Haemagglutinin substitutions N125D, D127E, D222G and R223Q improve replicative fitness and vaccine effectiveness of an A/H1N1pdm09 live attenuated influenza vaccine virus by enhancing α-2,6 receptor binding.

During 2013-14 and 2015-16, A/H1N1pdm09 live attenuated influenza vaccine (LAIV) viruses replicated inefficiently in primary human nasal epithelial cells (hNEC). This led to reduced vaccine effectiveness (VE) in quadrivalent formulations, mediated by inter-strain competition. By mutating the haemagglutinin (HA) protein, we aimed to enhance hNEC replication of a novel A/H1N1pdm09 vaccine strain to overcome competition and improve VE. Combinations of N125D, D127E, D222G and R223Q substitutions were introduced to the HA protein of A/Slovenia/2903/2015 (A/SLOV15). A/SLOV15 S13, containing all four HA substitutions, produced approximately 1000-fold more virus than parental V1 during hNEC infection. Immunogenicity in ferrets was increased by approximately 10-fold, without compromising yield in eggs or antigenic match to wild-type (wt) reference strains. Despite S13 and V1 being antigenically similar, only S13 protected ferrets from wt virus shedding and fever post-challenge. Crucially, these data suggested that enhanced fitness allowed S13 to overcome inter-strain competition in quadrivalent LAIV (QLAIV). This improved efficacy was later validated by real-world VE data. S13 displayed increased binding avidity to a mammalian-like α-2,6 receptor analogue (6-SLN), relative to V1, while maintaining avian-like 3-SLN avidity. In silico modelling of the HA receptor binding site revealed additional interactions in the S13:6-SLN binding network and a mild increase in 6-SLN binding energy, indicating a possible mechanism for increased α-2,6 receptor-binding avidity. These data confirm that rational HA mutagenesis can be used to optimise hNEC replication and VE for A/H1N1pdm09 LAIV viruses.

Coexistence of Ammonium Transporter and Channel Mechanisms in Amt-Mep-Rh Twin-His Variants Impairs the Filamentation Signaling Capacity of Fungal Mep2 Transceptors.

Ammonium translocation through biological membranes, by the ubiquitous Amt-Mep-Rh family of transporters, plays a key role in all domains of life. Two highly conserved histidine residues protrude into the lumen of the pore of these transporters, forming the family's characteristic Twin-His motif. It has been hypothesized that the motif is essential to confer the selectivity of the transport mechanism. Here, using a combination of in vitro electrophysiology on Escherichia coli AmtB, in silico molecular dynamics simulations, and in vivo yeast functional complementation assays, we demonstrate that variations in the Twin-His motif trigger a mechanistic switch between a specific transporter, depending on ammonium deprotonation, to an unspecific ion channel activity. We therefore propose that there is no selective filter that governs specificity in Amt-Mep-Rh transporters, but the inherent mechanism of translocation, dependent on the fragmentation of the substrate, ensures the high specificity of the translocation. We show that coexistence of both mechanisms in single Twin-His variants of yeast Mep2 transceptors disrupts the signaling function and so impairs fungal filamentation. These data support a signaling process driven by the transport mechanism of the fungal Mep2 transceptors. IMPORTANCE Fungal infections represent a significant threat to human health and cause huge damage to crop yields worldwide. The dimorphic switch between yeast and filamentous growth is associated with the virulence of pathogenic fungi. Of note, fungal Mep2 proteins of the conserved Amt-Mep-Rh family play a transceptor role in the induction of filamentation; however, the signaling mechanism remains largely unknown. Amt-Mep-Rh proteins ensure the specific scavenging of NH4+ through a mechanism relying on substrate deprotonation, thereby preventing competition and translocation of similar-sized K+. Our multidisciplinary approaches using E. coli AmtB, Saccharomyces cerevisiae, and Candida albicans Mep2 show that double variation of the family-defining Twin-His motif triggers a mechanistic switch from a specific transporter to an unspecific ion channel with both mechanisms still coexisting in single variants. Moreover, we show that this mechanistic alteration is associated with loss of signaling ability of Mep2, supporting a transport mechanism-driven process in filamentation induction.

An efficient system for bioconjugation based on a widely applicable engineered O-glycosylation tag.

Bioconjugates are an important class of therapeutic molecules. To date, O-glycan-based metabolic glycoengineering has had limited use in this field, due to the complexities of the endogenous O-glycosylation pathway and the lack of an O-glycosylation consensus sequence. Here, we describe the development of a versatile on-demand O-glycosylation system that uses a novel, widely applicable 5 amino acid O-glycosylation tag, and a metabolically engineered UDP-galactose-4-eperimase (GALE) knock-out cell line. Optimization of the primary sequence of the tag enables the production of Fc-based proteins with either single or multiple O-glycans with complexity fully controlled by media supplementation. We demonstrate how the uniformly labeled proteins containing exclusively N-azido-acetylgalactosamine are used for CLICK chemistry-based bioconjugation to generate site-specifically fluorochrome-labeled antibodies, dual-payload molecules, and bioactive Fc-peptides for applications in basic research and drug discovery. To our knowledge, this is the first description of generating a site-specific O-glycosylation system by combining an O-glycosylation tag and a metabolically engineered cell line.

A two-lane mechanism for selective biological ammonium transport.

The transport of charged molecules across biological membranes faces the dual problem of accommodating charges in a highly hydrophobic environment while maintaining selective substrate translocation. This has been the subject of a particular controversy for the exchange of ammonium across cellular membranes, an essential process in all domains of life. Ammonium transport is mediated by the ubiquitous Amt/Mep/Rh transporters that includes the human Rhesus factors. Here, using a combination of electrophysiology, yeast functional complementation and extended molecular dynamics simulations, we reveal a unique two-lane pathway for electrogenic NH4+ transport in two archetypal members of the family, the transporters AmtB from Escherichia coli and Rh50 from Nitrosomonas europaea. The pathway underpins a mechanism by which charged H+ and neutral NH3 are carried separately across the membrane after NH4+ deprotonation. This mechanism defines a new principle of achieving transport selectivity against competing ions in a biological transport process.

The lipid environment determines the activity of the Escherichia coli ammonium transporter AmtB.

The movement of ammonium across biologic membranes is a fundamental process in all living organisms and is mediated by the ubiquitous ammonium transporter/methylammonium permease/rhesus protein (Amt/Mep/Rh) family of transporters. Recent structural analysis and coupled mass spectrometry studies have shown that the Escherichia coli ammonium transporter AmtB specifically binds 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG). Upon POPG binding, several residues of AmtB undergo a small conformational change, which stabilizes the protein against unfolding. However, no studies have so far been conducted, to our knowledge, to explore whether POPG binding to AmtB has functional consequences. Here, we used an in vitro experimental assay with purified components, together with molecular dynamics simulations, to characterize the relation between POPG binding and AmtB activity. We show that the AmtB activity is electrogenic. Our results indicate that the activity, at the molecular level, of Amt in archaebacteria and eubacteria may differ. We also show that POPG is an important cofactor for AmtB activity and that, in the absence of POPG, AmtB cannot complete the full translocation cycle. Furthermore, our simulations reveal previously undiscovered POPG binding sites on the intracellular side of the lipid bilayer between the AmtB subunits. Possible molecular mechanisms explaining the functional role of POPG are discussed.-Mirandela, G. D., Tamburrino, G., Hoskisson, P. A., Zachariae, U., Javelle, A. The lipid environment determines the activity of the Escherichia coli ammonium transporter AmtB.

Merging In-Solution X-ray and Neutron Scattering Data Allows Fine Structural Analysis of Membrane-Protein Detergent Complexes.

In-solution small-angle X-ray and neutron scattering (SAXS/SANS) have become popular methods to characterize the structure of membrane proteins, solubilized by either detergents or nanodiscs. SANS studies of protein-detergent complexes usually require deuterium-labeled proteins or detergents, which in turn often lead to problems in their expression or purification. Here, we report an approach whose novelty is the combined analysis of SAXS and SANS data from an unlabeled membrane protein complex in solution in two complementary ways. First, an explicit atomic analysis, including both protein and detergent molecules, using the program WAXSiS, which has been adapted to predict SANS data. Second, the use of MONSA which allows one to discriminate between detergent head- and tail-groups in an ab initio approach. Our approach is readily applicable to any detergent-solubilized protein and provides more detailed structural information on protein-detergent complexes from unlabeled samples than SAXS or SANS alone.

Modulation of the Neisseria gonorrhoeae drug efflux conduit MtrE.

Widespread antibiotic resistance, especially of Gram-negative bacteria, has become a severe concern for human health. Tripartite efflux pumps are one of the major contributors to resistance in Gram-negative pathogens, by efficiently expelling a broad spectrum of antibiotics from the organism. In Neisseria gonorrhoeae, one of the first bacteria for which pan-resistance has been reported, the most expressed efflux complex is MtrCDE. Here we present the electrophysiological characterisation of the outer membrane component MtrE and the membrane fusion protein MtrC, obtained by a combination of planar lipid bilayer recordings and in silico techniques. Our in vitro results show that MtrE can be regulated by periplasmic binding events and that the interaction between MtrE and MtrC is sufficient to stabilize this complex in an open state. In contrast to other efflux conduits, the open complex only displays a slight preference for cations. The maximum conductance we obtain in the in vitro recordings is comparable to that seen in our computational electrophysiology simulations conducted on the MtrE crystal structure, indicating that this state may reflect a physiologically relevant open conformation of MtrE. Our results suggest that the MtrC/E binding interface is an important modulator of MtrE function, which could potentially be targeted by new efflux inhibitors.

Structural Mechanisms of Voltage Sensing in G Protein-Coupled Receptors.

G-protein-coupled receptors (GPCRs) form the largest superfamily of membrane proteins and one-third of all drug targets in humans. A number of recent studies have reported evidence for substantial voltage regulation of GPCRs. However, the structural basis of GPCR voltage sensing has remained enigmatic. Here, we present atomistic simulations on the δ-opioid and M2 muscarinic receptors, which suggest a structural and mechanistic explanation for the observed voltage-induced functional effects. The simulations reveal that the position of an internal Na(+) ion, recently detected to bind to a highly conserved aqueous pocket in receptor crystal structures, strongly responds to voltage changes. The movements give rise to gating charges in excellent agreement with previous experimental recordings. Furthermore, free energy calculations show that these rearrangements of Na(+) can be induced by physiological membrane voltages. Due to its role in receptor function and signal bias, the repositioning of Na(+) has important general implications for signal transduction in GPCRs.