Michael L. Oldham

Crystal structure of a catalytic intermediate of the maltose transporter

The maltose uptake system of Escherichia coli is a well-characterized member of the ATP-binding cassette transporter superfamily. Here we present the 2.8-Å crystal structure of the intact maltose transporter in complex with the maltose-binding protein, maltose and ATP. This structure, stabilized by a mutation that prevents ATP hydrolysis, captures the ATP-binding cassette dimer in a closed, ATP-bound conformation. Maltose is occluded within a solvent-filled cavity at the interface of the two transmembrane subunits, about halfway into the lipid bilayer. The binding protein docks onto the entrance of the cavity in an open conformation and serves as a cap to ensure unidirectional translocation of the sugar molecule. These results provide direct evidence for a concerted mechanism of transport in which solute is transferred from the binding protein to the transmembrane subunits when the cassette dimer closes to hydrolyse ATP.

Crystal structure of the multidrug transporter P-glycoprotein from Caenorhabditis elegans

P-glycoprotein (P-gp) is an ATP-binding cassette transporter that confers multidrug resistance in cancer cells. It also affects the absorption, distribution and clearance of cancer-unrelated drugs and xenobiotics. For these reasons, the structure and function of P-gp have been studied extensively for decades. Here we present biochemical characterization of P-gp from Caenorhabditis elegans and its crystal structure at a resolution of 3.4 ångströms. We find that the apparent affinities of P-gp for anticancer drugs actinomycin D and paclitaxel are approximately 4,000 and 100 times higher, respectively, in the membrane bilayer than in detergent. This affinity enhancement highlights the importance of membrane partitioning when a drug accesses the transporter in the membrane. Furthermore, the transporter in the crystal structure opens its drug pathway at the level of the membrane’s inner leaflet. In the helices flanking the opening to the membrane, we observe extended loops that may mediate drug binding, function as hinges to gate the pathway or both. We also find that the interface between the transmembrane and nucleotide-binding domains, which couples ATP hydrolysis to transport, contains a ball-and-socket joint and salt bridges similar to the ATP-binding cassette importers, suggesting that ATP-binding cassette exporters and importers may use similar mechanisms to achieve alternating access for transport. Finally, a model of human P-gp derived from the structure of C. elegans P-gp not only is compatible with decades of biochemical analysis, but also helps to explain perplexing functional data regarding the Phe335Ala mutant. These results increase our understanding of the structure and function of this important molecule.

Alternating access in maltose transporter mediated by rigid-body rotations.

ATP-binding cassette transporters couple ATP hydrolysis to substrate translocation through an alternating access mechanism, but the nature of the conformational changes in a transport cycle remains elusive. Previously we reported the structure of the maltose transporter MalFGK(2) in an outward-facing conformation in which the transmembrane (TM) helices outline a substrate-binding pocket open toward the periplasmic surface and ATP is poised for hydrolysis along the closed nucleotide-binding dimer interface. Here we report the structure of the nucleotide-free maltose transporter in which the substrate binding pocket is only accessible from the cytoplasm and the nucleotide-binding interface is open. Comparison of the same transporter crystallized in two different conformations reveals that alternating access involves rigid-body rotations of the TM subdomains that are coupled to the closure and opening of the nucleotide-binding domain interface. The comparison also reveals that point mutations enabling binding protein-independent transport line dynamic interfaces in the TM region.

Structural insights into ABC transporter mechanism

ATP-binding cassette (ABC) transporters utilize the energy from ATP hydrolysis to transport substances across the membrane. In recent years, crystal structures of several ABC transporters have become available. These structures show that both importers and exporters oscillate between two conformations: an inward-facing conformation with the substrate translocation pathway open to the cytoplasm and an outward-facing conformation with the translocation pathway facing the opposite side of the membrane. In this review, conformational differences found in the structures of homologous ABC transporters are analyzed to understand how alternating-access is achieved. It appears that rigid-body rotations of the transmembrane subunits, coinciding with the opening and closing of the nucleotide-binding subunits, couples ATP hydrolysis to substrate translocation.

Crystal structure of the maltose transporter in a pretranslocation intermediate state.

An intermediate structure provides insight into how a transport substrate allosterically activates adenosine triphosphatase activity. Adenosine triphosphate (ATP)–binding cassette (ABC) transporters convert chemical energy from ATP hydrolysis to mechanical work for substrate translocation. They function by alternating between two states, exposing the substrate-binding site to either side of the membrane. A key question that remains to be addressed is how substrates initiate the transport cycle. Using x-ray crystallography, we have captured the maltose transporter in an intermediate step between the inward- and outward-facing states. We show that interactions with substrate-loaded maltose-binding protein in the periplasm induce a partial closure of the MalK dimer in the cytoplasm. ATP binding to this conformation then promotes progression to the outward-facing state. These results, interpreted in light of biochemical and functional studies, provide a structural basis to understand allosteric communication in ABC transporters.

Snapshots of the maltose transporter during ATP hydrolysis

ATP-binding cassette transporters are powered by ATP, but the mechanism by which these transporters hydrolyze ATP is unclear. In this study, four crystal structures of the full-length wild-type maltose transporter, stabilized by adenosine 5′-(β,γ-imido)triphosphate or ADP in conjunction with phosphate analogs , , or , were determined to 2.2- to 2.4-Å resolution. These structures led to the assignment of two enzymatic states during ATP hydrolysis and demonstrate specific functional roles of highly conserved residues in the nucleotide-binding domain, suggesting that ATP-binding cassette transporters catalyze ATP hydrolysis via a general base mechanism.

Structure and function of a HECT domain ubiquitin-binding site

The Rsp5 ubiquitin ligase contains a non‐covalent binding site for ubiquitin within the amino‐terminal lobe (N‐lobe) of the HECT domain, and the X‐ray crystal structure of the HECT–ubiquitin complex has been determined. Hydrophobic patch residues of ubiquitin (L8, I44, V70) were crucial for interaction with Rsp5, and amino‐acid alterations at the Rsp5‐binding interface resulted in defects in polyubiquitination. Our results support a model in which the N‐lobe‐binding site acts to localize and orient the distal end of the ubiquitin chain to promote conjugation of the next ubiquitin molecule.

Carbon catabolite repression of the maltose transporter revealed by X-ray crystallography

Efficient carbon utilization is critical to the survival of microorganisms in competitive environments. To optimize energy usage, bacteria have developed an integrated control system to preferentially uptake carbohydrates that support rapid growth. The availability of a preferred carbon source, such as glucose, represses the synthesis and activities of proteins necessary for the transport and metabolism of secondary carbon sources. This regulatory phenomenon is defined as carbon catabolite repression. In enteric bacteria, the key player of carbon catabolite repression is a component of the glucose-specific phosphotransferase system, enzyme IIA (EIIAGlc). It is known that unphosphorylated EIIAGlc binds to and inhibits a variety of transporters when glucose is available. However, understanding the underlying molecular mechanism has been hindered by the complete absence of structures for any EIIAGlc–transporter complexes. Here we present the 3.9 Å crystal structure of Escherichia coli EIIAGlc in complex with the maltose transporter, an ATP-binding cassette (ABC) transporter. The structure shows that two EIIAGlc molecules bind to the cytoplasmic ATPase subunits, stabilizing the transporter in an inward-facing conformation and preventing the structural rearrangements necessary for ATP hydrolysis. We also show that the half-maximal inhibitory concentrations of the full-length EIIAGlc and an amino-terminal truncation mutant differ by 60-fold, consistent with the hypothesis that the amino-terminal region, disordered in the crystal structure, functions as a membrane anchor to increase the effective EIIAGlc concentration at the membrane. Together these data suggest a model of how the central regulatory protein EIIAGlc allosterically inhibits maltose uptake in E. coli.

A mechanism of viral immune evasion revealed by cryo-EM analysis of the TAP transporter

Cellular immunity against viral infection and tumour cells depends on antigen presentation by major histocompatibility complex class I (MHC I) molecules. Intracellular antigenic peptides are transported into the endoplasmic reticulum by the transporter associated with antigen processing (TAP) and then loaded onto the nascent MHC I molecules, which are exported to the cell surface and present peptides to the immune system. Cytotoxic T lymphocytes recognize non-self peptides and program the infected or malignant cells for apoptosis. Defects in TAP account for immunodeficiency and tumour development. To escape immune surveillance, some viruses have evolved strategies either to downregulate TAP expression or directly inhibit TAP activity. So far, neither the architecture of TAP nor the mechanism of viral inhibition has been elucidated at the structural level. Here we describe the cryo-electron microscopy structure of human TAP in complex with its inhibitor ICP47, a small protein produced by the herpes simplex virus I. Here we show that the 12 transmembrane helices and 2 cytosolic nucleotide-binding domains of the transporter adopt an inward-facing conformation with the two nucleotide-binding domains separated. The viral inhibitor ICP47 forms a long helical hairpin, which plugs the translocation pathway of TAP from the cytoplasmic side. Association of ICP47 precludes substrate binding and prevents nucleotide-binding domain closure necessary for ATP hydrolysis. This work illustrates a striking example of immune evasion by persistent viruses. By blocking viral antigens from entering the endoplasmic reticulum, herpes simplex virus is hidden from cytotoxic T lymphocytes, which may contribute to establishing a lifelong infection in the host.

Structure of the transporter associated with antigen processing trapped by herpes simplex virus.

The transporter associated with antigen processing (TAP) is an ATP-binding cassette (ABC) transporter essential to cellular immunity against viral infection. Some persistent viruses have evolved strategies to inhibit TAP so that they may go undetected by the immune system. The herpes simplex virus for example evades immune surveillance by blocking peptide transport with a small viral protein ICP47. In this study, we determined the structure of human TAP bound to ICP47 by electron cryo-microscopy (cryo-EM) to 4.0 Å. The structure shows that ICP47 traps TAP in an inactive conformation distinct from the normal transport cycle. The specificity and potency of ICP47 inhibition result from contacts between the tip of the helical hairpin and the apex of the transmembrane cavity. This work provides a clear molecular description of immune evasion by a persistent virus. It also establishes the molecular structure of TAP to facilitate mechanistic studies of the antigen presentation process. DOI: http://dx.doi.org/10.7554/eLife.21829.001