Lutz Schmitt

A structural classification of substrate-binding proteins.

Substrate‐binding proteins (SBP) are associated with a wide variety of protein complexes. The proteins are part of ATP‐binding cassette transporters for substrate uptake, ion gradient driven transporters, DNA‐binding proteins, as well as channels and receptors from both pro‐ and eukaryotes. A wealth of structural and functional data is available on SBPs, with over 120 unique entries in the Protein Data Bank (PDB). Over a decade ago these proteins were divided into three structural classes, but based on the currently available wealth of structural data, we propose a new classification into six clusters, based on features of their three‐dimensional structure.

H662 is the linchpin of ATP hydrolysis in the nucleotide-binding domain of the ABC transporter HlyB

The ABC transporter HlyB is a central element of the HlyA secretion machinery, a paradigm of Type I secretion. Here, we describe the crystal structure of the HlyB‐NBD (nucleotide‐binding domain) with H662 replaced by Ala in complex with ATP/Mg2+. The dimer shows a composite architecture, in which two intact ATP molecules are bound at the interface of the Walker A motif and the C‐loop, provided by the two monomers. ATPase measurements confirm that H662 is essential for activity. Based on these data, we propose a model in which E631 and H662, highly conserved among ABC transporters, form a catalytic dyad. Here, H662 acts as a ‘linchpin’, holding together all required parts of a complicated network of interactions between ATP, water molecules, Mg2+, and amino acids both in cis and trans, necessary for intermonomer communication. Based on biochemical experiments, we discuss the hypothesis that substrate‐assisted catalysis, rather than general base catalysis might operate in ABC‐ATPases.

Structure and mechanism of ABC transporters

ATP-binding cassette (ABC) transporters are central to many physiological processes, including the uptake of nutrients, the non-classical secretion of signaling molecules and toxins, multidrug resistance and the development of human disease. As one might expect from this spectrum of translocation events, these ubiquitous, ATP-dependent pumps or channels are capable of transporting an enormous variety of substrates, ranging from small ions to large proteins. Recently determined structures of full-length ABC transporters and isolated ABC domains have increased our understanding of the functional mechanism of these proteins.

Type 1 protein secretion in bacteria, the ABC-transporter dependent pathway (Review)

The relatively simple type 1 secretion system in Gram-negative bacteria is nevertheless capable of transporting polypeptides of up to 800 kDa across the cell envelope in a few seconds. The translocator is composed of an ABC-transporter, providing energy through ATP hydrolysis (and perhaps the initial channel across the inner membrane), linked to a multimeric Membrane Fusion Protein (MFP) spanning the initial part of the periplasm and forming a continuous channel to the surface with an outer membrane trimeric protein. Proteins targeted to the translocator carry an (uncleaved), poorly conserved secretion signal of approximately 50 residues. In E. coli the HlyA toxin interacts with both the MFP (HlyD) and the ABC protein HlyB, (a half transporter) triggering, via a conformational change in HlyD, recruitment of the third component, TolC, into the transenvelope complex. In vitro, HlyA, through its secretion signal, binds to the nucleotide binding domain (NBD or ABC-ATPase) of HlyB in a reaction reversible by ATP that may mimic initial movement of HlyA into the translocation channel. HlyA is then transported rapidly, apparently in an unfolded form, to the cell surface, where folding and release takes place. Whilst recent structural studies of TolC and MFP-like proteins are providing atomic detail of much of the transport path, structural analysis of the HlyB NBD and other ABC ATPases, have revealed details of the catalytic cycle within an NBD dimer and a glimpse of how the action of HlyB is coupled to the translocation of HlyA.

Crystal Structure of the Nucleotide-binding Domain of the ABC-transporter Haemolysin B: Identification of a Variable Region Within ABC Helical Domains

The ABC-transporter haemolysin B is a central component of the secretion machinery that translocates the toxin, haemolysin A, in a Sec-independent fashion across both membranes of E. coli. Here, we report the X-ray crystal structure of the nucleotide-binding domain (NBD) of HlyB. The molecule shares the common overall architecture of ABC-transporter NBDs. However, the last three residues of the Walker A motif adopt a 3(10) helical conformation, stabilized by a bound anion. In consequence, this results in an unusual interaction between the Walker A lysine residue and the Walker B glutamate residue. As these residues are normally required to be available for ATP binding, for catalysis and for dimer formation of ABC domains, we suggest that this conformation may represent a latent monomeric form of the NBD. Surprisingly, comparison of available NBD structures revealed a structurally diverse region (SDR) of about 30 residues within the helical arm II domain, unique to each of the eight NBDs analyzed. As this region interacts with the transmembrane part of ABC-transporters, the SDR helps to explain the selectivity and/or targeting of different NBDs to their cognate transmembrane domains.

A structural analysis of asymmetry required for catalytic activity of an ABC-ATPase domain dimer

The ATP‐binding cassette (ABC)‐transporter haemolysin (Hly)B, a central element of a Type I secretion machinery, acts in concert with two additional proteins in Escherichia coli to translocate the toxin HlyA directly from the cytoplasm to the exterior. The basic set of crystal structures necessary to describe the catalytic cycle of the isolated HlyB‐NBD (nucleotide‐binding domain) has now been completed. This allowed a detailed analysis with respect to hinge regions, functionally important key residues and potential energy storage devices that revealed many novel features. These include a structural asymmetry within the ATP dimer that was significantly enhanced in the presence of Mg2+, indicating a possible functional asymmetry in the form of one open and one closed phosphate exit tunnel. Guided by the structural analysis, we identified two amino acids, closing one tunnel by an apparent salt bridge. Mutation of these residues abolished ATP‐dependent cooperativity of the NBDs. The implications of these new findings for the coupling of ATP binding and hydrolysis to functional activity are discussed.

The motor domains of ABC-transporters

The transport of substrates across a cellular membrane is a vitally important biological function essential for cell survival. ATP-binding cassette (ABC) transporters constitute one of the largest subfamilies of membrane proteins, accomplishing this task. Mutations in genes encoding for ABC transporters cause different diseases, for example, Adrenoleukodystrophy, Stargardt disease or Cystic Fibrosis. Furthermore, some ABC transporters are responsible for multidrug resistance, presenting a major obstacle in modern cancer chemotherapy. In order to translocate the enormous variety of substrates, ranging from ions, nutrients, small peptides to large toxins, different ABC-transporters utilize the energy gained from ATP binding and hydrolysis. The ATP binding cassette, also called the motor domain of ABC transporters, is highly conserved among all ABC transporters. The ability to purify this domain rather easily presents a perfect possibility to investigate the mechanism of ATP hydrolysis, thus providing us with a detailed picture of this process. Recently, many crystal structures of the ATP-binding domain and the full-length structures of two ABC transporters have been solved. Combining these structural data, we have now the opportunity to analyze the hydrolysis event on a molecular level. This review provides an overview of the structural investigations of the ATP-binding domains, highlighting molecular changes upon ATP binding and hydrolysis.

A mutation of the H-loop selectively affects rhodamine transport by the yeast multidrug ABC transporter Pdr5

The yeast ABC transporter Pdr5 plays a major role in drug resistance against a large number of structurally unrelated compounds. Although Pdr5 has been extensively studied, many important aspects regarding its molecular mechanisms remain unresolved. For example, a striking degeneration of conserved amino acid residues exists in the nucleotide binding domains (NBDs), but their functional relevance is unknown. Here, we performed in vivo and in vitro experiments to address the functional asymmetry of NBDs. It became evident by ATPase activity and drug transport studies that catalysis at only one of the two NBD composite sites is crucial for protein function. Furthermore, mutations of the proposed “catalytic carboxylate” (E1036) and the “catalytic dyad histidine” (H1068) were characterized. Although a mutation of the glutamate abolished ATPase activity and substrate transport, mutation of H1068 had no influence on ATP consumption. However, the H1068A mutation abolished rhodamine transport in vivo and in vitro, while leaving the transport of other substrates unaffected. By contrast to mammalian P-glycoprotein (P-gp), the ATPase activity of yeast Pdr5 is not stimulated by the addition of substrates, indicating that Pdr5 is an uncoupled ABC transporter that constantly hydrolyses ATP to ensure active substrate transport. Taken together, our data provide important insights into the molecular mechanism of Pdr5 and suggest that not solely the transmembrane domains dictate substrate selection.

De novo bile salt transporter antibodies as a possible cause of recurrent graft failure after liver transplantation: A novel mechanism of cholestasis

Progressive familial intrahepatic cholestasis type 2 (PFIC‐2) is caused by mutations of the bile salt export pump (BSEP [ABCB11]), an ATP‐binding cassette (ABC)‐transporter exclusively expressed at the canalicular membrane of hepatocytes. An absence of BSEP from the canalicular membrane causes cholestasis and leads to liver cirrhosis, which may necessitate liver transplantation in early childhood. We report on the first case of a child with PFIC‐2 suffering from repeated posttransplant recurrence of progressive intrahepatic cholestasis due to autoantibodies against BSEP. These antibodies occurred after transplantation and were detected in the patients serum and at the canalicular membrane of two consecutive liver transplants. The antibodies were reactive toward the first extracellular loop of BSEP, were of high affinity, and inhibited transport activity of BSEP, thus causing severe cholestasis. The patient had three homozygous, missense changes in the BSEP gene. Their combination resulted in the complete absence of BSEP, which explains the lack of tolerance, a prerequisite of autoantibody formation toward BSEP. The findings illustrate a novel disease mechanism due to a new class of functionally relevant autoantibodies resulting in cholestasis and subsequent liver failure. (HEPATOLOGY 2009;50:510–517.)

Multidrug efflux pumps: substrate selection in ATP-binding cassette multidrug efflux pumps--first come, first served?

Multidrug resistance is a major challenge in the therapy of cancer and pathogenic fungal infections. More than three decades ago, P‐glycoprotein was the first identified multidrug transporter. It has been studied extensively at the genetic and biochemical levels ever since. Pdr5, the most abundant ATP‐binding cassette transporter in Saccharomyces cerevisiae, is highly homologous to azole‐resistance‐mediating multidrug transporters in fungal pathogens, and a focus of clinical drug resistance research. Despite functional equivalences, P‐glycoprotein and Pdr5 exhibit striking differences in their architecture and mechanisms. In this minireview, we discuss the mechanisms of substrate selection and multidrug transport by comparing the fraternal twins P‐glycoprotein and Pdr5. We propose that substrate selection in eukaryotic multidrug ATP‐binding cassette transporters is not solely determined by structural features of the transmembrane domains but also by their dynamic behavior.