Johanna Heuveling

Genome-Wide Analysis of the General Stress Response Network in Escherichia coli: σS-Dependent Genes, Promoters, and Sigma Factor Selectivity

The sigmaS (or RpoS) subunit of RNA polymerase is the master regulator of the general stress response in Escherichia coli. While nearly absent in rapidly growing cells, sigmaS is strongly induced during entry into stationary phase and/or many other stress conditions and is essential for the expression of multiple stress resistances. Genome-wide expression profiling data presented here indicate that up to 10% of the E. coli genes are under direct or indirect control of sigmaS and that sigmaS should be considered a second vegetative sigma factor with a major impact not only on stress tolerance but on the entire cell physiology under nonoptimal growth conditions. This large data set allowed us to unequivocally identify a sigmaS consensus promoter in silico. Moreover, our results suggest that sigmaS-dependent genes represent a regulatory network with complex internal control (as exemplified by the acid resistance genes). This network also exhibits extensive regulatory overlaps with other global regulons (e.g., the cyclic AMP receptor protein regulon). In addition, the global regulatory protein Lrp was found to affect sigmaS and/or sigma70 selectivity of many promoters. These observations indicate that certain modules of the sigmaS-dependent general stress response can be temporarily recruited by stress-specific regulons, which are controlled by other stress-responsive regulators that act together with sigma70 RNA polymerase. Thus, not only the expression of genes within a regulatory network but also the architecture of the network itself can be subject to regulation.

DBD Dyes as Fluorescence Lifetime Probes to Study Conformational Changes in Proteins

Previously, [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD)-based fluorophores used as highly sensitive fluorescence lifetime probes reporting on their microenvironmental polarity have been described. Now, a new generation of DBD dyes has been developed. Although they are still sensitive to polarity, in contrast to the former DBD dyes, they have extraordinary spectroscopic properties even in aqueous surroundings. They are characterized by long fluorescence lifetimes (10-20 ns), large Stokes shifts (≈100 nm), high photostabilities, and high quantum yields (>0.56). Here, the spectroscopic properties and synthesis of functionalized derivatives for labeling biological targets are described. Furthermore, thio-reactive maleimido derivatives of both DBD generations show strong intramolecular fluorescence quenching. This mechanism has been investigated and is found to undergo a photoelectron transfer (PET) process. After reaction with a thiol group, this fluorescence quenching is prevented, indicating successful bonding. Being sensitive to their environmental polarity, these compounds have been used as powerful fluorescence lifetime probes for the investigation of conformational changes in the maltose ATP-binding cassette transporter through fluorescence lifetime spectroscopy. The differing tendencies of the fluorescence lifetime change for both DBD dye generations promote their combination as a powerful toolkit for studying microenvironments in proteins.

Structural basis for substrate specificity of an amino acid ABC transporter

Significance Here we report the crystal structures of an amino acid ATP-binding cassette (ABC) importer either in its apo form or in complex with substrates (Arg, His) and/or ATPs. Interestingly, each transmembrane domain has a negatively charged pocket, allowing amino acids carrying positively charged groups to pass through. Functional analyses of the transporter in proteoliposomes indicate its capability to undergo substrate-dependent conformational changes resulting in stimulated ATPase activity. Taken together, we identified a previously undefined substrate binding mode of ABC transporters and shed light on the mechanism underlying how ABC transporters select and translocate their substrates. ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate a variety of substrates, ranging from ions to macromolecules, either out of or into the cytosol (hence defined as importers or exporters, respectively). It has been demonstrated that ABC exporters and importers function through a common mechanism involving conformational switches between inward-facing and outward-facing states; however, the mechanism underlying their functions, particularly substrate recognition, remains elusive. Here we report the structures of an amino acid ABC importer Art(QN)2 from Thermoanaerobacter tengcongensis composed of homodimers each of the transmembrane domain ArtQ and the nucleotide-binding domain ArtN, either in its apo form or in complex with substrates (Arg, His) and/or ATPs. The structures reveal that the straddling of the TMDs around the twofold axis forms a substrate translocation pathway across the membrane. Interestingly, each TMD has a negatively charged pocket that together create a negatively charged internal tunnel allowing amino acids carrying positively charged groups to pass through. Our structural and functional studies provide a better understanding of how ABC transporters select and translocate their substrates.

Conformational changes of the bacterial type I ATP-binding cassette importer HisQMP2 at distinct steps of the catalytic cycle.

Prokaryotic solute binding protein-dependent ATP-binding cassette import systems are divided into type I and type II and mechanistic differences in the transport process going along with this classification are under intensive investigation. Little is known about the conformational dynamics during the catalytic cycle especially concerning the transmembrane domains. The type I transporter for positively charged amino acids from Salmonella enterica serovar Typhimurium (LAO-HisQMP2) was studied by limited proteolysis in detergent solution in the absence and presence of co-factors including ATP, ADP, LAO/arginine, and Mg(2+) ions. Stable peptide fragments could be obtained and differentially susceptible cleavage sites were determined by mass spectrometry as Lys-258 in the nucleotide-binding subunit, HisP, and Arg-217/Arg-218 in the transmembrane subunit, HisQ. In contrast, transmembrane subunit HisM was gradually degraded but no stable fragment could be detected. HisP and HisQ were equally resistant under pre- and post-hydrolysis conditions in the presence of arginine-loaded solute-binding protein LAO and ATP/ADP. Some protection was also observed with LAO/arginine alone, thus reflecting binding to the transporter in the apo-state and transmembrane signaling. Comparable digestion patterns were obtained with the transporter reconstituted into proteoliposomes and nanodiscs. Fluorescence lifetime spectroscopy confirmed the change of HisQ(R218) to a more apolar microenvironment upon ATP binding and hydrolysis. Limited proteolysis was subsequently used as a tool to study the consequences of mutations on the transport cycle. Together, our data suggest similar conformational changes during the transport cycle as described for the maltose ABC transporter of Escherichia coli, despite distinct structural differences between both systems.

One Intact Transmembrane Substrate Binding Site Is Sufficient for the Function of the Homodimeric Type I ATP-Binding Cassette Importer for Positively Charged Amino Acids Art(MP)2 of Geobacillus stearothermophilus

ATP-binding cassette (ABC) transport systems comprise two transmembrane domains/subunits that form a translocation path and two nucleotide-binding domains/subunits that bind and hydrolyze ATP. Prokaryotic canonical ABC import systems require an extracellular substrate-binding protein for function. Knowledge of substrate-binding sites within the transmembrane subunits is scarce. Recent crystal structures of the ABC importer Art(QN)2 for positively charged amino acids of Thermoanerobacter tengcongensis revealed the presence of one substrate molecule in a defined binding pocket in each of the transmembrane subunits, ArtQ (J. Yu, J. Ge, J. Heuveling, E. Schneider, and M. Yang, Proc Natl Acad Sci U S A 112:5243-5248, 2015, https://doi.org/10.1073/pnas.1415037112). This finding raised the question of whether both sites must be loaded with substrate prior to initiation of the transport cycle. To address this matter, we first explored the role of key residues that form the binding pocket in the closely related Art(MP)2 transporter of Geobacillus stearothermophilus, by monitoring consequences of mutations in ArtM on ATPase and transport activity at the level of purified proteins embedded in liposomes. Our results emphasize that two negatively charged residues (E153 and D160) are crucial for wild-type function. Furthermore, the variant Art[M(L67D)P]2 exhibited strongly impaired activities, which is why it was considered for construction of a hybrid complex containing one intact and one impaired substrate-binding site. Activity assays clearly revealed that one intact binding site was sufficient for function. To our knowledge, our study provides the first biochemical evidence on transmembrane substrate-binding sites of an ABC importer.IMPORTANCE Canonical prokaryotic ATP-binding cassette importers mediate the uptake of a large variety of chemicals, including nutrients, osmoprotectants, growth factors, and trace elements. Some also play a role in bacterial pathogenesis, which is why full understanding of their mode of action is of the utmost importance. One of the unsolved problems refers to the chemical nature and number of substrate binding sites formed by the transmembrane subunits. Here, we report that a hybrid amino acid transporter of G. stearothermophilus, encompassing one intact and one impaired transmembrane binding site, is fully competent in transport, suggesting that the binding of one substrate molecule is sufficient to trigger the translocation process.