Review. Structure and mechanism of ATP-binding cassette transporters

ATP-binding cassette (ABC) transporters constitute a large superfamily of integral membrane proteins that includes both importers and exporters. In recent years, several structures of complete ABC transporters have been determined by X-ray crystallography. These structures suggest a mechanism by which binding and hydrolysis of ATP by the cytoplasmic, nucleotide-binding domains control the conformation of the transmembrane domains and therefore which side of the membrane the translocation pathway is exposed to. A basic, conserved two-state mechanism can explain active transport of both ABC importers and ABC exporters, but various questions remain unresolved. In this article, I will review some of the crystal structures and the mechanistic insight gained from them. Future challenges for a better understanding of the mechanism of ABC transporters will be outlined.     

Inspiratory resistances facilitate the diaphragm response to transcranial stimulation in humans

Background  

Breathing in humans is dually controlled for metabolic (brainstem commands) and behavioral purposes (suprapontine commands) with reciprocal modulation through spinal integration. Whereas the ventilatory response to chemical stimuli arises from the brainstem, the compensation of mechanical loads in awake humans is thought to involve suprapontine mechanisms. The aim of this study was to test this hypothesis by examining the effects of inspiratory resistive loading on the response of the diaphragm to transcranial magnetic stimulation.     

The structures of BtuCD and MscS and their implications for transporter and channel function

The passage of most molecules across biological membranes is mediated by specialized integral membrane proteins known as channels and transporters. Although these transport families encompass a wide range of functions, molecular architectures and mechanisms, there are common elements that must be incorporated within their structures, namely the translocation pathway, ligand specificity elements and regulatory sensors to control the rate of ligand flow across the membrane. This minireview discusses aspects of the structure and mechanism of two bacterial transport systems, the stretch-activated mechanosensitive channel of small conductance (MscS) and the ATP-dependent vitamin B12 uptake system (BtuCD), emphasizing their general implications for transporter function.     

Structural/functional characterization of the alpha 2-plasmin inhibitor C-terminal peptide

The alpha(2)-plasmin inhibitor (A2PI) is a main physiological regulator of the trypsin-like serine proteinase plasmin. It is composed of an N-terminal 15 amino acid fibrin cross-linking polypeptide, a 382-residue serpin domain, and a flexible C-terminal segment. The latter, peptide Asn(398)-Lys(452), and its Lys452Ala mutant were expressed as recombinant proteins in Escherichia coli (r-A2PIC and r-A2PICmut, respectively). CD and NMR analyses indicate that r-A2PIC is flexible, loosely folded, and with low content of regular secondary structure. Functional characterization via intrinsic fluorescence ligand titrations shows that r-A2PIC interacts with the isolated plasminogen kringle 1 (r-K1) (K(a) approximately 69.9 mM(-)(1)), K4 (K(a) approximately 45.7 mM(-)(1)), K5 (K(a) approximately 4.3 mM(-)(1)), and r-K2 (K(a) approximately 3.2 mM(-)(1)), all of which are known to exhibit lysine-binding capability. The affinities of these kringles for r-A2PIC are consistently larger than those reported for the ligand N(alpha)-acetyllysine, a mimic of a C-terminal Lys residue. The r-A2PICmut, with a C-terminal Ala residue, also interacts with r-K1 and K4, although with approximately 5-fold lesser affinities relative to r-A2PIC, demonstrating that while Lys(452) plays a major role in the binding, internal residues in r-A2PIC tether the kringles. (1)H NMR spectroscopy shows that key aromatic residues within the K4 lysine-binding site (LBS), namely, Trp(25), Trp(62), Phe(64), Trp(72), and Tyr(74), selectively respond to the addition of r-A2PIC and r-A2PICmut, indicating that these interactions proceed via the kringles' canonical LBS. We conclude that r-A2PIC docks to kringles primarily through lysine side chains and that Lys(452) most definitely enhances the binding. This suggests that multiple Lys residues within A2PI could contribute, perhaps in a zipper-like fashion, to its binding to the in-tandem, multikringle array that configures the plasmin heavy chain.     

Immune responses in baboons vaccinated with HIV-2 genetic expression libraries

Immunization using genetic expression libraries may be an improvement over conventional DNA immunization using a single gene because more epitopes are simultaneously presented to the immune system. In this study, we evaluated the effectiveness of an HIV-2 vaccine made from a genomic expression library in baboons. We found that HIV-2 expression library immunization induced HIV-2-specific memory responses but low levels of CD8+ cell anti-viral responses and neutralizing antibodies. After intravenous virus challenge using a homologous pathogenic variant, HIV-2UC2/9429, viral loads were similar in the HIV-2-immunized and control baboons. We conclude that although immunization using HIV-2 expression libraries induces immune responses, this approach does not provide protection in baboons against intravenous challenge with HIV-2.     

Cell wall-bound trans- and cis-ferulic acids in growing maize roots

The levels of cell wall-bound trans - and cis -ferulic acids in roots of dark grown Zea mays cv. LG11 plants were measured. They were quantified after alkaline hydrolysis of purified cell walls by reversed phase HPLC using trans -cinnamic acid as internal standard. The total amount of ferulic acid ( trans - and cis -ferulic acid) in the root base was 3–4 times higher than in the root tip. Cis -ferulic acid represented between 2% (tip) and 18% (base) of the total ferulic acid content. The total content of trans - and cis -ferulic acids was approximately the same in the stele and the cortex, but the level of cis -ferulic acid in the stele was 5–6 times higher than in the cortex. Trans - and cis -ferulic acid levels as well as the percentage of cis -ferulic acid in the elongation zone were steady between 48 and 96 h after the beginning of germination. Slowly growing roots contained more wall-bound ferulic acids, particularly cis -ferulic acid, than fast growing roots. This relationship was found in the differentiation zone but not in the elongation zone. The importance of cell wall-bound trans - and cis -ferulic acids is discussed in the context of root growth and differentiation.     

Structure and mechanism of ABC transporters

ATP-binding cassette (ABC) transporters facilitate unidirectional translocation of chemically diverse substrates across cell or organelle membranes. The recently determined crystal structures of the vitamin B(12) importer BtuCD and its cognate binding protein BtuF have revealed critical architectural features that are probably shared by other ABC transporters. For example, the arrangement of the ABC domains and their interface with the membrane-spanning domains are probably conserved, whereas the number of transmembrane helices and their arrangement are not. Two distinct mechanistic schemes for how ABC engines couple ATP hydrolysis to substrate transport have been proposed recently and are being explored.     

DNA shuffling and screening strategies for improving vaccine efficacy

The efficacy of vaccines can be improved by increasing their immunogenicity, broadening their crossprotective range, as well as by developing immunomodulators that can be coadministered with the vaccine antigen. One technology that can be applied to each of these aspects of vaccine development is MolecularBreeding directed molecular evolution. Essentially, this technology is used to evolve genes in vitro through an iterative process consisting of recombinant generation followed by selection of the desired recombinants. We have used DNA shuffling and screening strategies to develop and improve vaccine candidates against several infectious pathogens including Plasmodium falciparum (a common cause of severe and fatal human malaria), dengue virus, encephalitic alphaviruses such as Venezuelan, western and eastern equine encephalitis viruses (VEEV, WEEV, and EEEV, respectively), human immunodeficiency virus-1 (HIV-1), and hepatitis B virus (HBV). By recombining antigen-encoding genes from different serovar isolates, new chimeras are selected for crossreactivity; these vaccine candidates are expected to provide broader crossprotection than vaccines based on a single serovar. Furthermore, the vaccine candidates can be selected for improved immunogenicity, which would also improve their efficacy. In addition to vaccine candidates, we have applied the technology to evolve several immunomodulators that when coadministered with vaccines can improve vaccine efficacy by fine-tuning the T cell response. Thus, DNA shuffling and screening technology is a promising strategy to facilitate vaccine efficacy.     

Mating frequency and resource allocation to male and female function in the simultaneous hermaphrodite land snail Arianta arbustorum

Sex allocation theory predicts that mating frequency and long‐term sperm storage affect the relative allocation to male and female function in simultaneous hermaphrodites. We examined the effect of mating frequency on male and female reproductive output (number of sperm delivered and eggs deposited) and on the resources allocated to the male and female function (dry mass, nitrogen and carbon contents of spermatophores and eggs) in individuals of the simultaneous hermaphrodite land snail Arianta arbustorum. Similar numbers of sperm were delivered in successive copulations. Consequently, the total number of sperm transferred increased with increasing number of copulations. In contrast, the total number of eggs produced was not influenced by the number of copulations. Energy allocation to gamete production expressed as dry mass, nitrogen or carbon content was highly female‐biased (>95% in all estimates). With increasing number of copulations the relative nitrogen allocation to the male function increased from 1.7% (one copulation) to 4.7% (three copulations), but the overall reproductive allocation remained highly female‐biased. At the individual level, we did not find any trade‐off between male and female reproductive function. In contrast, there was a significant positive correlation between the resources allocated to the male and female function. Snails that delivered many sperm also produced a large number of eggs. This finding contradicts current theory of sex allocation in simultaneous hermaphrodites.     

Validation of surface recordings of the diaphragm response to transcranial magnetic stimulation in humans.

The integrity of the central efferent motor pathways to the diaphragm can be assessed by using transcranial magnetic stimulation to measure the latency of the corresponding motor evoked potentials with surface electrodes. Because transcranial magnetic stimulation does not activate the diaphragm alone, signal contamination is a potential problem. To evaluate this issue, surface diaphragmatic motor-evoked potential latencies were compared with latencies recorded from diaphragm needle in 9 healthy volunteers. Surface latencies of muscles likely to contaminate the diaphragm signals (serratus anterior, pectoralis major, and tranversus abdominis) were also recorded. The latencies in response to nonfocal transcranial stimulation from surface electrodes were not significantly different from the needle ones (17 +/- 1.3 vs. 17.2 +/- 1.1 ms, respectively) but were significantly different from the latencies of the other muscles. In two cases, signal contamination appeared likely (serratus anterior in 1 case, abdominal muscles in 1 case). It is possible to reliably measure the latency of the diaphragm response to transcranial magnetic stimulation with adequately positioned surface electrodes.