Spotlight pubs.acs.org/acschemicalbiology
■
■
TETHERED INHIBITORS SELECTIVELY REGULATE PROTEIN FUNCTION
Adapted by permission from Macmillan Publishers Ltd.: Nature Gouaux et al. 521, 322−327, copyright 2015.
Adapted by permission from Macmillan Publishers Ltd.: Nature Chem., advance online publication, June 2, 2015, DOI: 10.1038/ nchem.2253.
Neuronal signaling is controlled by chemistry occurring at synapses, the end-to-end junctions between nerve cells. A nerve relays a signal by releasing into the synapse neurotransmitting molecules that activate receptors on the nerve receiving the signal. The signal is commonly terminated by neurotransmitter transporters on the presynaptic nerve cell that import the neurotransmitters from the extracellular space back into the cytosol. Molecules that inhibit neurotransmitter transporters, which include antidepressant pharmaceuticals as well as illicit psychostimulants, induce a buildup of neurotransmitter in the synapse, effectively prolonging the signal and leading to elevation of mood or a stimulating “high.” For example, the accumulation of the neurotransmitter dopamine in the brain is associated with a “reward” response that can be induced by dopamine transporter (DAT) inhibitors such as cocaine or methamphetamine. The differences in the binding of DAT substrates, which can be taken up through the transporter, and DAT inhibitors, which lodge into and deactivate the transporter, have recently been revealed for the first time in X-ray crystal structures solved by Howard Hughes Medical Institute investigators Gouaux and co-workers (Nature 2015, 521, 322−327). The team cocrystallized Drosophila melanogaster DAT with its natural substrate dopamine and an oxidation-resistant dopamine analog 3,4-dichlorophenethylamine (DCP) and compared the corresponding crystal structures with those of DAT bound to psychostimulant substrates (amphetamine and methamphetamine) or inhibitors (cocaine and nortriptyline). The structures indicated that the ligands fit into a binding site in the center of the transporter such that their amino groups interacted with carboxylate groups and carbonyl oxygens at the hinges of helical gates at the core of the transporter while their aromatic moieties were stabilized in a hydrophobic cleft oriented toward scaffolding regions of the binding site. Cocaine and nortriptyline appeared to wedge DAT into outward-open inactive conformations due to their bulky tropane and linear polycyclic side chains, respectively. The authors anticipate that these crystal structures will enable determining the mechanistic distinction between addictive and nonaddictive cocaine analogs in future studies. Heidi A. Dahlmann
Scientists can deduce a lot about a protein’s function in cells by watching what happens when the protein is no longer active. One way to knock down a protein’s activity is by suppressing expression at a genetic level. Although this method is frequently employed to create model cell lines and organisms, it can backfire if the disruption is developmentally lethal or if the cells compensate by upregulating complementary proteins. Alternatively, application of small molecule inhibitors can be used to temporally knock down protein activity in a dose-dependent manner. However, it can be very tricky to inhibit specific proteins in cells when other members of the protein family are present. A new combination of techniques reported by Jason W. Chin and co-workers at the University of Cambridge offers an unprecedented level of control over small molecule inhibition of specific target proteins in cells (Nature Chem. 2015, DOI: 10.1038/nchem.2253). The team separately engineered cell lines to express modified versions of two closely related kinases, MEK1 and MEK2, such that the kinases contained artificial amino acids bearing a functional group capable of participating in bio-orthogonal coupling reactions. The team also designed MEK inhibitors linked to a complementary bio-orthogonal coupling partner. Although the inhibitors could theoretically reversibly bind to either MEK1 or MEK2, specificity was achieved by inducing the bio-orthogonal reaction to irreversibly link the inhibitor to its specific modified target isoform. The inclusion of a photosensitive functional group in the linker, which could toggle between an active trans conformation or an inactive cis conformation depending on exposure to UV or blue light, allowed the research team to control the access of the inhibitor to its binding site on the target protein. The authors expect that their technique will assist in studies of proteins that cannot currently be specifically targeted with small molecule inhibitors. Heidi A. Dahlmann © 2015 American Chemical Society
NEW VIEWS OF DOPAMINE TRANSPORTER BINDING
Published: June 19, 2015 1355
DOI: 10.1021/acschembio.5b00413 ACS Chem. Biol. 2015, 10, 1355−1357
ACS Chemical Biology
■
■
UNPRECEDENTED PEPTIDE CYCLIZATION MOTIF DISCOVERED
Spotlight
ANOTHER STEP TOWARD UNIVERSAL BLOOD
Reprinted with permission from Kwan et al. J. Am. Chem. Soc. 2015, 137, 5696−5705. Copyright 2015 American Chemical Society.
Adapted by permission from Macmillan Publishers Ltd.: Nat. Chem. Seyedsayamdost et al., 7, 431−437, copyright 2015.
A, B, and O blood types are determined by the antigens that decorate the surface of red blood cells, and successful blood transfusions require the right match of donor blood to the patient. When the patient’s blood type is not known, doctors use O-type blood, the universal donor, to avoid a dangerous immune response. But over the past decade, researchers have been looking for enzymes that can trim the saccharide antigens off the surface of A- and B-type blood cells and eliminate this compatibility problem. Now Kwan et al. (J. Am. Chem. Soc. 2015, 137, 5695−5705) report a new step toward converting all donated blood to a universal type. They started with an endo-β-galactosidase from Streptococcus pneumoniae (an EABase) that chops off a trisaccharide portion of the surface saccharides, including the A and B antigens and two other sugars. The resulting surface saccharide lacks any blood type antigenA, B, or O. This wildtype EABase only cleaves Galβ-1,4−GlcNAc linkages efficiently, but some of the trisaccharides on the surface of blood cells are attached via various β-1,3 linkages. Using directed evolution and high throughput screening, the researchers found variants that also clip Galβ-1,3−GlcNAc bonds while maintaining their ability to snip β-1,4 linkages. They then used X-ray structure data, sitedirected mutagenesis, and a further round of directed evolution to improve the efficiency of the EABase to 170-fold greater for the Galβ-1,3−GlcNAc bonds than the wild-type enzyme. Kwan et al. then showed that the resulting enzyme cleaves both the β-1,4 and Galβ-1,3−GlcNAc linkage in saccharides on the surfaces of cells. Two remaining types of β-1,3 linkages that can be found in sugars on the surface of blood cells are not cleaved by this enzyme, but this initial work suggests that it should be possible to further broaden the specificity of EABases. In addition, the Galβ-1,3−GlcNAc linkages that can now be clipped, known as type 1, also occur on epithelial cells of organs, which suggests that this type of enzyme might hold promise as a way to make organs more compatible for transplants. Sarah A. Webb
When it comes to generating cyclic peptides, biosynthetic steps for closing the ring systems abound, ranging from amide bond formation between the N- and C-termini to cross-links between side chains. With respect to the latter category, an unprecedented macrocyclic peptide closure has recently been reported by Mohammad R. Seyedsayamdost and co-workers at Princeton University (Nat. Chem. 2015, 7, 431−437). Previous studies indicated the existence of a gene cluster controlled by the shp/rgg quorum sensing pathway of Streptococcus thermophilus that produced a cyclic 9-mer peptide (N-AKGDGWKVM-C) named streptide from a 30-mer precursor. This gene cluster (now called str), was known to encode the 30-mer peptide (StrA), a radical S-adenosylmethionine (SAM) enzyme (StrB), and a putative transporter (StrC). The Seyedsayamdost group used large-scale cultures to obtain enough streptide to carry out extensive 1- and 2-D NMR studies and obtain the first detailed structure of the macrocyclic peptide. The group identified a never-before-seen linkage occurring between unactivated carbons on lysine and tryptophan, the second and sixth residues in the 9-mer streptide chain. Turning their attention to the biosynthesis of streptide, the research team also determined that the radical SAM enzyme StrB contained two [4Fe-4S] clusters, one pertaining to the classic radical SAM enzyme active site and the other belonging to an extended C-terminal domain characteristic of the recently classified SPASM domain subfamily of radical SAM enzymes. Both metal clusters were shown to be essential for the lysineto-tryptophan cross-linking within the 30-mer StrA peptide, which was afterward trimmed by a yet-unidentified protease to form streptide. The research team also scanned the genomes of other bacterial species for the str cluster and found that it was widespread, enabling the authors to predict other species, including pathogenic streptococci, that could produce streptide. Heidi A. Dahlmann 1356
DOI: 10.1021/acschembio.5b00413 ACS Chem. Biol. 2015, 10, 1355−1357
ACS Chemical Biology
■
Spotlight
CAGING CAS9
Reprinted with permission from Hemphill et al. J. Am. Chem. Soc. 2015, DOI: 10.1021/ja512664v. Copyright 2015 American Chemical Society.
The general field of genome editing continues to gain momentum, as the CRISPR-Cas9 system is shown to function in a wide variety of organisms. Since the field is quite young, researchers continue to tease apart the mechanistic details of how this gene editing system functions. Others are already deploying CRISPR-Cas9 as a shortcut to making knockout plants and animals or for large-scale functional genomics screens. Besides the litany of ethnical questions, researchers are also discussing how to make this powerful gene editing system inducible in some manner. This would unlock new lines of experimentation where developmental or temporal disruption of a gene is desired. Now, Hemphill et al. (J. Am. Chem. Soc. 2015, DOI: 10.1021/ ja512664v) have introduced a conditionally controlled Cas9 protein that can be switched on in mammalian cells using ultraviolet (UV) light. To accomplish this task, the group turned to a suppressor approach wherein a stop codon is reprogrammed to incorporate a synthetic amino acid. Their choice of amino acid was a photocaged lysine residue, which is essentially a lysine side chain modified with a photocleavable protecting group. They tested the approach by individually changing a set of evolutionarily conserved Cas9 lysines located close to the guide RNA or genomic DNA interface to the photocaged lysine. Two of the caged Cas9 proteins demonstrated no detectable cleavage activity in cell lines until being treated with UV light. Those proteins with restored activity once treated with UV light were chosen for further experiments. While the initial screen for the inducible Cas9 activity involved cleaving a transgene, the utility of the photoactivated Cas9 for silencing an endogenous gene was next tested using a series of short guide RNAs against the receptor CD71. The result was a loss of CD71 protein only when the cells were irradiated with UV light, indicating that this caged Cas9 system could be widely applicable for inducible silencing of genes. This study points to an exciting direction for users of the CRISPR-Cas9 system, affording a new level of spatial and temporal control through a simple push of a UV light switch. Jason G. Underwood
1357
DOI: 10.1021/acschembio.5b00413 ACS Chem. Biol. 2015, 10, 1355−1357
