Christopher F. Higgins

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.

Histone-like protein H1 (H-NS), DNA supercoiling, and gene expression in bacteria

Changes in DNA supercoiling in response to environmental signals such as osmolarity, temperature, or anaerobicity appear to play an underlying role in the regulation of gene expression in bacteria. Extensive genetic analyses have implicated the osmZ gene in this regulatory process: osmZ mutations are highly pleiotropic and alter the topology of cellular DNA. We have shown that the product of the osmZ gene is the histone-like protein H1 (H-NS). Protein H1 is one of the most abundant components of bacterial chromatin and binds to DNA in a relatively nonspecific fashion. These data imply a regulatory role for one of the major components of bacterial chromatin and provide support for the notion that changes in DNA topology and/or chromatin structure play a role in regulating gene expression.

The homodimeric ATP‐binding cassette transporter LmrA mediates multidrug transport by an alternating two‐site (two‐cylinder engine) mechanism

The bacterial LmrA protein and the mammalian multidrug resistance P‐glycoprotein are closely related ATP‐binding cassette (ABC) transporters that confer multidrug resistance on cells by mediating the extrusion of drugs at the expense of ATP hydrolysis. The mechanisms by which transport is mediated, and by which ATP hydrolysis is coupled to drug transport, are not known. Based on equilibrium binding experiments, photoaffinity labeling and drug transport assays, we conclude that homodimeric LmrA mediates drug transport by an alternating two‐site transport (two‐cylinder engine) mechanism. The transporter possesses two drug‐binding sites: a transport‐competent site on the inner membrane surface and a drug‐release site on the outer membrane surface. The interconversion of these two sites, driven by the hydrolysis of ATP, occurs via a catalytic transition state intermediate in which the drug transport site is occluded. The mechanism proposed for LmrA may also be relevant for P‐glycoprotein and other ABC transporters.

The molecular interaction of the high affinity reversal agent XR9576 with P‐glycoprotein

The kinetics and nature of equilibrium binding were used to characterize the molecular interaction of the anthranilic acid derivative [3H]‐XR9576 with the multidrug resistance P‐glycoprotein (P‐gp). XR9576 displayed specific high‐affinity binding to P‐gp (Bmax=275u2003pmolu2003mg−1, Kd=5.1u2003nM). The transport substrates [3H]‐vinblastine and [3H]‐paclitaxel displayed 4 fold and 20 fold lower affinity respectively for P‐gp. The duration of action of XR9576 with P‐gp was increased in comparison to that of vinblastine which displayed a slower rate of association and a faster dissociation rate. The relative affinities of several modulators and transport substrates to interact with P‐gp were determined from displacement drug equilibrium binding assays. Vinblastine and paclitaxel could only fractionally displace [3H]‐XR9576 binding, displaying Ki values significantly different from their measured Kd values. This suggests a non‐competitive interaction between XR9576 and the P‐gp substrates vinblastine and paclitaxel. XR9576 was shown to be a potent modulator of P‐gp mediated [3H]‐vinblastine and [3H]‐paclitaxel transport as it increased the steady‐state accumulation of these cytotoxics in CHrB30 cells to levels observed in non‐P‐gp‐expressing AuxB1 cells (EC50=487±50u2003nM). This inhibition of drug transport is not mediated through competition for transport since [3H]‐XR9576 accumulation was not influenced by P‐gp expression or function. These results demonstrate that the P‐gp modulator XR9576 exhibits greater selectivity, duration of inhibition and potency of interaction with this transporter than any other reported modulators. Several lines of evidence suggest that XR9576 inhibits P‐gp function by binding at a site which is distinct from the site of interaction of transport substrates. The two sites may be classified as serving modulatory or transport functions.

H-NS Oligomerization Domain Structure Reveals the Mechanism for High Order Self-association of the Intact Protein

H-NS plays a role in condensing DNA in the bacterial nucleoid. This 136 amino acid protein comprises two functional domains separated by a flexible linker. High order structures formed by the N-terminal oligomerization domain (residues 1-89) constitute the basis of a protein scaffold that binds DNA via the C-terminal domain. Deletion of residues 57-89 or 64-89 of the oligomerization domain precludes high order structure formation, yielding a discrete dimer. This dimerization event represents the initial event in the formation of high order structure. The dimers thus constitute the basic building block of the protein scaffold. The three-dimensional solution structure of one of these units (residues 1-57) has been determined. Activity of these structural units is demonstrated by a dominant negative effect on high order structure formation on addition to the full length protein. Truncated and site-directed mutant forms of the N-terminal domain of H-NS reveal how the dimeric unit self-associates in a head-to-tail manner and demonstrate the importance of secondary structure in this interaction to form high order structures. A model is presented for the structural basis for DNA packaging in bacterial cells.

The importance of cholesterol in maintenance of P-glycoprotein activity and its membrane perturbing influence

Abstract. In tumour cell lines that display multidrug resistance, expression of P-glycoprotein (P-gp) alters many aspects of biomembrane organization in addition to its well-characterized drug transport activity. We have developed a reconstitution system to directly investigate the effect of purified P-gp on the biophysical properties of lipid bilayers. Using a mixed detergent system it was possible to efficiently reconstitute P-gp at lipid:protein ratios as low as 2.5 (w/w) by removal of detergent using adsorption to SM-2 BioBeads. P-gp was able to alter many biophysical parameters associated with lipid organization within bilayers. For example, the changes in overall fluidity and excimer formation by lipid analogues indicate modified packing organization of bilayer constituents. Surprisingly, given its role in conferring drug resistance, P-gp insertion into bilayers also caused significantly increased permeability to aqueous compounds, also reflecting a modified phospholipid environment. Translocation of various phospholipid species between leaflets of the bilayer was increased in the presence of P-gp; however, the effect was not dependent on ATP hydrolysis by the protein. Physiological concentrations of cholesterol modified P-gp function and the degree to which it perturbed bilayer organization. The basal ATPase activity of P-gp was increased in a dose-dependent fashion by the incorporation of cholesterol in PC:PE liposomes. In addition, the degree to which the modulator verapamil was able to stimulate this basal ATPase activity was reduced by the presence of cholesterol in proteoliposomes. However, the potency of verapamil was unaltered, suggesting a specific effect, not simply caused by lower drug penetration into the cholesterol containing bilayers. In summary, P-gp is able to cause perturbation in the organization of bilayer constituents. Cholesterol imparted stability to this perturbation of bilayer organization by P-gp and moreover this led to altered protein function.

Oligomerization of the chromatin-structuring protein H-NS

H‐NS is a major component of the bacterial nucleoid, involved in condensing and packaging DNA and modulating gene expression. The mechanism by which this is achieved remains unclear. Genetic data show that the biological properties of H‐NS are influenced by its oligomerization properties. We have applied a variety of biophysical techniques to study the structural basis of oligomerization of the H‐NS protein from Salmonella typhimurium. The N‐terminal 89 amino acids are responsible for oligomerization. The first 64 residues form a trimer dominated by an α‐helix, likely to be in coiled–coil conformation. Extending this polypeptide to 89 amino acids generated higher order, heterodisperse oligomers. Similarly, in the full‐length protein no single, defined oligomeric state is adopted. The C‐terminal 48 residues do not participate in oligomerization and form a monomeric, DNA‐binding domain. These N‐ and C‐terminal domains are joined via a flexible linker which enables them to function independently within the context of the full‐length protein. This novel mode of oligomerization may account for the unusual binding properties of H‐NS.

The nucleoid-associated protein StpA binds curved DNA, has a greater DNA-binding affinity than H-NS and is present in significant levels in hns mutants

The StpA protein is closely related to H-NS, the well-characterised global regulator of gene expression which is a major component of eubacterial chromatin. Despite sharing a very high degree of sequence identify and having biochemical properties in common with H-NS, the physiological function of StpA remains unknown. We show that StpA exhibits similar DNA-binding activities to H-NS. Although both display a strong preference for binding to curved DNA, StpA binds DNA with a four-fold higher affinity than H-NS, with K(d)s of 0.7 microM and 2.8 microM, respectively. It has previously been reported that expression of stpA is derepressed in an hns mutant. We have quantified the amount of StpA protein produced under this condition and find it to be only one-tenth the level of H-NS protein in wild-type cells. Our findings explain why the presence of StpA does not compensate for the lack of H-NS in an hns mutant, and why the characteristic pleiotropic hns mutant phenotype is observed.

Detailed characterization of cysteine‐less P‐glycoprotein reveals subtle pharmacological differences in function from wild‐type protein

Subtle alterations in the coupling of drug binding to nucleotide hydrolysis were observed following mutation of all seven endogenous cysteine residues to serines in the human multidrug resistance transporter, P‐glycoprotein. Wild‐type (wt) and the mutant (cys‐less) forms of P‐gp were expressed in Trichoplusia ni (High Five) cells and purified by metal affinity chromatography in order to undertake functional studies. No significant differences were observed in substrate ([3H]‐azidopine) binding to wt or cys‐less P‐gp. Furthermore, neither the transported substrate vinblastine, nor the modulator nicardipine, differed in their respective potencies to displace [3H]‐azidopine from the wt or cys‐less P‐gp. These results suggest that respective binding sites for these drugs were unaffected by the introduced cysteine to serine substitutions. The Michaelis‐Menten characteristics of basal ATP hydrolysis of the two isoforms of P‐gp were identical. The maximal ATPase activity in the presence of vinblastine was marginally reduced whilst the Km was unchanged in cys‐less P‐gp compared to control. However, cys‐less P‐gp displayed lower overall maximal ATPase activity (62%), a decreased Km and a lower degree of stimulation (76%) in the presence of the modulator nicardipine. Therefore, the serine to cysteine mutations in P‐gp may suggest that vinblastine and nicardipine transduce their effects on ATP hydrolysis through distinct conformational pathways. The wt and cys‐less P‐gp isoforms display similarity in their fundamental kinetic properties thereby validating the use of cys‐less P‐gp as a template for future cysteine‐directed structure/function analysis.

Stability and degradation of mRNA.

Differential mRNA stability plays an important role in the regulation of gene expression. Several recent advances have helped to define the general pathways by which mRNA is degraded in prokaryotic cells, although many details remain to be elucidated. Much less is known about the pathways of degradation in eukaryotic cells, but recent studies on specific systems have highlighted both differences from and similarities to prokaryotic pathways.