerized peptidoglycan sacculi and soluble peptidoglycan fragments elucidated contributions of individual amino acid residues, metal binding, and disulfide bond formation to catalysis. The structure of product-bound Rv3717 suggested a mechanism that limits this enzyme's activity on polymerized sacculi.Peptidoglycan D,D-peptidases Rv2911, Rv3330 and Rv3627 are low molecular weight penicillin-binding proteins that participate in peptidoglycan maturation and degradation. Surprisingly, these three enzymes were inactive on peptidoglycan sacculi or peptidoglycan fragments, yet were active on beta-lactams meropenem and Bocillin. The structure of Rv3330 solved in complex with meropenem revealed a potential peptide-binging groove distant from the active site. The observed lack of activity of low molecular weight penicillin-binding proteins suggests a requirement for an activator. Discovery of such factors will significantly advance our understanding of Mtb peptidoglycan homeostasis.PknB is an essential sensor kinase that controls cell wall biosynthesis. Its homologs in Gram-positive bacteria have been implicated in binding peptidoglycan fragments and in mediating bacterial responses to cell wall stress. To investigate the mechanism of peptidoglycan recognition by PknB, the structure of its extracellular sensor domain was solved. It consists of four 70-amino acid PASTA repeat domains that adopt an extended conformation. The last repeat domain contains a hydrophobic pocket with a conserved tryptophan, a signature of a ligand-binding site. The structure of PknB extracellular domain suggests that ligand-dependent localization and oligomerization control kinase activity

Intraspecies diversity reveals a subset of highly variable plant immune receptors and predicts their binding sites

Evolution of recognition specificities by the immune system depends on the generation of receptor diversity, and connecting binding of new antigens with initiation of downstream signalling. In plant immunity, these functions are enabled by the family of innate Nucleotide-Binding Leucine Rich Repeat (NLR) receptors. In this paper we surveyed the NLR complements of 62 ecotypes of Arabidopsis thaliana and 54 lines of Brachypodium distachyon and identified a limited number of NLR subfamilies responsible for generation of new receptor specificities. We show that the predicted specificity-determining residues cluster on the surfaces of Leucine Rich Repeat domains, but the location of the clusters varies between NLR subfamilies. By comparing NLR phylogeny, allelic diversity, and known functions of the Arabidopsis NLRs, we formulate a hypothesis for emergence of direct and indirect pathogen sensing receptors, and of the autoimmune NLRs. These findings reveal the recurring patterns of evolution of innate immunity and inform NLR engineering efforts

Analysis of intraspecies diversity reveals a subset of highly variable plant immune receptors and predicts their binding sites.

The evolution of recognition specificities by the immune system depends on the generation of receptor diversity and on connecting the binding of new antigens with the initiation of downstream signaling. In plant immunity, the innate Nucleotide-Binding Leucine-Rich Repeat (NLR) receptor family enables antigen binding and immune signaling. In this study, we surveyed the NLR complements of 62 ecotypes of Arabidopsis thaliana and 54 lines of Brachypodium distachyon and identified a limited number of NLR subfamilies that show high allelic diversity. We show that the predicted specificity-determining residues cluster on the surfaces of Leucine-Rich Repeat domains, but the locations of the clusters vary among NLR subfamilies. By comparing NLR phylogeny, allelic diversity, and known functions of the Arabidopsis NLRs, we formulate a hypothesis for the emergence of direct and indirect pathogen-sensing receptors and of the autoimmune NLRs. These findings reveal the recurring patterns of evolution of innate immunity and can inform NLR engineering efforts