Ina L. Urbatsch

Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding.

P-glycoprotein (P-gp) detoxifies cells by exporting hundreds of chemically unrelated toxins but has been implicated in multidrug resistance (MDR) in the treatment of cancers. Substrate promiscuity is a hallmark of P-gp activity, thus a structural description of poly-specific drug-binding is important for the rational design of anticancer drugs and MDR inhibitors. The x-ray structure of apo P-gp at 3.8 angstroms reveals an internal cavity of ∼6000 angstroms cubed with a 30 angstrom separation of the two nucleotide-binding domains. Two additional P-gp structures with cyclic peptide inhibitors demonstrate distinct drug-binding sites in the internal cavity capable of stereoselectivity that is based on hydrophobic and aromatic interactions. Apo and drug-bound P-gp structures have portals open to the cytoplasm and the inner leaflet of the lipid bilayer for drug entry. The inward-facing conformation represents an initial stage of the transport cycle that is competent for drug binding.

THE CATALYTIC CYCLE OF P-GLYCOPROTEIN

P‐glycoprotein is a plasma‐membrane glycoprotein which confers multidrug‐resistance on cells and displays ATP‐driven drug‐pumping in vitro. It contains two nucleotide‐binding domains, and its structure places it in the ‘ABC transporter’ family. We review recent evidence that both nucleotide‐sites bind and hydrolyse Mg‐ATP. The two catalytic sites interact strongly. A minimal scheme for the MgATP hydrolysis reaction is presented. An alternating catalytic sites scheme is proposed, in which drug transport is coupled to relaxation of a high‐energy catalytic site conformation generated by the hydrolysis step. Other ABC transporters may show similar catalytic features.

Both P-glycoprotein Nucleotide-binding Sites Are Catalytically Active

The technique of vanadate trapping of nucleotide was used to study catalytic sites of P-glycoprotein (Pgp) in plasma membranes from multidrug-resistant Chinese hamster ovary cells. Vanadate trapping of Mg- or Co-8-azido-nucleotide (1 mol/mol of Pgp) caused complete inhibition of Pgp ATPase activity, with reactivation rates at 37°C of 1.4 × 10-3 s−1 (t1/2 = 8 min) or 3.3 × 10−4 s−1 (t1/2 = 35 min), respectively. UV irradiation of the inhibited Pgp yielded permanent inactivation of ATPase activity and specific photolabeling of Pgp. Mild trypsin digestion showed that the two nucleotide sites were labeled in equal proportion. The results show that both nucleotide sites in Pgp are capable of nucleotide hydrolysis, that vanadate trapping of nucleotide at either site completely prevents hydrolysis at both sites, and that vanadate trapping of nucleotide in the N- or C-terminal nucleotide sites occurs non-selectively. A minimal scheme is presented to explain inhibition by vanadate trapping of nucleotide and to describe the normal catalytic pathway. The inhibited Pgp·Mg-nucleotide·vanadate complex is probably an analog of the catalytic transition state, implying that when one nucleotide site assumes the catalytic transition state conformation the other site cannot do so and suggesting that the two sites may alternate in catalysis.

Structures of P-glycoprotein reveal its conformational flexibility and an epitope on the nucleotide-binding domain.

P-glycoprotein (P-gp) is one of the best-known mediators of drug efflux-based multidrug resistance in many cancers. This validated therapeutic target is a prototypic, plasma membrane resident ATP-Binding Cassette transporter that pumps xenobiotic compounds out of cells. The large, polyspecific drug-binding pocket of P-gp recognizes a variety of structurally unrelated compounds. The transport of these drugs across the membrane is coincident with changes in the size and shape of this pocket during the course of the transport cycle. Here, we present the crystal structures of three inward-facing conformations of mouse P-gp derived from two different crystal forms. One structure has a nanobody bound to the C-terminal side of the first nucleotide-binding domain. This nanobody strongly inhibits the ATP hydrolysis activity of mouse P-gp by hindering the formation of a dimeric complex between the ATP-binding domains, which is essential for nucleotide hydrolysis. Together, these inward-facing conformational snapshots of P-gp demonstrate a range of flexibility exhibited by this transporter, which is likely an essential feature for the binding and transport of large, diverse substrates. The nanobody-bound structure also reveals a unique epitope on P-gp.

Understanding polyspecificity of multidrug ABC transporters: closing in on the gaps in ABCB1

Multidrug ABC transporters can transport a wide range of drugs from the cell. Ongoing studies of the prototype mammalian multidrug resistance ATP-binding cassette transporter P-glycoprotein (ABCB1) have revealed many intriguing functional and biochemical features. However, a gap remains in our knowledge regarding the molecular basis of its broad specificity for structurally unrelated ligands. Recently, the first crystal structures of ligand-free and ligand-bound ABCB1 showed ligand binding in a cavity between its two membrane domains, and earlier observations on polyspecificity can now be interpreted in a structural context. Comparison of the new ABCB1 crystal structures with structures of bacterial homologs suggests a critical role for an axial rotation of transmembrane helices for high-affinity binding and low-affinity release of ligands during transmembrane transport.

Projection structure of P-glycoprotein by electron microscopy. Evidence for a closed conformation of the nucleotide binding domains.

The structure of P-glycoprotein (Pgp) from mouse has been studied by electron microscopy and image analysis. Two-dimensional crystals of Pgp in a lipid bilayer were generated by reconstituting pure, detergent-solubilized protein containing a C-terminal six-histidine tag using the lipid monolayer technique. The crystals belong to plane group P1 with a = b = 104 ± 2 Å and γ = 90 ± 4°. The projection structure of Pgp calculated at a resolution of 22 Å shows two closely interacting protein domains that can be interpreted as the N- and C-terminal halves of the protein. The projection structure of Pgp is consistent with the recently published x-ray structure of MsbA, a lipid A flippase from Escherichia coli with high sequence homology to Pgp but only when the two MsbA subunits are rotated to bring their nucleotide binding domains together.

Nucleotide-induced Structural Changes in P-glycoprotein Observed by Electron Microscopy

P-glycoprotein (Pgp) is an ATP hydrolysis driven multidrug efflux pump, which, when overexpressed in the plasma membrane of certain cancers, can lead to the failure of chemotherapy. Previously, we have presented a projection structure of nucleotide-free mouse Pgp from electron microscopic images of lipid monolayer-generated two-dimensional crystals ( Lee, J. Y., Urbatsch, I. L., Senior, A. E., and Wilkens, S. (2002) J. Biol. Chem. 277, 40125-40131 ). Here we have analyzed the structure of cysteine-free human Pgp from two-dimensional crystals that were generated with the same lipid-monolayer technique in the absence and presence of various nucleotides. The images show that human Pgp has a similar structure to the mouse protein. Furthermore, the analysis of projection structures obtained under different nucleotide conditions suggests that Pgp can exist in at least two major conformations, one of which shows a central cavity between the N- and C-terminal halves of the molecule and another in which the two halves have moved sideways, thereby closing the central cavity. Intermediate conformations were observed for some nucleotide/vanadate combinations. A low-resolution, three-dimensional model of human Pgp was calculated from tilted specimen crystallized in the presence of the non-hydrolyzable nucleotide analog, adenosine 5′-O-(thiotriphosphate). The structural analysis presented here adds to the emerging picture that multidrug ABC transporters function by switching between two major conformations in a nucleotide-dependent manner.

Snapshots of ligand entry, malleable binding and induced helical movement in P-glycoprotein

Co-crystal structures of P-glycoprotein with a series of engineered ligands reveal multiple ligand-binding modes, a ligand-binding site on the outer surface of the transporter and a conformational change that may couple to ATP hydrolysis.

A Gene Optimization Strategy that Enhances Production of Fully Functional P-Glycoprotein in Pichia pastoris

Background Structural and biochemical studies of mammalian membrane proteins remain hampered by inefficient production of pure protein. We explored codon optimization based on highly expressed Pichia pastoris genes to enhance co-translational folding and production of P-glycoprotein (Pgp), an ATP-dependent drug efflux pump involved in multidrug resistance of cancers. Methodology/Principal Findings Codon-optimized “Opti-Pgp” and wild-type Pgp, identical in primary protein sequence, were rigorously analyzed for differences in function or solution structure. Yeast expression levels and yield of purified protein from P. pastoris (∼130 mg per kg cells) were about three-fold higher for Opti-Pgp than for wild-type protein. Opti-Pgp conveyed full in vivo drug resistance against multiple anticancer and fungicidal drugs. ATP hydrolysis by purified Opti-Pgp was strongly stimulated ∼15-fold by verapamil and inhibited by cyclosporine A with binding constants of 4.2±2.2 µM and 1.1±0.26 µM, indistinguishable from wild-type Pgp. Maximum turnover number was 2.1±0.28 µmol/min/mg and was enhanced by 1.2-fold over wild-type Pgp, likely due to higher purity of Opti-Pgp preparations. Analysis of purified wild-type and Opti-Pgp by CD, DSC and limited proteolysis suggested similar secondary and ternary structure. Addition of lipid increased the thermal stability from Tm ∼40°C to 49°C, and the total unfolding enthalpy. The increase in folded state may account for the increase in drug-stimulated ATPase activity seen in presence of lipids. Conclusion The significantly higher yields of protein in the native folded state, higher purity and improved function establish the value of our gene optimization approach, and provide a basis to improve production of other membrane proteins.

Crystal structure of the human sterol transporter ABCG5/ABCG8.

ATP binding cassette (ABC) transporters play critical roles in maintaining sterol balance in higher eukaryotes. The ABCG5/ABCG8 heterodimer (G5G8) mediates excretion of neutral sterols in liver and intestines. Mutations disrupting G5G8 cause sitosterolaemia, a disorder characterized by sterol accumulation and premature atherosclerosis. Here we use crystallization in lipid bilayers to determine the X-ray structure of human G5G8 in a nucleotide-free state at 3.9 Å resolution, generating the first atomic model of an ABC sterol transporter. The structure reveals a new transmembrane fold that is present in a large and functionally diverse superfamily of ABC transporters. The transmembrane domains are coupled to the nucleotide-binding sites by networks of interactions that differ between the active and inactive ATPases, reflecting the catalytic asymmetry of the transporter. The G5G8 structure provides a mechanistic framework for understanding sterol transport and the disruptive effects of mutations causing sitosterolaemia.