Kenneth J. Linton

The ATP switch model for ABC transporters

ABC transporters mediate active translocation of a diverse range of molecules across all cell membranes. They comprise two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Recent biochemical, structural and genetic studies have led to the ATP-switch model in which ATP binding and ATP hydrolysis, respectively, induce formation and dissociation of an NBD dimer. This provides an exquisitely regulated switch that induces conformational changes in the TMDs to mediate membrane transport.

THE ESCHERICHIA COLI ATP-BINDING CASSETTE (ABC) PROTEINS

The recent completion of the Escherichia coli genome sequence (Blattner et al., 1997) has permitted an analysis of the complement of genomically encoded ATP‐binding cassette (ABC) proteins. A total of 79 ABC proteins makes this the largest paralogous family of proteins in E. coli. These 79 proteins include 97 ABC domains (as some proteins include more than one ABC domain) and are components of 69 independent functional systems (as many systems involve more than one ABC domain). The ABC domains are often, but not exclusively, the energy‐generating domains of multicomponent membrane‐bound transporters. Thus, 57 of the 69 systems are ABC transporters, of which 44 are periplasmic‐binding protein‐dependent uptake systems and 13 are presumed exporters. The genes encoding these ABC transporters occupy almost 5% of the genome. Of the 12 systems that are not obviously transport related, the function of only one, the excision repair protein UvrA, is known. A phylogenetic analysis suggests that the majority of ABC proteins can be assigned to 10 subfamilies. Together with statistical and, importantly, biological evidence, this analysis provides insight into the evolution and function of the ABC proteins.

Repacking of the transmembrane domains of P-glycoprotein during the transport ATPase cycle

P‐glycoprotein (P‐gp) is an ABC (ATP‐binding cassette) transporter, which hydrolyses ATP and extrudes cytotoxic drugs from mammalian cells. P‐gp consists of two transmembrane domains (TMDs) that span the membrane multiple times, and two cytoplasmic nucleotide‐binding domains (NBDs). We have determined projection structures of P‐gp trapped at different steps of the transport cycle and correlated these structures with function. In the absence of nucleotide, an ∼10 Å resolution structure was determined by electron cryo‐microscopy of two‐dimensional crystals. The TMDs form a chamber within the membrane that appears to be open to the extracellular milieu, and may also be accessible from the lipid phase at the interfaces between the two TMDs. Nucleotide binding causes a repacking of the TMDs and reduction in drug binding affinity. Thus, ATP binding, not hydrolysis, drives the major conformational change associated with solute translocation. A third distinct conformation of the protein was observed in the post‐hydrolytic transition state prior to release of ADP/Pi. Biochemical data suggest that these rearrangements may involve rotation of transmembrane α‐helices. A mechanism for transport is suggested.

Membrane phosphatidylserine distribution as a non-apoptotic signalling mechanism in lymphocytes

Phosphatidylserine (PS) exposure is normally associated with apoptosis and the removal of dying cells. We observed that PS is exposed constitutively at high levels on T lymphocytes that express low levels of the transmembrane tyrosine phosphatase CD45RB. CD45 was shown to be a negative regulator of PS translocation in response to various signals, including activation of the ATP receptor P2X7. Changes in PS distribution were shown to modulate several membrane activities: Ca2+ and Na+ uptake through the P2X7 cation channel itself; P2X7-stimulated shedding of the homing receptor CD62L; and reversal of activity of the multidrug transporter P-glycoprotein. The data identify a role for PS distribution changes in signal transduction, rapidly modulating the activities of several membrane proteins. This seems to be an all-or-none effect, coordinating the activity of most or all the molecules of a target protein in each cell. The data also suggest a new approach to circumventing multidrug resistance.

Contribution of Variant Alleles of ABCB11 to Susceptibility to Intrahepatic Cholestasis of Pregnancy.

Background: Intrahepatic cholestasis of pregnancy (ICP) has a complex aetiology with a significant genetic component. ABCB11 encodes the bile salt export pump (BSEP); mutations cause a spectrum of cholestatic disease, and are implicated in the aetiology of ICP. Methods: ABCB11 variation in ICP was investigated by screening for five mutant alleles (E297G, D482G, N591S, D676Y and G855R) and the V444A polymorphism (c.1331T>C, rs2287622) in two ICP cohorts (n = 333 UK, n = 158 continental Europe), and controls (n = 261) for V444A. PCR primers were used to amplify and sequence patient and control DNA. The molecular basis for the observed phenotypes was investigated in silico by analysing the equivalent residues in the structure of the homologous bacterial transporter Sav1866. Results: E297G was observed four times and D482G once. N591S was present in two patients; D676Y and G855R were not observed. The V444A polymorphism was associated with ICP (allelic analysis for C vs T: OR 1.7 (95% CI 1.4 to 2.1, p<0.001)). In addition, CC homozygotes were more likely to have ICP than TT homozygotes: OR 2.8 (95% CI 1.7 to 4.4 p<0.0001). Structural analyses suggest that E297G and D482G destabilise the protein fold of BSEP. The molecular basis of V444A and N591S was not apparent from the Sav1866 structure. Conclusions: Heterozygosity for the common ABCB11 mutations accounts for 1% of European ICP cases; these two mutants probably reduce the folding efficiency of BSEP. N591S is a recurrent mutation; however, the mechanism may be independent of protein stability or function. The V444A polymorphism is a significant risk factor for ICP in this population.

Missense mutations and single nucleotide polymorphisms in ABCB11 impair bile salt export pump processing and function or disrupt pre‐messenger RNA splicing

The gene encoding the human bile salt export pump (BSEP), ABCB11, is mutated in several forms of intrahepatic cholestasis. Here we classified the majority (63) of known ABCB11 missense mutations and 21 single‐nucleotide polymorphisms (SNPs) to determine whether they caused abnormal ABCB11 pre‐messenger RNA splicing, abnormal processing of BSEP protein, or alterations in BSEP protein function. Using an in vitro minigene system to analyze splicing events, we found reduced wild‐type splicing for 20 mutations/SNPs, with normal mRNA levels reduced to 5% or less in eight cases. The common ABCB11 missense mutation encoding D482G enhanced aberrant splicing, whereas the common SNP A1028A promoted exon skipping. Addition of exogenous splicing factors modulated several splicing defects. Of the mutants expressed in vitro in CHO‐K1 cells, most appeared to be retained in the endoplasmic reticulum and degraded. A minority had BSEP levels similar to wild‐type. The SNP variant A444 had reduced levels of protein compared with V444. Treatment with glycerol and incubation at reduced temperature overcame processing defects for several mutants, including E297G. Taurocholate transport by two assessed mutants, N490D and A570T, was reduced compared with wild‐type. Conclusion: This work is a comprehensive analysis of 80% of ABCB11 missense mutations and single‐nucleotide polymorphisms at pre‐mRNA splicing and protein processing/functional levels. We show that aberrant pre‐mRNA splicing occurs in a considerable number of cases, leading to reduced levels of normal mRNA. Thus, primary defects at either the protein or the mRNA level (or both) contribute significantly to BSEP deficiency. These results will help to develop mutation‐specific therapies for children and adults suffering from intrahepatic cholestasis due to BSEP deficiency. (HEPATOLOGY 2008.)

Evidence for a Sav1866-like architecture for the human multidrug transporter P-glycoprotein

The recently reported structures of the bacterial multidrug exporter Sav1866 suggest a domain architecture in which both nucleotide‐binding domains (NBDs) of this ATP binding cassette (ABC) transporter contact both transmembrane domains (TMDs). Such a domain arrangement is particularly unexpected because it is not found in the structures of three solute importers BtuCD, HI1470/1, and ModBC from the same protein family. There is also no precedent for such an arrangement from biochemical studies with any ABC transporter. Sav1866 is homologous with the clinically relevant human P‐glycoprotein (ABCB1). If the structure proposed for Sav1866 is physiologically relevant, the long intracellular loops of P‐glycoprotein TMD2 should contact NBD1. We have tested this by using cysteine mutagenesis and chemical cross‐linking to verify proximal relationships of the introduced sulfhydryls across the proposed interdomain interface. We report the first biochemical evidence in support of the domain arrangement proposed for the multidrug resistance class of ABC transporters. With a domain arrangement distinctly different from the three solute importers it seems likely that the TMDs of ABC importers and exporters have evolved different mechanisms to couple to common conformational changes at conserved NBDs.— Zolnerciks, J. K., Wooding, C., Linton, K. J. Evidence for a Sav1866‐like architecture for the human multidrug transporter P‐glycoprotein. FASEB J. 21, 3937–3948 (2007)

An atomic detail model for the human ATP binding cassette transporter P-glycoprotein derived from disulfide cross-linking and homology modeling

The multidrug resistance P‐glycoprotein mediates the extrusion of chemotherapeutic drugs from cancer cells. Characterization of the drug binding and ATPase activities of the protein have made it the paradigm ATP binding cassette (ABC) transporter. P‐glycoprotein has been imaged at low resolution by electron cryo‐microscopy and extensively analyzed by disulphide cross‐linking, but a high resolution structure solved ab initio remains elusive. Homology models of P‐glycoprotein were generated using the structure of a related prokaryotic ABC transporter, the lipid A transporter MsbA, as a template together with structural data describing the dimer interface of the nucleotide binding domains (NBDs). The first model, which maintained the NBD:transmembrane domain (TMD) interface of MsbA, did not satisfy previously published cross‐linking data. This suggests that either P‐glycoprotein has a very different structure from MsbA or that the published E. coli MsbA structure does not reflect a physiological state. To distinguish these alternatives, we mapped the interface between the two TMDs of P‐glycoprotein experimentally by chemical cross‐linking of introduced triple‐cysteine residues. Based on these data, a plausible atomic model of P‐glycoprotein could be generated using the MsbA template, if the TMDs of MsbA are reoriented with respect to the NBDs. This model will be important for understanding the mechanism of P‐glycoprotein and other ABC transporters.

P-glycoprotein in blood-brain barrier endothelial cells: Interaction and oligomerization with caveolins

P‐glycoprotein (P‐gp), an adenosine triphosphate (ATP)‐binding cassette transporter which acts as a drug efflux pump, is highly expressed at the blood–brain barrier (BBB) where it plays an important role in brain protection. Recently, P‐gp has been reported to be located in the caveolae of multidrug‐resistant cells. In this study, we investigated the localization and the activity of P‐gp in the caveolae of endothelial cells of the BBB. We used an in vitro model of the BBB which is formed by co‐culture of bovine brain capillary endothelial cells (BBCEC) with astrocytes. Caveolar microdomains isolated from BBCEC are enriched in P‐gp, cholesterol, caveolin‐1, and caveolin‐2. Moreover, P‐gp interacts with caveolin‐1 and caveolin‐2; together, they form a high molecular mass complex. P‐gp in isolated caveolae is able to bind its substrates, and the caveolae‐disrupting agents filipin III and nystatin decrease P‐gp transport activity. In addition, mutations in the caveolin‐binding motif present in P‐gp reduced the interaction of P‐gp with caveolin‐1 and increased the transport activity of P‐gp. Thus, P‐gp expressed at the BBB is mainly localized in caveolae and its activity may be modulated by interaction with caveolin‐1.

ABCB4 gene sequence variation in women with intrahepatic cholestasis of pregnancy

Intrahepatic cholestasis of pregnancy (ICP), also known as obstetric cholestasis, is a liver disease of pregnancy that complicates 0.7% of pregnancies in the UK.1,2 ICP causes maternal pruritus and hepatic impairment and can cause fetal death, spontaneous prematurity, and sudden intrauterine death.3–6 A diagnosis of ICP is made by the demonstration of abnormal liver function test results, and in particular the serum bile acids are raised.7–10 This is thought to be a consequence of abnormal bile transport across the hepatocyte canalicular membrane. Clinical features are heterogeneous and the aetiology is likely to be complex. Insights to the genetic aetiology of ICP have come from studies of the childhood liver disease progressive familial intrahepatic cholestasis (PFIC), a condition which is divided into three subtypes. Children with PFIC1 and 2 have low concentrations of biliary bile acids and low to normal gamma-glutamyl transpeptidase (GGT) in the serum. PFIC3 patients have high serum levels of GGT and bile which lacks phospholipid but has a normal biliary bile acid concentration.11 Homozygous mutations of the ABCB4 (also called MDR3 or mdr2 in the mouse) gene have been described in pedigrees with PFIC3.11–13 The ABCB4 protein is a member of the ATP binding cassette (ABC) family of membrane transporters.14–16 One of the normal functions of ABCB4 is to transport phosphatidylcholine across the hepatocyte canalicular membrane. The fact that expression is not only found in hepatocytes but also in B lymphocytes, heart, and muscle suggests that it may also transport other substrates. However, homozygous knockouts of the homologous (>90% identity at the amino acid level) murine mdr2 only had hepatic effects.17 Several heterozygous mothers of children with PFIC3 have symptoms consistent with ICP.11–13 In a large consanguineous pedigree with coexisting PFIC3 and ICP, …