G. Berridge

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.

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=275 pmol mg−1, Kd=5.1 nM). 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±50 nM). 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.

Structures of ABCB10, a human ATP-binding cassette transporter in apo- and nucleotide-bound states

ABCB10 is one of the three ATP-binding cassette (ABC) transporters found in the inner membrane of mitochondria. In mammals ABCB10 is essential for erythropoiesis, and for protection of mitochondria against oxidative stress. ABCB10 is therefore a potential therapeutic target for diseases in which increased mitochondrial reactive oxygen species production and oxidative stress play a major role. The crystal structure of apo-ABCB10 shows a classic exporter fold ABC transporter structure, in an open-inwards conformation, ready to bind the substrate or nucleotide from the inner mitochondrial matrix or membrane. Unexpectedly, however, ABCB10 adopts an open-inwards conformation when complexed with nonhydrolysable ATP analogs, in contrast to other transporter structures which adopt an open-outwards conformation in complex with ATP. The three complexes of ABCB10/ATP analogs reported here showed varying degrees of opening of the transport substrate binding site, indicating that in this conformation there is some flexibility between the two halves of the protein. These structures suggest that the observed plasticity, together with a portal between two helices in the transmembrane region of ABCB10, assist transport substrate entry into the substrate binding cavity. These structures indicate that ABC transporters may exist in an open-inwards conformation when nucleotide is bound. We discuss ways in which this observation can be aligned with the current views on mechanisms of ABC transporters.

Dissection of PIM serine/threonine kinases in FLT3-ITD-induced leukemogenesis reveals PIM1 as regulator of CXCL12-CXCR4-mediated homing and migration.

FLT3-ITD–mediated leukemogenesis is associated with increased expression of oncogenic PIM serine/threonine kinases. To dissect their role in FLT3-ITD–mediated transformation, we performed bone marrow reconstitution assays. Unexpectedly, FLT3-ITD cells deficient for PIM1 failed to reconstitute lethally irradiated recipients, whereas lack of PIM2 induction did not interfere with FLT3-ITD–induced disease. PIM1-deficient bone marrow showed defects in homing and migration and displayed decreased surface CXCR4 expression and impaired CXCL12–CXCR4 signaling. Through small interfering RNA–mediated knockdown, chemical inhibition, expression of a dominant-negative mutant, and/or reexpression in knockout cells, we found PIM1 activity to be essential for proper CXCR4 surface expression and migration of cells toward a CXCL12 gradient. Purified PIM1 led to the phosphorylation of serine 339 in the CXCR4 intracellular domain in vitro, a site known to be essential for normal receptor recycling. In primary leukemic blasts, high levels of surface CXCR4 were associated with increased PIM1 expression, and this could be significantly reduced by a small molecule PIM inhibitor in some patients. Our data suggest that PIM1 activity is important for homing and migration of hematopoietic cells through modification of CXCR4. Because CXCR4 also regulates homing and maintenance of cancer stem cells, PIM1 inhibitors may exert their antitumor effects in part by interfering with interactions with the microenvironment.

THE FUNCTIONAL PURIFICATION OF P-GLYCOPROTEIN IS DEPENDENT ON MAINTENANCE OF A LIPID-PROTEIN INTERFACE

P-Glycoprotein (P-gp) is a 180-kDa membrane-bound transporter which can confer the multi-drug resistance phenotype on tumor cells. We have examined the factors required to preserve activity of P-gp during its purification. The starting material for purification was plasma membranes from Chinese hamster ovary (CHrB30) cells, overexpressing P-glycoprotein. These membranes displayed drug stimulated ATPase activity (Vm = 897 +/- 55 nmol min(-1) mg(-1); Km = 1.8 +/- 0.4 mM) and high affinity binding of [3H]vinblastine (Kd = 36 +/- 5 nM; Bm = 161 +/- 11 pmol/mg). Several non-ionic detergents which readily solubilized P-glycoprotein significantly inhibited ATPase activity and drug binding at concentrations well below their respective CMC values. This inactivation was prevented by excess crude lipid mixtures, with the greatest protection afforded against dodecyl-maltoside. Furthermore, the significantly reduced binding affinity and capacity of solubilized P-gp was partly reversed by the addition of lipids. A combination of anion-exchange and hydroxyapatite chromatography were used to purify P-gp with high yield to greater than 90%. The purified, reconstituted P-gp displayed high ATPase activity (Vm = 2137 +/- 309; Km = 2.9 +/- 0.9 mM) which was stimulated by verapamil (EC50 = 3.8 +/- 0.6 microM) and inhibited by orthovanadate (3.1 +/- 0.8 microM). Pure P-gp also displayed high affinity vinblastine binding (Kd = 64 +/- 9 nM) with a capacity of 2320 +/- 192 pmol/mg. This purification scheme yields the highest P-gp activity reported to date, and indicates a dependence of function on maintaining a lipid-protein interface.

The multi-drug resistance reversal agent SR33557 and modulation of vinca alkaloid binding to P-glycoprotein by an allosteric interaction.

1 The interaction of the indolizin sulfone SR33557 with the multidrug resistance P‐glycoprotein (P‐gp), was used to explore the nature of drug binding site(s) on this transporter. The steady‐state accumulation of [3H]‐vinblastine in P‐gp expressing CHrB30 cells was increased by SR33557 with greater potency than verapamil. Furthermore, SR33557 potentiated the affinity of verapamil to modulate vinblastine transport when added simultaneously. 2 Verapamil elicited a 1.5 to 2.5 fold stimulation of basal ATPase activity in CHrB30 membranes, whereas SR33557 and vinblastine inhibited activity, but only at relatively high concentrations. However, SR33557 and vinblastine decreased the Vmax but not the Km for verapamil stimulation of ATPase activity. This is indicative of a non‐competitive interaction, most likely at distinct sites. 3 The specific [3H]‐vinblastine binding to P‐gp in CHrB30 cell membranes was displaced by SR33557 with an IC50 of 8.3±4.5 nM. Moreover, SR33557 caused a 3 fold increase in the dissociation rate of vinblastine binding to P‐gp indicating a negative allosteric effect on the vinca alkaloid acceptor site. 4 These results demonstrate that SR33557 interacts with a site on P‐gp which is distinct from, but allosterically linked to the vinca alkaloid site. The apparent broad substrate specificity displayed by P‐gp may be explained by a multiple drug binding site model.

Dissection of PIM serine/threonine kinases in FLT3-ITD–induced leukemogenesis reveals PIM1 as regulator of CXCL12–CXCR4-mediated homing and migration

FLT3-ITD-mediated leukemogenesis is associated with increased expression of oncogenic PIM serine/threonine kinases. To dissect their role in FLT3-ITD-mediated transformation, we performed bone marrow reconstitution assays. Unexpectedly, FLT3-ITD cells deficient for PIM1 failed to reconstitute lethally irradiated recipients, whereas lack of PIM2 induction did not interfere with FLT3-ITD-induced disease. PIM1-deficient bone marrow showed defects in homing and migration and displayed decreased surface CXCR4 expression and impaired CXCL12-CXCR4 signaling. Through small interfering RNA-mediated knockdown, chemical inhibition, expression of a dominant-negative mutant, and/or reexpression in knockout cells, we found PIM1 activity to be essential for proper CXCR4 surface expression and migration of cells toward a CXCL12 gradient. Purified PIM1 led to the phosphorylation of serine 339 in the CXCR4 intracellular domain in vitro, a site known to be essential for normal receptor recycling. In primary leukemic blasts, high levels of surface CXCR4 were associated with increased PIM1 expression, and this could be significantly reduced by a small molecule PIM inhibitor in some patients. Our data suggest that PIM1 activity is important for homing and migration of hematopoietic cells through modification of CXCR4. Because CXCR4 also regulates homing and maintenance of cancer stem cells, PIM1 inhibitors may exert their antitumor effects in part by interfering with interactions with the microenvironment.

High throughput production of recombinant human proteins for crystallography.

This chapter presents in detail the process used in high throughput bacterial production of recombinant human proteins for crystal structure determination. The core principles are: (1) Generating at least 10 truncated constructs from each target gene. (2) Ligation-independent cloning (LIC) into a bacterial expression vector. All proteins are expressed with an N-terminal, TEV protease cleavable fusion peptide. (3) Small-scale test expression to identify constructs producing soluble protein. (4) Liter-scale production in shaker flasks. (5) Purification by Ni-affinity chromatography and gel filtration. (6) Protein characterization and preparation for crystallography. The chapter also briefly presents alternative procedures, to be applied based on specific knowledge of protein families or when the core protocol is unsatisfactory. This scheme has been applied to more than 550 human proteins (>10,000 constructs) and has resulted in the deposition of 112 unique structures. The methods presented do not depend on specialized equipment or robotics; hence, they provide an effective approach for handling individual proteins in a regular research lab.

The Structural Basis of ZMPSTE24-Dependent Laminopathies

Lamin Loppers The nuclear lamina provides mechanical stability to the nuclear envelope and is involved in regulation of cellular processes such as DNA replication. Defects in the nuclear lamina lead to diseases such as progeria and metabolic disorders. One of the components of the nuclear lamina, lamin A, undergoes a complex maturation process. A key player is an inner nuclear membrane zinc metalloprotease (ZMP) that is responsible for two proteolysis steps (see the Perspective by Michaelis and Hrycyna). Quigley et al. (p. 1604) report the crystal structure of human ZMPSTE24 and Pryor et al. (p. 1600) that of the yeast homolog Ste24p. The structures provide insight into the mechanism of catalysis and into why mutations in ZMPSTE24 lead to laminopathies. Structures of two transmembrane zinc proteases reveal a barrel of seven helices surrounding a large cavity. [Also see Perspective by Michaelis and Hrycyna] Mutations in the nuclear membrane zinc metalloprotease ZMPSTE24 lead to diseases of lamin processing (laminopathies), such as the premature aging disease progeria and metabolic disorders. ZMPSTE24 processes prelamin A, a component of the nuclear lamina intermediate filaments, by cleaving it at two sites. Failure of this processing results in accumulation of farnesylated, membrane-associated prelamin A. The 3.4 angstrom crystal structure of human ZMPSTE24 has a seven transmembrane α-helical barrel structure, surrounding a large, water-filled, intramembrane chamber, capped by a zinc metalloprotease domain with the catalytic site facing into the chamber. The 3.8 angstrom structure of a complex with a CSIM tetrapeptide showed that the mode of binding of the substrate resembles that of an insect metalloprotease inhibitor in thermolysin. Laminopathy-associated mutations predicted to reduce ZMPSTE24 activity map to the zinc metalloprotease peptide–binding site and to the bottom of the chamber.

The PHD and chromo domains regulate the ATPase activity of the human chromatin remodeler CHD4.

The NuRD (nucleosome remodeling and deacetylase) complex serves as a crucial epigenetic regulator of cell differentiation, proliferation, and hematopoietic development by coupling the deacetylation and demethylation of histones, nucleosome mobilization, and the recruitment of transcription factors. The core nucleosome remodeling function of the mammalian NuRD complex is executed by the helicase-domain-containing ATPase CHD4 (Mi-2β) subunit, which also contains N-terminal plant homeodomain (PHD) and chromo domains. The mode of regulation of chromatin remodeling by CHD4 is not well understood, nor is the role of its PHD and chromo domains. Here, we use small-angle X-ray scattering, nucleosome binding ATPase and remodeling assays, limited proteolysis, cross-linking, and tandem mass spectrometry to propose a three-dimensional structural model describing the overall shape and domain interactions of CHD4 and discuss the relevance of these for regulating the remodeling of chromatin by the NuRD complex.