Stephan Wilkens

Structure and mechanism of ABC transporters

All living organisms depend on primary and secondary membrane transport for the supply of external nutrients and removal or sequestration of unwanted (toxic) compounds. Due to the chemical diversity of cellular molecules, it comes as no surprise that a significant part of the proteome is dedicated to the active transport of cargo across the plasma membrane or the membranes of subcellular organelles. Transport against a chemical gradient can be driven by, for example, the free energy change associated with ATP hydrolysis (primary transport), or facilitated by the potential energy of the chemical gradient of another molecule (secondary transport). Primary transporters include the rotary motor ATPases (F-, A-, and V-ATPases), P-type ATPases and a large family of integral membrane proteins referred to as “ABC” (ATP binding cassette) transporters. ABC transporters are widespread in all forms of life and are characterized by two nucleotide-binding domains (NBD) and two transmembrane domains (TMDs). ATP hydrolysis on the NBD drives conformational changes in the TMD, resulting in alternating access from inside and outside of the cell for unidirectional transport across the lipid bilayer. Common to all ABC transporters is a signature sequence or motif, LSGGQ, that is involved in nucleotide binding. Both importing and exporting ABC transporters are found in bacteria, whereas the majority of eukaryotic family members function in the direction of export. Recent progress with the X-ray crystal structure determination of a variety of bacterial and eukaryotic ABC transporters has helped to advance our understanding of the ATP hydrolysis-driven transport mechanism but has also illustrated the large structural and functional diversity within the family.

Structure of the Yeast Vacuolar ATPase

The subunit architecture of the yeast vacuolar ATPase (V-ATPase) was analyzed by single particle transmission electron microscopy and electrospray ionization (ESI) tandem mass spectrometry. A three-dimensional model of the intact V-ATPase was calculated from two-dimensional projections of the complex at a resolution of 25 Å. Images of yeast V-ATPase decorated with monoclonal antibodies against subunits A, E, and G position subunit A within the pseudo-hexagonal arrangement in the V1, the N terminus of subunit G in the V1-V0 interface, and the C terminus of subunit E at the top of the V1 domain. ESI tandem mass spectrometry of yeast V1-ATPase showed that subunits E and G are most easily lost in collision-induced dissociation, consistent with a peripheral location of the subunits. An atomic model of the yeast V-ATPase was generated by fitting of the available x-ray crystal structures into the electron microscopy-derived electron density map. The resulting atomic model of the yeast vacuolar ATPase serves as a framework to help understand the role the peripheral stalk subunits are playing in the regulation of the ATP hydrolysis driven proton pumping activity of the vacuolar ATPase.

Structural analysis of membrane-bound retrovirus capsid proteins

We have developed a system for analysis of histidine‐tagged (His‐tagged) retrovirus core (Gag) proteins, assembled in vitro on lipid monolayers consisting of egg phosphatidylcholine (PC) plus the novel lipid DHGN. DHGN was shown to chelate nickel by atomic absorption spectrometry, and DHGN‐containing monolayers specifically bound gold conjugates of His‐tagged proteins. Using PC+DHGN monolayers, we examined membrane‐bound arrays of an N‐terminal His‐tagged Moloney murine leukemia virus (M‐MuLV) capsid (CA) protein, His‐MoCA, and in vivo studies suggest that in vitro‐derived His‐MoCA arrays reflect some of the Gag protein interactions which occur in assembling virus particles. The His‐MoCA proteins formed extensive two‐dimensional (2D) protein crystals, with reflections out to 9.5 Å resolution. The image‐analyzed 2D projection of His‐MoCA arrays revealed a distinct cage‐like network. The asymmetry of the individual building blocks of the network led to the formation of two types of hexamer rings, surrounding protein‐free cage holes. These results predict that Gag hexamers constitute a retrovirus core substructure, and that cage hole sizes define an exclusion limit for entry of retrovirus envelope proteins, or other plasma membrane proteins, into virus particles. We believe that the 2D crystallization method will permit the detailed analysis of retroviral Gag proteins and other His‐tagged proteins.

Stoichiometry of the Peripheral Stalk Subunits E and G of Yeast V1-ATPase Determined by Mass Spectrometry

The stoichiometry of yeast V1-ATPase peripheral stalk subunits E and G was determined by two independent approaches using mass spectrometry (MS). First, the subunit ratio was inferred from measuring the molecular mass of the intact V1-ATPase complex and each of the individual protein components, using native electrospray ionization-MS. The major observed intact complex had a mass of 593,600 Da, with minor components displaying masses of 553,550 and 428,300 Da, respectively. Second, defined amounts of V1-ATPase purified from yeast grown on 14N-containing medium were titrated with defined amounts of 15N-labeled E and G subunits as internal standards. Following protease digestion of subunit bands, 14N- and 15N-containing peptide pairs were used for quantification of subunit stoichiometry using matrix-assisted laser desorption/ionization-time of flight MS. Results from both approaches are in excellent agreement and reveal that the subunit composition of yeast V1-ATPase is A3B3DE3FG3H.

On the Origin of Large Flexibility of P-glycoprotein in the Inward-Facing State

Background: P-glycoprotein relies on largely unknown structural changes for its transport function. Results: EPR spectroscopy and simulations capture large-amplitude structural fluctuations for inward-facing P-glycoprotein. Conclusion: The characterized distinct dynamics of P-glycoprotein suggests mechanistic diversity of ATP-coupled transport in ABC transporters. Significance: Characterizing structural dynamics is a key step toward understanding the mechanism of this multidrug resistance transporter. P-glycoprotein (Pgp) is one of the most biomedically relevant transporters in the ATP binding cassette (ABC) superfamily due to its involvement in developing multidrug resistance in cancer cells. Employing molecular dynamics simulations and double electron-electron resonance spectroscopy, we have investigated the structural dynamics of membrane-bound Pgp in the inward-facing state and found that Pgp adopts an unexpectedly wide range of conformations, highlighted by the degree of separation between the two nucleotide-binding domains (NBDs). The distance between the two NBDs in the equilibrium simulations covers a range of at least 20 Å, including, both, more open and more closed NBD configurations than the crystal structure. The double electron-electron resonance measurements on spin-labeled Pgp mutants also show wide distributions covering both longer and shorter distances than those observed in the crystal structure. Based on structural and sequence analyses, we propose that the transmembrane domains of Pgp might be more flexible than other structurally known ABC exporters. The structural flexibility of Pgp demonstrated here is not only in close agreement with, but also helps rationalize, the reported high NBD fluctuations in several ABC exporters and possibly represents a fundamental difference in the transport mechanism between ABC exporters and ABC importers. In addition, during the simulations we have captured partial entrance of a lipid molecule from the bilayer into the lumen of Pgp, reaching the putative drug binding site. The location of the protruding lipid suggests a putative pathway for direct drug recruitment from the membrane.

Dynamic ligand induced conformational rearrangements in P-glycoprotein as probed by fluorescence resonance energy transfer spectroscopy

Background: P-glycoprotein is an ATP-binding cassette transporter involved in multidrug resistance. Results: The two nucleotide binding domains are found to be in close association during the catalytic cycle as determined by fluorescence spectroscopy. Conclusion: Small distance changes were observed during ATP hydrolysis supporting an alternating site mechanism. Significance: Understanding the mechanism of P-glycoprotein is pertinent for developing inhibitors aimed at overcoming multidrug resistance. P-glycoprotein (Pgp), a member of the ATP-binding cassette transporter family, functions as an ATP hydrolysis-driven efflux pump to rid the cell of toxic organic compounds, including a variety of drugs used in anticancer chemotherapy. Here, we used fluorescence resonance energy transfer (FRET) spectroscopy to delineate the structural rearrangements the two nucleotide binding domains (NBDs) are undergoing during the catalytic cycle. Pairs of cysteines were introduced into equivalent regions in the N- and C-terminal NBDs for labeling with fluorescent dyes for ensemble and single-molecule FRET spectroscopy. In the ensemble FRET, a decrease of the donor to acceptor (D/A) ratio was observed upon addition of drug and ATP. Vanadate trapping further decreased the D/A ratio, indicating close association of the two NBDs. One of the cysteine mutants was further analyzed using confocal single-molecule FRET spectroscopy. Single Pgp molecules showed fast fluctuations of the FRET efficiencies, indicating movements of the NBDs on a time scale of 10–100 ms. Populations of low, medium, and high FRET efficiencies were observed during drug-stimulated MgATP hydrolysis, suggesting the presence of at least three major conformations of the NBDs during catalysis. Under conditions of vanadate trapping, most molecules displayed high FRET efficiency states, whereas with cyclosporin, more molecules showed low FRET efficiency. Different dwell times of the FRET states were found for the distinct biochemical conditions, with the fastest movements during active turnover. The FRET spectroscopy observations are discussed in context of a model of the catalytic mechanism of Pgp.

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.

An Endoplasmic Reticulum (ER) Membrane Complex Composed of SPFH1 and SPFH2 Mediates the ER-associated Degradation of Inositol 1,4,5-Trisphosphate Receptors

How endoplasmic reticulum (ER) proteins that are substrates for the ER-associated degradation (ERAD) pathway are recognized for polyubiquitination and proteasomal degradation is largely unresolved. Inositol 1,4,5-trisphosphate receptors (IP3Rs) form tetrameric calcium channels in ER membranes, whose primary role is to control the release of ER calcium stores, but whose levels are also regulated, in an activation-dependent manner, by the ERAD pathway. Here we report that the ER membrane protein SPFH1 and its homolog SPFH2 form a heteromeric ∼2 MDa complex that binds to IP3R tetramers immediately after their activation and is required for their processing. The complex is ring-shaped (diameter ∼250Å), and RNA interference-mediated depletion of SPFH1 and SPFH2 blocks IP3R polyubiquitination and degradation. We propose that this novel SPFH1/2 complex is a recognition factor that targets IP3Rs and perhaps other substrates for ERAD.

Functional 20S proteasomes in mature human red blood cells

The purpose of the present study was to investigate whether functional 20S and/or 26S proteasomes are present within mature human red blood cells (RBCs; depleted of reticulocytes and leukocytes). Double-immunofluorescence confocal microscopy showed the presence of immunoreactive 20S and 19S proteasomal subunit proteins and their partial co-localization within mature RBCs. Proteasomes isolated from mature RBCs displayed 20S activity in vitro; atomic-force and transmission electron microscopy of isolated proteasomes revealed abundant 20S core particles and very few 26S particles. A two-dimensional differential in-gel electrophoresis (2D-DIGE) approach was used to determine if proteasome-dependent protein degradation occurs within mature RBCs. Twenty-eight proteins were identified with altered protein content in response to lactacystin. Seven cytosolic proteins showed an increase and 16 showed a decrease; five membrane proteins showed a decrease. We conclude that the proteins showing increased abundance are either primary or secondary targets of the 20S proteasome and that putatively degraded proteins are secondary targets. Therefore, functional 20S proteasomes exist within mature RBCs. Our study did not detect 26S proteasome activity using the 2D-DIGE approach.

P-glycoprotein retains drug-stimulated ATPase activity upon covalent linkage of the two nucleotide binding domains at their C-terminal ends.

P-glycoprotein (Pgp), a member of the ABC transporter family, functions as an ATP hydrolysis-driven efflux pump to rid the cell of toxic organic compounds, including a variety of drugs used in anti-cancer chemotherapy. We have recently obtained EM projection images of lipid-bound Pgp without nucleotide and transport substrate that showed the two halves of the transporter separated by a central cavity (Lee, J. Y., Urbatsch, I. L., Senior, A. E., and Wilkens, S. (2002) J. Biol. Chem. 277, 40125–40131). Addition of nucleotide and/or substrate lead to a close association of the two halves of the transporter, thereby closing the central cavity (Lee, J. Y., Urbatsch, I. L., Senior, A. E., and Wilkens, S. (2008) J. Biol. Chem. 283, 5769–5779). Here, we used cysteine-mediated disulfide cross-linking to further delineate the structural rearrangements of the two nucleotide binding domains (NBD1 and NBD2) that take place during catalysis. Cysteines introduced at or near the C-terminal ends of NBD1 and NBD2 allowed for spontaneous disulfide cross-linking under nonreducing conditions. For mutant A627C/S1276C, disulfide formation was with high efficiency and cross-linked Pgp retained 30–68% drug-stimulated ATPase activity compared with reduced or cysteine-less Pgp. Two other cysteine pairs (K615C/S1276C and A627C/K1260C) also formed a disulfide but to a lesser extent, and the cross-linked form of these two mutants had lower drug-stimulated ATPase activity. The data suggest that the C-terminal ends of the two NBDs of Pgp are not required to undergo significant motion with respect to one another during the catalytic cycle.