May 2009 news and views

Research highlights

PSI-SGKB [doi:10.1038/nchembio0509-280]

Antibody double feature

Antibodies typically recognize a single antigen with high affinity and specificity. Is 'one antibody, one antigen epitope' a fixed feature of antibody recognition, or could one antibody recognize two binding sites? To test this question, Bostrom et al. started with the therapeutic monoclonal antibody trastuzumab, which recognizes human epidermal growth factor receptor 2 (HER2). Using phage display, the light chain of the complementarity-determining region was randomized, and variants were selected that bound to vascular endothelial growth factor (VEGF). In some cases, the selected antibodies had lost their affinity for HER2, whereas in other cases, the resulting antibodies had high affinity for both antigens. Solving the crystal structure of an antibody-HER2 complex and an antibody-VEGF complex for a dual-specificity antibody revealed substantial overlap in the binding sites. However, based on alanine scanning mutagenesis, the regions that contributed most significantly to binding energy were largely distinct. An optimized HER2-VEGF antibody with low-nanomolar affinity for both antigens effectively blocked HER2- and VEGF-mediated cell proliferation and tumor growth in a mouse xenograft model. These results highlight an unexpected plasticity in antibody recognition that is reminiscent of the promiscuity sometimes seen in protein interaction domains and that could have important biological and therapeutic implications. ( Science 323, 1610–1614, 2009) JK

Cycles of four

β-turns mediate many protein-protein interactions. Although larger scaffolds have been designed to present four amino acid side chains in the characteristic geometry of β-turns, the most obvious mimetics, cyclic tetrapeptides, have not been systematically examined because of the synthetically difficult cyclization reaction. Beierle et al. now report a synthetic route in which macrocyclization by copper(I)-mediated [3+2] Huisgen dipolar cycloaddition results in cyclic pseudotetrapeptides containing one or two triazole moieties. Using this reaction, all 16 stereoisomers of the somatostatin pharmacophore, Phe-Trp-Lys-Thr, were synthesized. NMR structures revealed that the cyclized peptides formed rigid rectangular scaffolds, very similar in shape to native β-turns, with predictable side chain conformations. Despite being composed of the identical linear amino acid sequence, the 16 somatostatin-based peptides exhibited activities ranging from no detectable binding to any of the somatostatin receptor subtypes to binding receptor subtypes selectively or nonselectively. In an accompanying paper, Horne et al. use this synthetic approach to demonstrate that a naturally occurring cyclic tetrapeptide histone deacetylase inhibitor is most potent in the cis-trans-trans-trans conformation, not the all-trans conformation favored in solution. The new synthetic accessibility of structurally well-defined β-turn mimetics should prove useful for developing chemical tools for this class of protein-protein interactions and more generally for defining three-dimensional pharmacophores. ( Angew. Chem. Int. Ed., published online 5 March 2009, doi:10.1002/anie.200805901; Angew. Chem. Int. Ed., published online 6 March 2009, doi:10.1002/anie.200805900) JK

Sensing positivity

Kumaran Ramamurthi

Asymmetric protein localization along an axis is the basis of developmental programs in all organisms, including single-celled ones such as the rod-shaped bacterium Bacillus subtilis. SpoVM (VM) is a 26-residue B. subtilis peptide produced during sporulation that is involved in the assembly of a protein coat around an internal membrane (termed the forespore) generated from the mother cell. A key proline residue in the amphipathic α-helix formed by the VM peptide is critical for VM to localize to the forespore surface. To test further requirements for VM membrane localization, Ramamurthi et al. monitored VM in B. subtilis mutants that have membrane protrusions and bulges with various degrees of curvature. Combined with localization patterns of VM in the heterologous host organisms Escherichia coli and Saccharomyces cerevisiae and in vitro on unilamellar phospholipid vesicles of various sizes, the data suggested that VM binds to membranes with a positive (convex) curvature. Assuming a length of 40 Å for VM, it seemed improbable that a single VM molecule lying flat on the surface of a sphere the size of a forespore (approximately 1 μm in diameter) could be influenced by such a small degree of curvature. Based on experiments where they varied the concentration of VM in the in vitro assay, the authors noted a range of VM-compatible sizes (less than 4 μm), which suggests that several VM molecules may act cooperatively to sense curvature. ( Science 323, 1354–1357, 2009) MB

I'm added to your chain

Investigations of peptides containing non-natural amino acids have led to a deeper understanding of protein conformation and have provided access to new scaffolds for inhibitor development. Though synthetic and some limited genetic reprogramming protocols to access these sequences are available, complementary methods are needed to improve yields and versatility. The Suga group has previously developed a tRNA aminoacylation ribozyme called flexizyme that can load almost any single amino acid onto an initiator tRNA for use in cell-free protein expression. This technology was limited, however, in only being able to include one non-natural residue, restricted to the N terminus, in the final peptide. Goto et al. now significantly expand this process with the unexpected observation that peptides can be used to initiate ribosomal protein production. The peptide sequences, ranging from two to five residues and incorporating one to three non-natural residues, were successfully loaded onto the initiator tRNA in 30–85% yields. Elongation of the peptides was less consistent, with yields of 12–133% relative to a standard methionine initiator. These variations could not be traced to any particular property of the peptides, but they point to unexpected promiscuity of the initiation machinery in regards to both length and composition of the starter sequence. Further investigation should provide continued increases in biosynthetic flexibility and may lead to more subtle information about how the initiation machinery operates. ( J. Am. Chem. Soc., published online 20 March 2009, doi:10.1021/ja900597d) CG

Peptides LURE the pollen tube

Satohiro Okuda

Fertilization in flowering plants is mediated by the pollen tube, which grows from the pollen grain to the ovaries and provides a route for sperm cell migration. Current models suggest that chemoattractant molecules secreted by female gametophytic cells are responsible for guiding pollen tube growth toward the ovules. Now, Okuda et al. report the isolation of LUREs, a class of cysteine-rich polypeptides (CRPs) that have these pollen tube–guidance properties. Earlier studies had suggested that synergid cells flanking the ovules are a likely source of chemoattractants. The authors devised a method to isolate synergid cells from the embryo sacs of Torenia fournieri. Expressed sequence tag analysis showed that a group of CRPs was particularly abundant in these cells. The authors focused on two of these proteins, LURE1 and LURE2, and showed by immunoblotting that these small proteins are secreted and localized to the filiform apparatus of synergid cells. LUREs were able to redirect the growth of pollen tubes from T. fournieri in a dose-dependent manner but did not serve as a chemoattractant for pollen tubes of other plant species. The identification of LUREs advances a view that CRPs might have diverse signaling roles in plants and highlights that these systems are now open to molecular-level interrogation. ( Nature 458, 357–361, 2009) TLS

Fucose forced out

Glycosylation is a prevalent post-translational modification, but gaining insight into the role of these carbohydrates or harnessing them for practical applications is complicated by the extensive diversity and overlapping functionality of natural sugars. Introduction of modified sugars, such as azido sialic acids, has allowed significant advances in understanding eukaryotic systems. However, though bacterial glycosylation is important in adhesion and infection, corresponding tools for these species are limited. Yi et al. now utilize a bacterial fucose salvage pathway to incorporate a range of C6-modified sugars into Escherichia coli. The authors first removed the only intrinsic GDP-fucose biosynthesis pathway by disrupting the genes for GDP-mannose dehydratase and GDP-fucose synthetase. They then introduced Fkp, a protein from Bacterioides fragilis capable of converting fucose into phosphofucose and then GDP-fucose. Fkp was able to restore the wild-type phenotype, thereby confirming that the salvage pathway is functional. Feeding of ten different fucose analogs resulted in their incorporation into cell surface glycans, with reactive groups such as azides, aldehydes and amines appropriately positioned for further labeling. The method is complicated by the fact that downstream enzymes may not be as promiscuous in their usage of the modified sugars as Fkp, as exemplified by the authors' observation of shortened LPS chains. However, this technique, and its likely extension to other carbohydrate scaffolds, represents an exciting means to interrogate glycosylation and may enable the generation of polysaccharide-based vaccines. (Proc. Natl. Acad. Sci. USA 106, 4207–4212, 2009) CG

Sensing its fat

The hepatocyte nuclear factor 4 (HNF4) nuclear receptors are a family of transcription factors for genes involved in glucose and lipid metabolism. They can bind long-chain fatty acids (LCFAs), which seems to be required but not sufficient for their transcriptional activity. Palanker et al. generated mutants lacking dHNF4, the only HNF4 family member in Drosophila melanogaster, after finding that this paralog has an expression pattern in tissues similar to that in tissues where the two mammalian HNF4 paralogs are found—those involved in nutrient metabolism and storage, lipid mobilization, and excretory functions.The dHNF4 mutants are highly sensitive to starvation, which suggests that they are unable to properly access stored forms of energy. Starved dHNF4 mutants have defects in lipid droplet morphology, as well as higher levels of triglycerides (TAGs) and free LCFAs, which suggests that dHNF4 is involved in mobilization of lipid stores. From a microarray study, the authors found that the dHNF4 mutants affected the expression of numerous genes involved in mitochondrial β-oxidation, in which fatty acids are converted into high-energy electron donors for energy production. Reduced β-oxidation can cause an accumulation of TAGs and LCFAs, explaining the phenotypes seen in the dHNF4 mutant flies. The authors also found that fatty acids activate dHNF4, with evidence that binding of the fatty acids to dHNF4 is required for its activation. These results form a model where, like other nuclear receptors, HNF4 is involved in a feedforward pathway that maintains lipid homeostasis through the breakdown of TAGs and the sensing and utilization of LCFAs for energy production via β-oxidation. (Cell Metab. 9, 228–239, 2009) MB

Float on through to the other side

Nature Structural & Molecular Biology

Glutamine amidotransferase (GATase) domains are found in many biosynthetic multifunctional enzymes; GATases catalyze the hydrolysis of glutamine, producing glutamate and ammonia, which is the substrate for the reaction catalyzed by a synthase/synthetase domain. LaRonde-LeBlanc et al. recently reported the X-ray crystal structure of a glutamine-dependent NAD⁺ synthetase from M. tuberculosis. This protein has an N-terminal GATase domain and a C-terminal synthetase domain, which synthesizes NAD⁺ using ATP/Mg²⁺, NaAD⁺ and the ammonia produced by the GATase domain. Kinetic experiments indicate that the catalytic activities of the two domains are tightly coupled: formation of the NaAD-AMP intermediate in the synthetase domain activates the GATase domain (resulting in the hydrolysis of glutamine and the production of ammonia). The structure reveals two tunnels in the enzyme: a 33-Å-long tunnel that glutamine uses to reach the active site of the GATase domain, and a 40-Å tunnel that enables ammonia to reach the activated carboxyl group of NaAD-AMP in the synthetase domain. This is one of the longest ammonia tunnels reported thus far, and it is lined with hydrophilic and hydrophobic amino acids, unlike the ammonia tunnels of other GATase-containing proteins, which contain only hydrophilic or hydrophobic amino acids. It may be possible to exploit the structural and mechanistic differences between this enzyme and human NAD⁺ synthetase for the design of highly selective drugs to combat tuberculosis. ( Nat. Struct. Mol. Biol., published online 8 March 2009, doi:10.1038/nsmb.1567) JMF

Written by Mirella Bucci, Joshua M. Finkelstein, Catherine Goodman, Joanne Kotz & Terry L. Sheppard