Nature-inspired engineering of an artificial ligase enzyme by domain fusion

The function of most proteins is accomplished through the interplay of two or more protein domains and fine-tuned by natural evolution. In contrast, artificial enzymes have often been engineered from a single domain scaffold and frequently have lower catalytic activity than natural enzymes. We previously generated an artificial enzyme that catalyzed an RNA ligation by >2 million-fold but was likely limited in its activity by low substrate affinity. Inspired by nature''s concept of domain fusion, we fused the artificial enzyme to a series of protein domains known to bind nucleic acids with the goal of improving its catalytic activity. The effect of the fused domains on catalytic activity varied greatly, yielding severalfold increases but also reductions caused by domains that previously enhanced nucleic acid binding in other protein engineering projects. The combination of the two better performing binding domains improved the activity of the parental ligase by more than an order of magnitude. These results demonstrate for the first time that nature''s successful evolutionary mechanism of domain fusion can also improve an unevolved primordial-like protein whose structure and function had just been created in the test tube. The generation of multi-domain proteins might therefore be an ancient evolutionary process.     

Warming and Elevated CO<Subscript>2</Subscript> Interact to Alter Seasonality and Reduce Variability of Soil Water in a Semiarid Grassland

Global changes that alter soil water availability may have profound effects on semiarid ecosystems. Although both elevated CO₂ (eCO₂) and warming can alter water availability, often in opposite ways, few studies have measured their combined influence on the amount, timing, and temporal variability of soil water. Here, we ask how free air CO₂ enrichment (to 600 ppmv) and infrared warming (+?1.5 °C day, +?3 °C night) effects on soil water vary within years and across wet-dry periods in North American mixed-grass prairie. We found that eCO₂ and warming interacted to influence soil water and that those interactions varied by season. In the spring, negative effects of warming on soil water largely offset positive effects of eCO₂. As the growing season progressed, however, warming reduced soil water primarily (summer) or only (autumn) in plots treated with eCO₂. These interactions constrained the combined effect of eCO₂ and warming on soil water, which ranged from neutral in spring to positive in autumn. Within seasons, eCO₂ increased soil water under drier conditions, and warming decreased soil water under wetter conditions. By increasing soil water under dry conditions, eCO₂ also reduced temporal variability in soil water. These temporal patterns explain previously observed plant responses, including reduced leaf area with warming in summer, and delayed senescence with eCO₂ plus warming in autumn. They also suggest that eCO₂ and warming may favor plant species that grow in autumn, including winter annuals and C₃ graminoids, and species able to remain active under the dry conditions moderated by eCO₂.     

The innate immune molecule,NOD1, regulates direct killing of Helicobacter pylori by antimicrobial peptides

The cytosolic innate immune molecule, NOD1, recognizes peptidoglycan (PG) delivered to epithelial cells via the Helicobacter pylori cag pathogenicity island (cagPAI), and has been implicated in host defence against cagPAI⁺H. pylori bacteria. To further clarify the role of NOD1 in host defence, we investigated NOD1‐dependent regulation of human β‐defensins (DEFBs) in two epithelial cell lines. Our findings identify that NOD1 activation, via either cagPAI⁺ bacteria or internalized PG, was required for DEFB4 and DEFB103 expression in HEK293 cells. To investigate cell type‐specific induction of DEFB4 and DEFB103, we generated stable NOD1‘knockdown’ (KD) and control AGS cells. Reporter gene assay and RT‐PCR analyses revealed that only DEFB4 was induced in an NOD1‐/cagPAI‐dependent fashion in AGS cells. Moreover, culture supernatants from AGS control, but not AGS NOD1 KD cells, stimulated with cagPAI⁺H. pylori, significantly reduced H. pylori bacterial numbers. siRNA studies confirmed that human β‐defensin 2 (hBD‐2), but not hBD‐3, contributes to the antimicrobial activity of AGS cell supernatants against H. pylori. This study demonstrates, for the first time, the involvement of NOD1 and hBD‐2 in direct killing of H. pylori bacteria by epithelial cells and confirms the importance of NOD1 in host defence mechanisms against cagPAI⁺H. pylori infection.     

Redefining the structure–activity relationships of 2,6-methano-3-benzazocines. Part 9: Synthesis,characterization and molecular modeling of pyridinyl isosteres of N-BPE-8-CAC (1), a high affinity ligand for opioid receptors

Derivatives of the lead compound N-BPE-8-CAC (1) where each CH of the biphenyl group was individually replaced by N were prepared in hopes of identifying high affinity ligands with improved aqueous solubility. Compared to 1, binding affinities of the five possible pyridinyl derivatives for the μ opioid receptor were between threefold lower to fivefold higher with the K_i of the most potent compound being 0.064 nM. Docking of 8-CAC (2) into the unliganded binding site of the mouse μ opioid receptor (pdb: 4DKL) revealed that 8-CAC and β-FNA (from 4DKL) make nearly identical interactions with the receptor. However, for 1 and the new pyridinyl derivatives 4–8, binding is not tolerated in the 8-CAC binding mode due to the steric constraints of the large N-substituents. Either an alternative binding mode or rearrangement of the protein to accommodate these modifications may account for their high binding affinity.     

Functionally Important Carboxyls in a Bacterial Homologue of the Vesicular Monoamine Transporter (VMAT)

Transporters essential for neurotransmission in mammalian organisms and bacterial multidrug transporters involved in antibiotic resistance are evolutionarily related. To understand in more detail the evolutionary aspects of the transformation of a bacterial multidrug transporter to a mammalian neurotransporter and to learn about mechanisms in a milieu amenable for structural and biochemical studies, we identified, cloned, and partially characterized bacterial homologues of the rat vesicular monoamine transporter (rVMAT2). We performed preliminary biochemical characterization of one of them, Brevibacillus brevis monoamine transporter (BbMAT), from the bacterium B. brevis. BbMAT shares substrates with rVMAT2 and transports them in exchange with >1H⁺, like the mammalian transporter. Here we present a homology model of BbMAT that has the standard major facilitator superfamily fold; that is, with two domains of six transmembrane helices each, related by 2-fold pseudosymmetry whose axis runs normal to the membrane and between the two halves. The model predicts that four carboxyl residues, a histidine, and an arginine are located in the transmembrane segments. We show here that two of the carboxyls are conserved, equivalent to the corresponding ones in rVMAT2, and are essential for H⁺-coupled transport. We conclude that BbMAT provides an excellent experimental paradigm for the study of its mammalian counterparts and bacterial multidrug transporters.     

Kinetic properties of bovine brain proteinl-isoaspartyl methyltransferase determined using a synthetic isoaspartyl peptide substrate

Proteinl-isoaspartyl methyltransferase, an enzyme enriched in brain, is implicated in the repair of age-damaged proteins containing atypical, isoaspartyl peptide bonds. We have investigated the kinetics of methylation using a synthetic peptide substrate having the structure Trp-Ala-Gly-Gly-isoAsp-Ala-Ser-Gly-Glu. Double-reciprocal plots of initial velocity versus concentration of S-adenosylmethionine (AdoMet) at different fixed concentrations of peptide gave straight lines converging at a positive 1/v value and a negative 1/AdoMet value. The product S-adenosylhomocysteine (AdoHcy) was a competitive inhibitor towards AdoMet and a linear mixed-type inhibitor towards peptide. These results are consistent with the rapid-equilibrium random sequential bi-bi mechanism previously proposed for the enzyme, but they also reveal the formation of the deadend, enzyme-peptide-AdoHcy, complex. The rate constants were:V _max=32–34 nmol/min/mg,K ^peptide=7.6–9.4 M,K ^AdoMet=1.9–2.2 M, =0.43–0.53,K ^AdoHcy=0.08 M, =2.9. The interaction factors and indicate that binding of enzyme to peptide increases its affinity for AdoMet and decreases its affinity for AdoHcy. Methylation was linear with time throughout the transfer of 2 mol of methyl groups/mol of enzyme. This absence of burst kinetics suggests that slow release of products cannot explain the low turnover number.Special issue dedicated to Dr. Paul Greengard     

Morphological and sensorimotor phenotypes in a zebrafish CHARGE syndrome model are domain-dependent

CHARGE syndrome is a heterogeneous disorder characterized by a spectrum of defects affecting multiple tissues and behavioral difficulties such as autism, attention-deficit/hyperactivity disorder, obsessive–compulsive disorder, anxiety, and sensory deficits. Most CHARGE cases arise from de novo, loss-of-function mutations in chromodomain-helicase-DNA-binding-protein-7 (CHD7). CHD7 is required for processes such as neuronal differentiation and neural crest cell migration, but how CHD7 affects neural circuit function to regulate behavior is unclear. To investigate the pathophysiology of behavioral symptoms in CHARGE, we established a mutant chd7 zebrafish line that recapitulates multiple CHARGE phenotypes including ear, cardiac, and craniofacial defects. Using a panel of behavioral assays, we found that chd7 mutants have specific auditory and visual behavior deficits that are independent of defects in sensory structures. Mauthner cell-dependent short-latency acoustic startle responses are normal in chd7 mutants, while Mauthner-independent long-latency responses are reduced. Responses to sudden decreases in light are also reduced in mutants, while responses to sudden increases in light are normal, suggesting that the retinal OFF pathway may be affected. Furthermore, by analyzing multiple chd7 alleles we observed that the penetrance of morphological and behavioral phenotypes is influenced by genetic background but that it also depends on the mutation location, with a chromodomain mutation causing the highest penetrance. This pattern is consistent with analysis of a CHARGE patient dataset in which symptom penetrance was highest in subjects with mutations in the CHD7 chromodomains. These results provide new insight into the heterogeneity of CHARGE and will inform future work to define CHD7-dependent neurobehavioral mechanisms.