Cryo-EM reconstruction of oleate hydratase bound to a phospholipid membrane bilayer.

Oleate hydratase (OhyA) is a bacterial peripheral membrane protein that catalyzes FAD-dependent water addition to membrane bilayer-embedded unsaturated fatty acids. The opportunistic pathogen Staphylococcus aureus uses OhyA to counteract the innate immune system and support colonization. Many Gram-positive and Gram-negative bacteria in the microbiome also encode OhyA. OhyA is a dimeric flavoenzyme whose carboxy terminus is identified as the membrane binding domain; however, understanding how OhyA binds to cellular membranes is not complete until the membrane-bound structure has been elucidated. All available OhyA structures depict the solution state of the protein outside its functional environment. Here, we employ liposomes to solve the cryo-electron microscopy structure of the functional unit: the OhyA•membrane complex. The protein maintains its structure upon membrane binding and slightly alters the curvature of the liposome surface. OhyA preferentially associates with 20-30 nm liposomes with multiple copies of OhyA dimers assembling on the liposome surface resulting in the formation of higher-order oligomers. Dimer assembly is cooperative and extends along a formed ridge of the liposome. We also solved an OhyA dimer of dimers structure that recapitulates the intermolecular interactions that stabilize the dimer assembly on the membrane bilayer as well as the crystal contacts in the lattice of the OhyA crystal structure. Our work enables visualization of the molecular trajectory of membrane binding for this important interfacial enzyme.

Principles of peptide selection by the transporter associated with antigen processing.

Our ability to fight pathogens relies on major histocompatibility complex class I (MHC-I) molecules presenting diverse antigens on the surface of diseased cells. The transporter associated with antigen processing (TAP) transports nearly the entire repertoire of antigenic peptides into the endoplasmic reticulum for MHC-I loading. How TAP transports peptides specific for MHC-I is unclear. In this study, we used cryo-EM to determine a series of structures of human TAP, both in the absence and presence of peptides with various sequences and lengths. The structures revealed that peptides of eight or nine residues in length bind in a similarly extended conformation, despite having little sequence overlap. We also identified two peptide-anchoring pockets on either side of the transmembrane cavity, each engaging one end of a peptide with primarily main chain atoms. Occupation of both pockets results in a global conformational change in TAP, bringing the two halves of the transporter closer together to prime it for isomerization and ATP hydrolysis. Shorter peptides are able to bind to each pocket separately but are not long enough to bridge the cavity to bind to both simultaneously. Mutations that disrupt hydrogen bonds with the N and C termini of peptides almost abolish MHC-I surface expression. Our findings reveal that TAP functions as a molecular caliper that selects peptides according to length rather than sequence, providing antigen diversity for MHC-I presentation.

The carboxy terminus causes interfacial assembly of oleate hydratase on a membrane bilayer.

The soluble flavoprotein oleate hydratase (OhyA) hydrates the 9-cis double bond of unsaturated fatty acids. OhyA substrates are embedded in membrane bilayers; OhyA must remove the fatty acid from the bilayer and enclose it in the active site. Here, we show that the positively charged helix-turn-helix motif in the carboxy terminus (CTD) is responsible for interacting with the negatively charged phosphatidylglycerol (PG) bilayer. Super-resolution microscopy of Staphylococcus aureus cells expressing green fluorescent protein fused to OhyA or the CTD sequence shows subcellular localization along the cellular boundary, indicating OhyA is membrane-associated and the CTD sequence is sufficient for membrane recruitment. Using cryo-electron microscopy, we solved the OhyA dimer structure and conducted 3D variability analysis of the reconstructions to assess CTD flexibility. Our surface plasmon resonance experiments corroborated that OhyA binds the PG bilayer with nanomolar affinity and we found the CTD sequence has intrinsic PG binding properties. We determined that the nuclear magnetic resonance structure of a peptide containing the CTD sequence resembles the OhyA crystal structure. We observed intermolecular NOE from PG liposome protons next to the phosphate group to the CTD peptide. The addition of paramagnetic MnCl2 indicated the CTD peptide binds the PG surface but does not insert into the bilayer. Molecular dynamics simulations, supported by site-directed mutagenesis experiments, identify key residues in the helix-turn-helix that drive membrane association. The data show that the OhyA CTD binds the phosphate layer of the PG surface to obtain bilayer-embedded unsaturated fatty acids.

A macrocyclic peptide inhibitor traps MRP1 in a catalytically incompetent conformation.

Adenosine triphosphate-binding cassette (ABC) transporters, such as multidrug resistance protein 1 (MRP1), protect against cellular toxicity by exporting xenobiotic compounds across the plasma membrane. However, constitutive MRP1 function hinders drug delivery across the blood-brain barrier, and MRP1 overexpression in certain cancers leads to acquired multidrug resistance and chemotherapy failure. Small-molecule inhibitors have the potential to block substrate transport, but few show specificity for MRP1. Here we identify a macrocyclic peptide, named CPI1, which inhibits MRP1 with nanomolar potency but shows minimal inhibition of a related multidrug transporter P-glycoprotein. A cryoelectron microscopy (cryo-EM) structure at 3.27 Å resolution shows that CPI1 binds MRP1 at the same location as the physiological substrate leukotriene C4 (LTC4). Residues that interact with both ligands contain large, flexible sidechains that can form a variety of interactions, revealing how MRP1 recognizes multiple structurally unrelated molecules. CPI1 binding prevents the conformational changes necessary for adenosine triphosphate (ATP) hydrolysis and substrate transport, suggesting it may have potential as a therapeutic candidate.

Structural basis of substrate recognition by a polypeptide processing and secretion transporter.

The peptidase-containing ATP-binding cassette transporters (PCATs) are unique members of the ABC transporter family that proteolytically process and export peptides and proteins. Each PCAT contains two peptidase domains that cleave off the secretion signal, two transmembrane domains forming a translocation pathway, and two nucleotide-binding domains that hydrolyze ATP. Previously the crystal structures of a PCAT from Clostridium thermocellum (PCAT1) were determined in the absence and presence of ATP, revealing how ATP binding regulates the protease activity and access to the translocation pathway. However, how the substrate CtA, a 90-residue polypeptide, is recognized by PCAT1 remained elusive. To address this question, we determined the structure of the PCAT1-CtA complex by electron cryo-microscopy (cryo-EM) to 3.4 Å resolution. The structure shows that two CtAs are bound via their N-terminal leader peptides, but only one is positioned for cleavage and translocation. Based on these results, we propose a model of how substrate cleavage, ATP hydrolysis, and substrate translocation are coordinated in a transport cycle.

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.

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.

Structural basis for substrate specificity in the Escherichia coli maltose transport system.

ATP-binding cassette (ABC) transporters are molecular pumps that harness the chemical energy of ATP hydrolysis to translocate solutes across the membrane. The substrates transported by different ABC transporters are diverse, ranging from small ions to large proteins. Although crystal structures of several ABC transporters are available, a structural basis for substrate recognition is still lacking. For the Escherichia coli maltose transport system, the selectivity of sugar binding to maltose-binding protein (MBP), the periplasmic binding protein, does not fully account for the selectivity of sugar transport. To obtain a molecular understanding of this observation, we determined the crystal structures of the transporter complex MBP-MalFGK2 bound with large malto-oligosaccharide in two different conformational states. In the pretranslocation structure, we found that the transmembrane subunit MalG forms two hydrogen bonds with malto-oligosaccharide at the reducing end. In the outward-facing conformation, the transmembrane subunit MalF binds three glucosyl units from the nonreducing end of the sugar. These structural features explain why modified malto-oligosaccharides are not transported by MalFGK2 despite their high binding affinity to MBP. They also show that in the transport cycle, substrate is channeled from MBP into the transmembrane pathway with a polarity such that both MBP and MalFGK2 contribute to the overall substrate selectivity of the system.

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 (EIIA(Glc)). It is known that unphosphorylated EIIA(Glc) 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 EIIA(Glc)-transporter complexes. Here we present the 3.9 Å crystal structure of Escherichia coli EIIA(Glc) in complex with the maltose transporter, an ATP-binding cassette (ABC) transporter. The structure shows that two EIIA(Glc) 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 EIIA(Glc) 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 EIIA(Glc) concentration at the membrane. Together these data suggest a model of how the central regulatory protein EIIA(Glc) allosterically inhibits maltose uptake in E. coli.

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.

Identification of a third osmoprotectant transport system, the osmU system, in Salmonella enterica.

The growth of Salmonella enterica serovar Typhimurium mutants lacking the ProP and ProU osmoprotectant transport systems is stimulated by glycine betaine in high-osmolarity media, suggesting that this organism has an additional osmoprotectant transport system. Bioinformatic analysis revealed that the genome of this organism contains a hitherto-unidentified operon, designated osmU, consisting of four genes whose products show high similarity to ABC-type transport systems for osmoprotectants in other bacteria. The osmU operon was inactivated by a site-directed deletion, which abolished the ability of glycine betaine to alleviate the inhibitory effect of high osmolarity and eliminated the accumulation of [(14)C]glycine betaine and [(14)C]choline-O-sulfate in high-osmolarity media in a strain lacking the ProP and ProU systems. Although the OsmU system can take up glycine betaine and choline-O-sulfate, these two osmoprotectants are recognized at low affinity by this transporter, suggesting that there might be more efficient substrates that are yet to be discovered. The transcription of osmU is induced 23-fold by osmotic stress (0.3 M NaCl). The osmU operon is present in the genomes of a number of Enterobacteriaceae, and orthologs of the OsmU system can be recognized in a wide variety of Bacteria and Archaea. The structure of the periplasmic binding protein component of this transporter, OsmX, was modeled on the crystallographic structure of the glycine betaine-binding protein ProX of Archaeoglobus fulgidus; the resultant model indicated that the amino acids that constitute substrate-binding site, including an "aromatic cage" made up of four tyrosines, are conserved between these two proteins.

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'-(ß,γ-imido)triphosphate or ADP in conjunction with phosphate analogs BeF(3)(-), VO(4)(3-), or AIF(4)(-), 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.

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

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.

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.

Dynamics of alpha-helical subdomain rotation in the intact maltose ATP-binding cassette transporter.

ATP-binding cassette (ABC) transporters are powered by a nucleotide-binding domain dimer that opens and closes during cycles of ATP hydrolysis. These domains consist of a RecA-like subdomain and an α-helical subdomain that is specific to the family. Many studies on isolated domains suggest that the helical subdomain rotates toward the RecA-like subdomain in response to ATP binding, moving the family signature motif into a favorable position to interact with the nucleotide across the dimer interface. Moreover, the transmembrane domains are docked into a cleft at the interface between these subdomains, suggesting a putative role of the rotation in interdomain communication. Electron paramagnetic resonance spectroscopy was used to study the dynamics of this rotation in the intact Escherichia coli maltose transporter MalFGK(2). This importer requires a periplasmic maltose-binding protein (MBP) that activates ATP hydrolysis by promoting the closure of the cassette dimer (MalK(2)). Whereas this rotation occurred during the transport cycle, it required not only trinucleotide, but also MBP, suggesting it is part of a global conformational change in the transporter. Interaction of AMP-PNP-Mg(2+) and a MBP that is locked in a closed conformation induced a transition from open MalK(2) to semiopen MalK(2) without significant subdomain rotation. Inward rotation of the helical subdomain and complete closure of MalK(2) therefore appear to be coupled to the reorientation of transmembrane helices and the opening of MBP, events that promote transfer of maltose into the transporter. After ATP hydrolysis, the helical subdomain rotates out as MalK(2) opens, resetting the transporter in an inward-facing conformation.

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 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-A 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 severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein dimerization domain reveals evolutionary linkage between corona- and arteriviridae.

The causative agent of severe acute respiratory syndrome (SARS) is the SARS-associated coronavirus, SARS-CoV. The nucleocapsid (N) protein plays an essential role in SARS-CoV genome packaging and virion assembly. We have previously shown that SARS-CoV N protein forms a dimer in solution through its C-terminal domain. In this study, the crystal structure of the dimerization domain, consisting of residues 270-370, is determined to 1.75A resolution. The structure shows a dimer with extensive interactions between the two subunits, suggesting that the dimeric form of the N protein is the functional unit in vivo. Although lacking significant sequence similarity, the dimerization domain of SARS-CoV N protein has a fold similar to that of the nucleocapsid protein of the porcine reproductive and respiratory syndrome virus. This finding provides structural evidence of the evolutionary link between Coronaviridae and Arteriviridae, suggesting that the N proteins of both viruses have a common origin.

The Prp19 U-box crystal structure suggests a common dimeric architecture for a class of oligomeric E3 ubiquitin ligases.

Prp19 is an essential splicing factor and a member of the U-box family of E3 ubiquitin ligases. Prp19 forms a tetramer via a central coiled-coil domain. Here, we show the U-box domain of Prp19 exists as a dimer within the context of the Prp19 tetramer. A high-resolution structure of the homodimeric state of the Prp19 U-box was determined by X-ray crystallography. Mutation of the U-box dimer interface abrogates U-box dimer formation and is lethal in vivo. The structure of the U-box dimer enables construction of a complete model of Prp19 providing insights into how the tetrameric protein functions as an E3 ligase. Finally, comparison of the Prp19 U-box homodimer with the heterodimeric complex of BRCA1/BARD1 RING-finger domains uncovers a common architecture for a family of oligomeric U-box and RING-finger E3 ubiquitin ligases, which has mechanistic implications for E3 ligase-mediated polyubiquitination and E4 polyubiquitin ligases.