Structural and functional insights into the Pseudomonas aeruginosa glycosyltransferase WaaG and the implications for lipopolysaccharide biosynthesis.

The glycosyltransferase WaaG in Pseudomonas aeruginosa (PaWaaG) is involved in the synthesis of the core region of lipopolysaccharides. It is a promising target for developing adjuvants that could help in the uptake of antibiotics. Herein, we have determined structures of PaWaaG in complex with the nucleotide-sugars UDP-glucose, UDP-galactose, and UDP-GalNAc. Structural comparison with the homolog from Escherichia coli (EcWaaG) revealed five key differences in the sugar-binding pocket. Solution-state NMR analysis showed that WT PaWaaG specifically hydrolyzes UDP-GalNAc and unlike EcWaaG, does not hydrolyze UDP-glucose. Furthermore, we found that a PaWaaG mutant (Y97F/T208R/N282A/T283A/T285I) designed to resemble the EcWaaG sugar binding site, only hydrolyzed UDP-glucose, underscoring the importance of the identified amino acids in substrate specificity. However, neither WT PaWaaG nor the PaWaaG mutant capable of hydrolyzing UDP-glucose was able to complement an E. coli ΔwaaG strain, indicating that more remains to be uncovered about the function of PaWaaG in vivo. This structural and biochemical information will guide future structure-based drug design efforts targeting PaWaaG.

Tips for efficiently maintaining pET expression plasmids.

pET expression plasmids are widely used for producing recombinant proteins in Escherichia coli. Selection and maintenance of cells harboring a pET plasmid are possible using either a Tn3.1-type genetic fragment (which encodes a ß-lactamase and confers resistance to ß-lactam antibiotics) or a Tn903.1-type genetic fragment (which encodes an aminoglycoside-3'-phosphotransferase and confers resistance aminoglycoside antibiotics). Herein we have investigated how efficiently pET plasmids are maintained using these two fragments. The study reveals that pET plasmids are efficiently maintained with both Tn3.1 and Tn903.1 genetic fragments prior to the induction of recombinant protein production, and over short induction times (i.e., 2 h). However, over longer induction times (i.e., 20 h), the efficiency of plasmid maintenance depends on the host strain used, and the type of antibiotic selection cassette used. Based on our collective observations, we have 2 general tips for efficiently maintaining pET plasmids during recombinant production experiments. Tip #1: Use a strain with lowered levels of the T7 RNA polymerase, such as C41(DE3). pET plasmids will be efficiently maintained over long induction times with both the Tn3.1 and Tn903.1 genetic fragments, regardless of whether antibiotics are present during cultivation. Tip #2: If a strain with higher levels of T7 RNA polymerase strain is necessary, such as BL21(DE3)), keep induction times short or use a plasmid containing a Tn903.1-type fragment and select with kanamycin.

Folding pathway of an Ig domain is conserved on and off the ribosome.

Proteins that fold cotranslationally may do so in a restricted configurational space, due to the volume occupied by the ribosome. How does this environment, coupled with the close proximity of the ribosome, affect the folding pathway of a protein? Previous studies have shown that the cotranslational folding process for many proteins, including small, single domains, is directly affected by the ribosome. Here, we investigate the cotranslational folding of an all-ß Ig domain, titin I27. Using an arrest peptide-based assay and structural studies by cryo-EM, we show that I27 folds in the mouth of the ribosome exit tunnel. Simulations that use a kinetic model for the force dependence of escape from arrest accurately predict the fraction of folded protein as a function of length. We used these simulations to probe the folding pathway on and off the ribosome. Our simulations-which also reproduce experiments on mutant forms of I27-show that I27 folds, while still sequestered in the mouth of the ribosome exit tunnel, by essentially the same pathway as free I27, with only subtle shifts of critical contacts from the C to the N terminus.

Increased production of periplasmic proteins in Escherichia coli by directed evolution of the translation initiation region.

BACKGROUND: Recombinant proteins are often engineered with an N-terminal signal peptide, which facilitates their secretion to the oxidising environment of the periplasm (gram-negative bacteria) or the culture supernatant (gram-positive bacteria). A commonly encountered problem is that the signal peptide influences the synthesis and secretion of the recombinant protein in an unpredictable manner. A molecular understanding of this phenomenon is highly sought after, as it could lead to improved methods for producing recombinant proteins in bacterial cell factories. RESULTS: Herein we demonstrate that signal peptides contribute to an unpredictable translation initiation region. A directed evolution approach that selects a new translation initiation region, whilst leaving the amino acid sequence of the signal peptide unchanged, can increase production levels of secreted recombinant proteins. The approach can increase production of single chain antibody fragments, hormones and other recombinant proteins in the periplasm of E. coli. CONCLUSIONS: The study demonstrates that signal peptide performance is coupled to the efficiency of the translation initiation region.

Spatial separation of FtsZ and FtsN during cell division.

The division of Escherichia coli is mediated by a collection of some 34 different proteins that are recruited to the division septum and are thought to assemble into a macromolecular complex known as 'the divisome'. Herein, we have endeavored to better understand the structure of the divisome by imaging two of its core components; FtsZ and FtsN. Super resolution microscopy (SIM and gSTED) indicated that both proteins are localized in large assemblies, which are distributed around the division septum (i.e., forming a discontinuous ring). Although the rings had similar radii prior to constriction, the individual densities were often spatially separated circumferentially. As the cell envelope constricted, the discontinuous ring formed by FtsZ moved inside the discontinuous ring formed by FtsN. The radial and circumferential separation observed in our images indicates that the majority of FtsZ and FtsN molecules are organized in different macromolecular assemblies, rather than in a large super-complex. This conclusion was supported by fluorescence recovery after photobleaching measurements, which indicated that the dynamic behavior of the two macromolecular assemblies was also fundamentally different. Taken together, the data indicates that constriction of the cell envelope is brought about by (at least) two spatially separated complexes.

Signal amplification of araC pBAD using a standardized translation initiation region.

araC pBAD is a genetic fragment that regulates the expression of the araBAD operon in bacteria, which is required for the metabolism of L-arabinose. It is widely used in bioengineering applications because it can drive regulatable and titratable expression of genes and genetic pathways in microbial cell factories. A notable limitation of araC pBAD is that it generates a low signal when induced with high concentrations of L-arabinose (the maximum ON state). Herein we have amplified the maximum ON state of araC pBAD by coupling it to a synthetically evolved translation initiation region (TIREVOL ). The coupling maintains regulatable and titratable expression from araC pBAD and yet increases the maximal ON state by >5-fold. The general principle demonstrated in the study can be applied to amplify the signal from similar genetic modules. Graphical Abstract.

Human cytomegalovirus hijacks host stress response fueling replication stress and genome instability.

Viral infections enhance cancer risk and threaten host genome integrity. Although human cytomegalovirus (HCMV) proteins have been detected in a wide spectrum of human malignancies and HCMV infections have been implicated in tumorigenesis, the underlying mechanisms remain poorly understood. Here, we employed a range of experimental approaches, including single-molecule DNA fiber analysis, and showed that infection by any of the four commonly used HCMV strains: AD169, Towne, TB40E or VR1814 induced replication stress (RS), as documented by host-cell replication fork asymmetry and formation of 53BP1 foci. The HCMV-evoked RS triggered an ensuing host DNA damage response (DDR) and chromosomal instability in both permissive and non-permissive human cells, the latter being particularly relevant in the context of tumorigenesis, as such cells can survive and proliferate after HCMV infection. The viral major immediate early enhancer and promoter (MIEP) that controls expression of the viral genes IE72 (IE-1) and IE86 (IE-2), contains transcription-factor binding sites shared by promoters of cellular stress-response genes. We found that DNA damaging insults, including those relevant for cancer therapy, enhanced IE72/86 expression. Thus, MIEP has been evolutionary shaped to exploit host DDR. Ectopically expressed IE72 and IE86 also induced RS and increased genomic instability. Of clinical relevance, we show that undergoing standard-of-care genotoxic radio-chemotherapy in patients with HCMV-positive glioblastomas correlated with elevated HCMV protein markers after tumor recurrence. Collectively, these results are consistent with our proposed concept of HCMV hijacking transcription-factor binding sites shared with host stress-response genes. We present a model to explain the potential oncomodulatory effects of HCMV infections through enhanced replication stress, subverted DNA damage response and induced genomic instability.

Implementing Novel Designs in pET Expression Plasmids that Increase Protein Production.

pET expression plasmids are widely used in the biotechnology, biopharmaceutical, and basic research sectors for the production of recombinant proteins. Typically, they are used off-the-shelf because they support high production titers; however, we have identified two design flaws in many pET plasmids that limit their production capacity. We used modern methods of DNA assembly and directed evolution to identify improved designs for these modules and demonstrated that these designs support higher protein production yields. Herein, we present two PCR protocols for implementing the designs and increasing protein production from existing pET expression plasmids. Graphic abstract: A simple workflow for implementing novel designs in pET expression plasmids.

Improved designs for pET expression plasmids increase protein production yield in Escherichia coli.

The pET series of expression plasmids are widely used for recombinant protein production in Escherichia coli. The genetic modules controlling transcription and translation in these plasmids were first described in the 1980s and have not changed since. Herein we report design flaws in these genetic modules. We present improved designs and demonstrate that, when incorporated into pET28a, they support increases in protein production. The improved designs are applicable to most of the 103 vectors in the pET series and can be easily implemented.

Determinants of Ligand Subtype-Selectivity at α1A-Adrenoceptor Revealed Using Saturation Transfer Difference (STD) NMR.

α1A- and α1B-adrenoceptors (α1A-AR and α1B-AR) are closely related G protein-coupled receptors (GPCRs) that modulate the cardiovascular and nervous systems in response to binding epinephrine and norepinephrine. The GPCR gene superfamily is made up of numerous subfamilies that, like α1A-AR and α1B-AR, are activated by the same endogenous agonists but may modulate different physiological processes. A major challenge in GPCR research and drug discovery is determining how compounds interact with receptors at the molecular level, especially to assist in the optimization of drug leads. Nuclear magnetic resonance spectroscopy (NMR) can provide great insight into ligand-binding epitopes, modes, and kinetics. Ideally, ligand-based NMR methods require purified, well-behaved protein samples. The instability of GPCRs upon purification in detergents, however, makes the application of NMR to study ligand binding challenging. Here, stabilized α1A-AR and α1B-AR variants were engineered using Cellular High-throughput Encapsulation, Solubilization, and Screening (CHESS), allowing the analysis of ligand binding with Saturation Transfer Difference NMR (STD NMR). STD NMR was used to map the binding epitopes of epinephrine and A-61603 to both receptors, revealing the molecular determinants for the selectivity of A-61603 for α1A-AR over α1B-AR. The use of stabilized GPCRs for ligand-observed NMR experiments will lead to a deeper understanding of binding processes and assist structure-based drug design.

Characterisation of a cell-free synthesised G-protein coupled receptor.

G-protein coupled receptors are the largest family of integral membrane proteins found within the human genome. They function as receptors and modulators to a wide range of ligands and responses which are crucial for human health. GPCR study, specifically the investigation of structure and interaction to cognate ligands, is of high priority. Limitations for structural study can be traced in part, to obtaining suitable quantities of recombinant protein. We sought to address the limitations of traditional recombinant technologies by utilising an Escherichia coli based cell-free protein synthesis (CFPS) approach for production of a thermostable neurotensin receptor 1 (en2NTS1). Initial results were promising, with a high amount (up to 2 mg/mL) of en2NTS1 produced, that had attained correct secondary structure. Meanwhile, concurrent experiments indicated that CFPS produced en2NTS1 showed non-competitive binding to the peptide ligand neurotensin8-13 when compared to E. coli produced en2NTS1. 1H-13C HMQC SOFAST NMR spectra were indicative of disrupted tertiary structure for CFPS produced 13CH3-methionine labelled en2NTS1. The results obtained, indicate CFPS produced en2NTS1 is not forming a discrete tertiary structure and that further development of the CFPS technique needs to be carried out.

Cell-free protein synthesis as a tool to study RXFP3-relaxin-3 protein interactions.

With the discovery of the relaxin family peptide receptors there is interest in obtaining a clearer understanding of the structure of these proteins and the molecular mechanism of receptor-ligand interaction. As G-protein coupled receptors, obtaining milligram quantities for structural investigations is hampered by the inherent instability of these integral membrane proteins. In the current context, understanding of GPCR structural biology has increased dramatically with crystal structures of several inactive and now active forms solved. In addition, the first nuclear magnetic resonance structure of a GPCR was obtained which is of crucial importance to studying these receptors in a more "biologically relevant" setting. However despite this expansion in the field, most structures have been solved on modified systems so as to increase stability and are not necessarily representative of the native receptors. In relation to the relaxin family peptide receptors, we chose to investigate relaxin-family peptide receptor-3 expressed by cell-free protein synthesis. In contrast to in-vivo expression, cell-free was capable of producing large amounts of native receptor which makes it amenable to demanding structural studies.

Recognition of mitochondrial targeting sequences by the import receptors Tom20 and Tom22.

The Tom20 and Tom22 receptor subunits of the TOM (translocase of the outer mitochondrial membrane) complex recognize N-terminal presequences of proteins that are to be imported into the mitochondrion. In plants, Tom20 is C-terminally anchored in the mitochondrial membrane, whereas Tom20 is N-terminally anchored in animals and fungi. Furthermore, the cytosolic domain of Tom22 in plants is smaller than its animal/fungal counterpart and contains fewer acidic residues. Here, NMR spectroscopy was used to explore presequence interactions with the cytosolic regions of receptors from the plant Arabidopsis thaliana and the fungus Saccharomyces cerevisiae (i.e., AtTom20, AtTom22, and ScTom22). It was found that AtTom20 possesses a discontinuous bidentate hydrophobic binding site for presequences. The presequences on plant mitochondrial proteins comprise two or more hydrophobic binding regions to match this bidentate site. NMR data suggested that while these presequences bind to ScTom22, they do not bind to AtTom22. AtTom22, however, binds to AtTom20 at the same binding site as presequences, suggesting that this domain competes with the presequences of imported proteins, thereby enabling their progression along the import pathway.

Membrane receptors: structure and function of the relaxin family peptide receptors.

The receptors for members of the relaxin peptide family have only recently been discovered and are G-protein-coupled receptors (GPCRs). Relaxin and insulin-like peptide 3 (INSL3) interact with the leucine-rich-repeat-containing GPCRs (LGRs) LGR7 and LGR8, respectively. These receptors show closest similarity to the glycoprotein hormone receptors and contain large ectodomains with 10 leucine-rich repeats (LRRs) but are unique members of the LGR family (class C) as they have an LDL class A (LDLa) module at their N-terminus. In contrast, relaxin-3 and INSL5 interact with another class of type I GPCRs which lack a large ectodomain, the peptide receptors GPCR135 and GPCR142, respectively. These receptors are now classified as relaxin family peptide (RXFP) receptors, RXFP1 (LGR7), RXFP2 (LGR8), RXFP3 (GPCR135) and RXFP4 (GPCR142). This review outlines the identification of the peptides and receptors, their expression profiles and physiological roles and the functional interactions of the peptides with their unique receptors.