Structural Basis of the Allosteric Inhibition of Human ABCG2 by Nanobodies.

ABCG2 is an ATP-binding cassette transporter that exports a wide range of xenobiotic compounds and has been recognized as a contributing factor for multidrug resistance in cancer cells. Substrate and inhibitor interactions with ABCG2 have been extensively studied and small molecule inhibitors have been developed that prevent the export of anticancer drugs from tumor cells. Here, we explore the potential for inhibitors that target sites other than the substrate binding pocket of ABCG2. We developed novel nanobodies against ABCG2 and used functional analyses to select three inhibitory nanobodies (Nb8, Nb17 and Nb96) for structural studies by single particle cryo-electron microscopy. Our results showed that these nanobodies allosterically bind to different regions of the nucleotide binding domains. Two copies of Nb8 bind to the apex of the NBDs preventing them from fully closing. Nb17 binds near the two-fold axis of the transporter and interacts with both NBDs. Nb96 binds to the side of the NBD and immobilizes a region connected to key motifs involved in ATP binding and hydrolysis. All three nanobodies prevent the transporter from undergoing conformational changes required for substrate transport. These findings advance our understanding of the molecular basis of modulation of ABCG2 by external binders, which may contribute to the development of a new generation of inhibitors. Furthermore, this is the first example of modulation of human multidrug resistance transporters by nanobodies.

Structures of ABCG2 under turnover conditions reveal a key step in the drug transport mechanism.

ABCG2 is a multidrug transporter that affects drug pharmacokinetics and contributes to multidrug resistance of cancer cells. In previously reported structures, the reaction cycle was halted by the absence of substrates or ATP, mutation of catalytic residues, or the presence of small-molecule inhibitors or inhibitory antibodies. Here we present cryo-EM structures of ABCG2 under turnover conditions containing either the endogenous substrate estrone-3-sulfate or the exogenous substrate topotecan. We find two distinct conformational states in which both the transport substrates and ATP are bound. Whereas the state turnover-1 features more widely separated NBDs and an accessible substrate cavity between the TMDs, turnover-2 features semi-closed NBDs and an almost fully occluded substrate cavity. Substrate size appears to control which turnover state is mainly populated. The conformational changes between turnover-1 and turnover-2 states reveal how ATP binding is linked to the closing of the cytoplasmic side of the TMDs. The transition from turnover-1 to turnover-2 is the likely bottleneck or rate-limiting step of the reaction cycle, where the discrimination of substrates and inhibitors occurs.

Structural Basis of Drug Recognition by the Multidrug Transporter ABCG2.

ABCG2 is an ATP-binding cassette (ABC) transporter whose function affects the pharmacokinetics of drugs and contributes to multidrug resistance of cancer cells. While its interaction with the endogenous substrate estrone-3-sulfate (E1S) has been elucidated at a structural level, the recognition and recruitment of exogenous compounds is not understood at sufficiently high resolution. Here we present three cryo-EM structures of nanodisc-reconstituted, human ABCG2 bound to anticancer drugs tariquidar, topotecan and mitoxantrone. To enable structural insight at high resolution, we used Fab fragments of the ABCG2-specific monoclonal antibody 5D3, which binds to the external side of the transporter but does not interfere with drug-induced stimulation of ATPase activity. We observed that the binding pocket of ABCG2 can accommodate a single tariquidar molecule in a C-shaped conformation, similar to one of the two tariquidar molecules bound to ABCB1, where tariquidar acts as an inhibitor. We also found single copies of topotecan and mitoxantrone bound between key phenylalanine residues. Mutagenesis experiments confirmed the functional importance of two residues in the binding pocket, F439 and N436. Using 3D variability analyses, we found a correlation between substrate binding and reduced dynamics of the nucleotide binding domains (NBDs), suggesting a structural explanation for drug-induced ATPase stimulation. Our findings provide additional insight into how ABCG2 differentiates between inhibitors and substrates and may guide a rational design of new modulators and substrates.

Tariquidar-related triazoles as potent, selective and stable inhibitors of ABCG2 (BCRP).

Tariquidar derivatives have been described as potent and selective ABCG2 inhibitors. However, their susceptibility to hydrolysis limits their applicability. The current study comprises the synthesis and characterization of novel tariquidar-related inhibitors, obtained by bioisosteric replacement of the labile moieties in our previous tariquidar analog UR-ME22-1 (9). CuAAC ("click" reaction) gave convenient access to a triazole core as a substitute for the labile amide group and the labile ester moiety was replaced by different acyl groups in a Sugasawa reaction. A stability assay proved the enhancement of the stability in blood plasma. Compounds UR-MB108 (57) and UR-MB136 (59) inhibited ABCG2 in a Hoechst 33342 transport assay with an IC50 value of about 80 nM and belong to the most potent ABCG2 inhibitors described so far. Compound 57 was highly selective, whereas its PEGylated analog 59 showed some potency at ABCB1. Both 57 and 59 produced an ABCG2 ATPase-depressing effect which is in agreement with our precedent cryo-EM study identifying 59 as an ATPase inhibitor that exerts its effect via locking the inward-facing conformation. Thermostabilization of ABCG2 by 57 and 59 can be taken as a hint to comparable binding to ABCG2. As reference substances, compounds 57 and 59 allow additional mechanistic studies on ABCG2 inhibition. Due to their stability in blood plasma, they are also applicable in vivo. The highly specific inhibitor 57 is suited for PET labeling, helping to further elucidate the (patho)physiological role of ABCG2, e.g. at the BBB.

Cryo-EM structures of a human ABCG2 mutant trapped in ATP-bound and substrate-bound states.

ABCG2 is a transporter protein of the ATP-binding-cassette (ABC) family that is expressed in the plasma membrane in cells of various tissues and tissue barriers, including the blood-brain, blood-testis and maternal-fetal barriers1-4. Powered by ATP, it translocates endogenous substrates, affects the pharmacokinetics of many drugs and protects against a wide array of xenobiotics, including anti-cancer drugs5-12. Previous studies have revealed the architecture of ABCG2 and the structural basis of its inhibition by small molecules and antibodies13,14. However, the mechanisms of substrate recognition and ATP-driven transport are unknown. Here we present high-resolution cryo-electron microscopy (cryo-EM) structures of human ABCG2 in a substrate-bound pre-translocation state and an ATP-bound post-translocation state. For both structures, we used a mutant containing a glutamine replacing the catalytic glutamate (ABCG2EQ), which resulted in reduced ATPase and transport rates and facilitated conformational trapping for structural studies. In the substrate-bound state, a single molecule of estrone-3-sulfate (E1S) is bound in a central, hydrophobic and cytoplasm-facing cavity about halfway across the membrane. Only one molecule of E1S can bind in the observed binding mode. In the ATP-bound state, the substrate-binding cavity has collapsed while an external cavity has opened to the extracellular side of the membrane. The ATP-induced conformational changes include rigid-body shifts of the transmembrane domains, pivoting of the nucleotide-binding domains (NBDs), and a change in the relative orientation of the NBD subdomains. Mutagenesis and in vitro characterization of transport and ATPase activities demonstrate the roles of specific residues in substrate recognition, including a leucine residue that forms a 'plug' between the two cavities. Our results show how ABCG2 harnesses the energy of ATP binding to extrude E1S and other substrates, and suggest that the size and binding affinity of compounds are important for distinguishing substrates from inhibitors.

Structural basis of small-molecule inhibition of human multidrug transporter ABCG2.

ABCG2 is an ATP-binding cassette (ABC) transporter that protects tissues against xenobiotics, affects the pharmacokinetics of drugs and contributes to multidrug resistance. Although many inhibitors and modulators of ABCG2 have been developed, understanding their structure-activity relationship requires high-resolution structural insight. Here, we present cryo-EM structures of human ABCG2 bound to synthetic derivatives of the fumitremorgin C-related inhibitor Ko143 or the multidrug resistance modulator tariquidar. Both compounds are bound to the central, inward-facing cavity of ABCG2, blocking access for substrates and preventing conformational changes required for ATP hydrolysis. The high resolutions allowed for de novo building of the entire transporter and also revealed tightly bound phospholipids and cholesterol interacting with the lipid-exposed surface of the transmembrane domains (TMDs). Extensive chemical modifications of the Ko143 scaffold combined with in vitro functional analyses revealed the details of ABCG2 interactions with this compound family and provide a basis for the design of novel inhibitors and modulators.

Structure of the human multidrug transporter ABCG2.

ABCG2 is a constitutively expressed ATP-binding cassette (ABC) transporter that protects many tissues against xenobiotic molecules. Its activity affects the pharmacokinetics of commonly used drugs and limits the delivery of therapeutics into tumour cells, thus contributing to multidrug resistance. Here we present the structure of human ABCG2 determined by cryo-electron microscopy, providing the first high-resolution insight into a human multidrug transporter. We visualize ABCG2 in complex with two antigen-binding fragments of the human-specific, inhibitory antibody 5D3 that recognizes extracellular loops of the transporter. We observe two cholesterol molecules bound in the multidrug-binding pocket that is located in a central, hydrophobic, inward-facing translocation pathway between the transmembrane domains. Combined with functional in vitro analyses, our results suggest a multidrug recognition and transport mechanism of ABCG2, rationalize disease-causing single nucleotide polymorphisms and the allosteric inhibition by the 5D3 antibody, and provide the structural basis of cholesterol recognition by other G-subfamily ABC transporters.

Mechanism of farnesylated CAAX protein processing by the intramembrane protease Rce1.

CAAX proteins have essential roles in multiple signalling pathways, controlling processes such as proliferation, differentiation and carcinogenesis. The ∼120 mammalian CAAX proteins function at cellular membranes and include the Ras superfamily of small GTPases, nuclear lamins, the γ-subunit of heterotrimeric GTPases, and several protein kinases and phosphatases. The proper localization of CAAX proteins to cell membranes is orchestrated by a series of post-translational modifications of the carboxy-terminal CAAX motifs (where C is cysteine, A is an aliphatic amino acid and X is any amino acid). These reactions involve prenylation of the cysteine residue, cleavage at the AAX tripeptide and methylation of the carboxyl-prenylated cysteine residue. The major CAAX protease activity is mediated by Rce1 (Ras and a-factor converting enzyme 1), an intramembrane protease (IMP) of the endoplasmic reticulum. Information on the architecture and proteolytic mechanism of Rce1 has been lacking. Here we report the crystal structure of a Methanococcus maripaludis homologue of Rce1, whose endopeptidase specificity for farnesylated peptides mimics that of eukaryotic Rce1. Its structure, comprising eight transmembrane α-helices, and catalytic site are distinct from those of other IMPs. The catalytic residues are located ∼10 Å into the membrane and are exposed to the cytoplasm and membrane through a conical cavity that accommodates the prenylated CAAX substrate. We propose that the farnesyl lipid binds to a site at the opening of two transmembrane α-helices, which results in the scissile bond being positioned adjacent to a glutamate-activated nucleophilic water molecule. This study suggests that Rce1 is the founding member of a novel IMP family, the glutamate IMPs.

Mechanism of isoprenylcysteine carboxyl methylation from the crystal structure of the integral membrane methyltransferase ICMT.

The posttranslational modification of C-terminal CAAX motifs in proteins such as Ras, most Rho GTPases, and G protein γ subunits, plays an essential role in determining their subcellular localization and correct biological function. An integral membrane methyltransferase, isoprenylcysteine carboxyl methyltransferase (ICMT), catalyzes the final step of CAAX processing after prenylation of the cysteine residue and endoproteolysis of the -AAX motif. We have determined the crystal structure of a prokaryotic ICMT ortholog, revealing a markedly different architecture from conventional methyltransferases that utilize S-adenosyl-L-methionine (SAM) as a cofactor. ICMT comprises a core of five transmembrane α helices and a cofactor-binding pocket enclosed within a highly conserved C-terminal catalytic subdomain. A tunnel linking the reactive methyl group of SAM to the inner membrane provides access for the prenyl lipid substrate. This study explains how an integral membrane methyltransferase achieves recognition of both a hydrophilic cofactor and a lipophilic prenyl group attached to a polar protein substrate.

Structure and genetic analysis of the arterivirus nonstructural protein 7alpha.

Arterivirus replicase polyproteins are cleaved into at least 13 mature nonstructural proteins (nsps), and in particular the nsp5-to-nsp8 region is subject to a complex processing cascade. The function of the largest subunit from this region, nsp7, which is further cleaved into nsp7α and nsp7ß, is unknown. Using nuclear magnetic resonance (NMR) spectroscopy, we determined the solution structure of nsp7α of equine arteritis virus, revealing an interesting unique fold for this protein but thereby providing little clue to its possible functions. Nevertheless, structure-based reverse genetics studies established the importance of nsp7/nsp7α for viral RNA synthesis, thus providing a basis for future studies.

Resonance assignment of nsp7α from arterivirus.

The (1)H, (15)N and (13)C resonance assignment of nsp7α, a non-structural protein of unknown function from the equine arteritis virus, is reported.

Papain-like protease 1 from transmissible gastroenteritis virus: crystal structure and enzymatic activity toward viral and cellular substrates.

Coronaviruses encode two classes of cysteine proteases, which have narrow substrate specificities and either a chymotrypsin- or papain-like fold. These enzymes mediate the processing of the two precursor polyproteins of the viral replicase and are also thought to modulate host cell functions to facilitate infection. The papain-like protease 1 (PL1(pro)) domain is present in nonstructural protein 3 (nsp3) of alphacoronaviruses and subgroup 2a betacoronaviruses. It participates in the proteolytic processing of the N-terminal region of the replicase polyproteins in a manner that varies among different coronaviruses and remains poorly understood. Here we report the first structural and biochemical characterization of a purified coronavirus PL1(pro) domain, that of transmissible gastroenteritis virus (TGEV). Its tertiary structure is compared with that of severe acute respiratory syndrome (SARS) coronavirus PL2(pro), a downstream paralog that is conserved in the nsp3's of all coronaviruses. We identify both conserved and unique structural features likely controlling the interaction of PL1(pro) with cofactors and substrates, including the tentative mapping of substrate pocket residues. The purified recombinant TGEV PL1(pro) was shown to cleave a peptide mimicking the cognate nsp2|nsp3 cleavage site. Like its PL2(pro) paralogs from several coronaviruses, TGEV PL1(pro) was also found to have deubiquitinating activity in an in vitro cleavage assay, implicating it in counteracting ubiquitin-regulated host cell pathways, likely including innate immune responses. In combination with the prior characterization of PL2(pro) from other alphacoronaviruses, e.g., human coronaviruses 229E and NL63, our results unequivocally establish that these viruses employ two PL(pro)s with overlapping specificities toward both viral and cellular substrates.

Structure of the X (ADRP) domain of nsp3 from feline coronavirus.

The structure of the X (or ADRP) domain of a pathogenic variant of feline coronavirus (FCoV) has been determined in tetragonal and cubic crystal forms to 3.1 and 2.2 A resolution, respectively. In the tetragonal crystal form, glycerol-3-phosphate was observed in the ADP-ribose-binding site. Both crystal forms contained large solvent channels and had a solvent content of higher than 70%. Only very weak binding of this domain to ADP-ribose was detected in vitro. However, the structure with ADP-ribose bound was determined in the cubic crystal form at 3.9 A resolution. The structure of the FCoV X domain had the expected macro-domain fold and is the first structure of this domain from a coronavirus belonging to subgroup 1a.

Structure of the C-terminal domain of nsp4 from feline coronavirus.

Coronaviruses are a family of positive-stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26-31 kb) encodes 15-16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication-transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes. In this report, a conserved C-terminal domain ( approximately 100 amino-acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library-based construct screening. A prototypical crystal structure at 2.8 A resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet-substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P4(3). The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS-derived composite model. The structure consists of a single domain with a predominantly alpha-helical content displaying a unique fold that could be engaged in protein-protein interactions.

Structural and biophysical characterization of the proteins interacting with the herpes simplex virus 1 origin of replication.

The C terminus of the herpes simplex virus type 1 origin-binding protein, UL9ct, interacts directly with the viral single-stranded DNA-binding protein ICP8. We show that a 60-amino acid C-terminal deletion mutant of ICP8 (ICP8DeltaC) also binds very strongly to UL9ct. Using small angle x-ray scattering, the low resolution solution structures of UL9ct alone, in complex with ICP8DeltaC, and in complex with a 15-mer double-stranded DNA containing Box I of the origin of replication are described. Size exclusion chromatography, analytical ultracentrifugation, and electrophoretic mobility shift assays, backed up by isothermal titration calorimetry measurements, are used to show that the stoichiometry of the UL9ct-dsDNA15-mer complex is 2:1 at micromolar protein concentrations. The reaction occurs in two steps with initial binding of UL9ct to DNA (Kd approximately 6 nM) followed by a second binding event (Kd approximately 0.8 nM). It is also shown that the stoichiometry of the ternary UL9ct-ICP8DeltaC-dsDNA15-mer complex is 2:1:1, at the concentrations used in the different assays. Electron microscopy indicates that the complex assembled on the extended origin, oriS, rather than Box I alone, is much larger. The results are consistent with a simple model whereby a conformational switch of the UL9 DNA-binding domain upon binding to Box I allows the recruitment of a UL9-ICP8 complex by interaction between the UL9 DNA-binding domains.

Structure of the C-terminal domain of human La protein reveals a novel RNA recognition motif coupled to a helical nuclear retention element.

The La protein is an important component of ribonucleoprotein complexes that acts mainly as an RNA chaperone to facilitate correct processing and maturation of RNA polymerase III transcripts, but can also stimulate translation initiation. We report here the structure of the C-terminal domain of human La, which comprises an atypical RNA recognition motif (La225-334) and a long unstructured C-terminal tail. The central beta sheet of La225-334 reveals novel features: the putative RNA binding surface is formed by a five-stranded beta sheet and, strikingly, is largely obscured by a long C-terminal alpha helix that encompasses a recently identified nuclear retention element. Contrary to previous observations, we find that the La protein does not contain a dimerization domain.