Band 404, Heft 7

Reconstitution of membrane proteins into liposomal membranes represents a key technique in enabling functional analysis under well-defined conditions. In this review, we provide a brief introduction to selected methods that have been developed to determine membrane protein orientation after reconstitution in liposomes, including approaches based on proteolytic digestion with proteases, site-specific labeling, fluorescence quenching and activity assays. In addition, we briefly highlight new strategies based on single vesicle analysis to address the problem of sample heterogeneity.

R epeats in T o X ins (RTX) protein family is a group of exoproteins secreted by Type 1 secretion system (T1SS) of several Gram-negative bacteria. The term RTX is derived from the characteristic nonapeptide sequence (GGxGxDxUx) present at the C-terminus of the protein. This RTX domain binds to calcium ions in the extracellular medium after being secreted out of the bacterial cells, and this facilitates folding of the entire protein. The secreted protein then binds to the host cell membrane and forms pores via a complex pathway, which eventually leads to the cell lysis. In this review, we summarize two different pathways in which RTX toxins interact with host cell membrane and discuss the possible reasons for specific and unspecific activity of RTX toxins to different types of host cells.

Na + /taurocholate cotransporting polypeptide (NTCP) is a member of the solute carrier (SLC) family 10 transporters (gene symbol SLC10A1 ) and is responsible for the sodium-dependent uptake of bile salts across the basolateral membrane of hepatocytes. In addition to its primary transporter function, NTCP is the high-affinity hepatic receptor for hepatitis B (HBV) and hepatitis D (HDV) viruses and, therefore, is a prerequisite for HBV/HDV virus entry into hepatocytes. The inhibition of HBV/HDV binding to NTCP and internalization of the virus/NTCP receptor complex has become a major concept in the development of new antiviral drugs called HBV/HDV entry inhibitors. Hence, NTCP has emerged as a promising target for therapeutic interventions against HBV/HDV infections in the last decade. In this review, recent findings on protein–protein interactions (PPIs) between NTCP and cofactors relevant for entry of the virus/NTCP receptor complex are summarized. In addition, strategies aiming to block PPIs with NTCP to dampen virus tropism and HBV/HDV infection rates are discussed. Finally, this article suggests novel directions for future investigations evaluating the functional contribution of NTCP-mediated PPIs in the development and progression of HBV/HDV infection and subsequent chronic liver disorders.

Mycobacteria, such as the pathogen M. tuberculosis , utilize up to five paralogous type VII secretion systems to transport proteins across their cell envelope. Since these proteins associate in pairs that depend on each other for transport to a different extent, the secretion pathway to the bacterial surface remained challenging to address. Structural characterization of the inner-membrane embedded secretion machineries along with recent advances on the substrates’ co-dependencies for transport allow for the first time more detailed and testable models for secretion.

Polymer-encapsulated nanodiscs enable membrane proteins to be investigated within a native-like lipid-bilayer environment. Unlike other bilayer-based membrane mimetics, these nanodiscs are equilibrium structures that permit lipid exchange on experimentally relevant timescales. Therefore, examining the kinetics and mechanisms of lipid exchange is of great interest. Since the high charge densities of existing anionic polymers can interfere with protein–protein and protein–lipid interactions as well as charge-sensitive analysis techniques, electroneutral nanodisc-forming polymers have been recently introduced. However, it has remained unclear how the electroneutrality of these polymers affects the lipid-exchange behavior of the nanodiscs. Here, we use time-resolved Förster resonance energy transfer to study the kinetics and the mechanisms of lipid exchange among nanodiscs formed by the electroneutral polymer Sulfo-DIBMA. We also examine the role of coulombic repulsion and specific counterion association in lipid exchange. Our results show that Sulfo-DIBMA nanodiscs exchange lipids on a similar timescale as DIBMA nanodiscs. In contrast with nanodiscs made from polyanionic DIBMA, however, the presence of mono- and divalent cations does not influence lipid exchange among Sulfo-DIBMA nanodiscs, as expected from their electroneutrality. The robustness of Sulfo-DIBMA nanodiscs against varying ion concentrations opens new possibilities for investigating charge-sensitive processes involving membrane proteins.

Substrate-binding proteins (SBPs) are part of solute transport systems and serve to increase substrate affinity and uptake rates. In contrast to primary transport systems, the mechanism of SBP-dependent secondary transport is not well understood. Functional studies have thus far focused on Na + -coupled Tripartite ATP-independent periplasmic (TRAP) transporters for sialic acid. Herein, we report the in vitro functional characterization of TAXIPm-PQM from the human pathogen Proteus mirabilis . TAXIPm-PQM belongs to a TRAP-subfamily using a different type of SBP, designated TRAP-associated extracytoplasmic immunogenic (TAXI) protein. TAXIPm-PQM catalyzes proton-dependent α-ketoglutarate symport and its SBP is an essential component of the transport mechanism. Importantly, TAXIPm-PQM represents the first functionally characterized SBP-dependent secondary transporter that does not rely on a soluble SBP, but uses a membrane-anchored SBP instead.

The essential Escherichia coli ATPase MsbA is a lipid flippase that serves as a prototype for multi drug resistant ABC transporters. Its physiological function is the transport of lipopolisaccharides to build up the outer membranes of Gram-negative bacteria. Although several structural and biochemical studies of MsbA have been conducted previously, a detailed picture of the dynamic processes that link ATP hydrolysis to allocrit transport remains elusive. We report here for the first time time-resolved Fourier transform infrared (FTIR) spectroscopic measurements of the ATP binding and ATP hydrolysis reaction of full-length MsbA and determined reaction rates at 288 K of k 1  = 0.49 ± 0.28 s −1 and k 2  = 0.014 ± 0.003 s −1 , respectively. We further verified these rates with photocaged NPE cg AppNHp where only nucleotide binding was observable and the negative mutant MsbA-H537A that showed slow hydrolysis ( k 2  < 2 × 10 −4  s −1 ). Besides single turnover kinetics, FTIR measurements also deliver IR signatures of all educts, products and the protein. ADP remains protein-bound after ATP hydrolysis. In addition, the spectral changes observed for the two variants MsbA-S378A and MsbA-S482A correlated with the loss of hydrogen bonding to the γ-phosphate of ATP. This study paves the way for FTIR-spectroscopic investigations of allocrite transport in full-length MsbA.