ErbB3 (HER3) is a member of the EGF receptor (EGFR) family of receptor tyrosine kinases, which, unlike the other three family members, contains a pseudo kinase in place of a tyrosine kinase domain. In cancer, ErbB3 activation is driven by a ligand-dependent mechanism through the formation of heterodimers with EGFR, ErbB2, or ErbB4 or via a ligand-independent process through heterodimerization with ErbB2 overexpressed in breast tumors or other cancers. Here we describe the crystal structure of the Fab fragment of an antagonistic monoclonal antibody KTN3379, currently in clinical development in human cancer patients, in complex with the ErbB3 extracellular domain. The structure reveals a unique allosteric mechanism for inhibition of ligand-dependent or ligand-independent ErbB3-driven cancers by binding to an epitope that locks ErbB3 in an inactive conformation. Given the similarities in the mechanism of ErbB receptor family activation, these findings could facilitate structure-based design of antibodies that inhibit EGFR and ErbB4 by an allosteric mechanism.cancer | surface receptor | cell signaling | therapeutic antibodies | crystal structure T he EGF receptor (EGFR)/ErbB family of receptor tyrosine kinases (RTKs) participates in a multitude of roles during embryonic development and in adult homeostasis. In healthy tissues, ErbB signal transduction is initiated through ligandinduced homo-or heterodimerization of receptor extracellular domains leading to the stimulation of tyrosine kinase activity and autophosphorylation of several tyrosine residues in the cytoplasmic domain followed by recruitment and activation of multiple downstream signaling pathways (1). In contrast, unregulated ErbB signaling through activating mutations, receptor overexpression, or aberrant autocrine ligand signaling loops can lead to cellular transformation and tumorigenesis (2). Thus, members of the ErbB receptor family (in particular EGFR and ErbB2) have become well-validated targets for the development of anticancer therapeutics, resulting in a number of Food and Drug Administration-approved and marketed monoclonal antibodies and small-molecule tyrosine kinase inhibitors used for treatment of different cancers (3).The activity of ErbB3 (also designated HER3) is normally regulated by the neuregulin (NRG) family of growth factors (4), but, unlike other members of the family, ErbB3 functions as an obligate heterodimer with other ErbB receptors because its cytoplasmic domain contains a pseudo kinase in place of a tyrosine kinase domain (5, 6). ErbB3 therefore takes part in heterodimer formation through ligand binding, whereas its coreceptor (most often ErbB2) provides enzymatic activity to phosphorylate multiple tyrosine residues located primarily in the ErbB3 C-terminal tail. The role of ErbB3 in cancer has been fully appreciated only within the last decade and has prompted the development of several monoclonal antibodies geared at inhibiting its action in solid tumors. ErbB3 phosphorylation and subsequent signaling have been associated wi...
Orphan riboswitch candidates are noncoding RNA motifs whose representatives are believed to function as genetic regulatory elements, but whose target ligands have yet to be identified. The study of certain orphans, particularly classes that have resisted experimental validation for many years, has led to the discovery of important biological pathways and processes once their ligands were identified. Previously, we highlighted details for four of the most common and intriguing orphan riboswitch candidates. This facilitated the validation of riboswitches for the signaling molecules c-di-AMP, ZTP, and ppGpp, the metal ion Mn, and the metabolites guanidine and PRPP. Such studies also yield useful linkages between the ligands sensed by the riboswitches and numerous biochemical pathways. In the current report, we describe the known characteristics of 30 distinct classes of orphan riboswitch candidates - some of which have remained unsolved for over a decade. We also discuss the prospects for uncovering novel biological insights via focused studies on these RNAs. Lastly, we make recommendations for experimental objectives along the path to finding ligands for these mysterious RNAs.
We recently implemented a bioinformatics pipeline that can uncover novel, but rare, riboswitch candidates as well as other noncoding RNA structures in bacteria. A prominent candidate revealed by our initial search efforts was called the ‘thiS motif’ because of its frequent association with a gene coding for the ThiS protein, which delivers sulfur to form the thiazole moiety of the thiamin precursor HET-P. In the current report, we describe biochemical and genetic data demonstrating that thiS motif RNAs function as sensors of the thiamin precursor HMP-PP, which is fused with HET-P ultimately to form the final active coenzyme thiamin pyrophosphate (TPP). HMP-PP riboswitches exhibit a distinctive architecture wherein an unusually small ligand-sensing aptamer is almost entirely embedded within an otherwise classic intrinsic transcription terminator stem. This arrangement yields remarkably compact genetic switches that bacteria use to tune the levels of thiamin precursors during the biosynthesis of this universally distributed coenzyme.
Comparative sequence analysis methods are highly effective for uncovering novel classes of structured noncoding RNAs (ncRNAs) from bacterial genomic DNA sequence datasets. Previously, we developed a computational pipeline to more comprehensively identify structured ncRNA representatives from individual bacterial genomes. This search process exploits the fact that genomic regions serving as templates for the transcription of structured RNAs tend to be present in longer than average noncoding 'intergenic regions' (IGRs) that are enriched in G and C nucleotides compared to the remainder of the genome. In the present study, we apply this computational pipeline to identify structured ncRNA candidates from 26 diverse bacterial species. Numerous novel structured ncRNA motifs were discovered, including several riboswitch candidates, one whose ligand has been identified and others that have yet to be experimentally validated. Our findings support recent predictions that hundreds of novel riboswitch classes and other ncRNAs remain undiscovered among the limited number of bacterial species whose genomes have been completely sequenced.
The Fibro-purF motif is a putative structured noncoding RNA domain that was discovered previously in species of Fibrobacter by using comparative sequence analysis methods. An updated bioinformatics search yielded a total of only 30 unique-sequence representatives, exclusively found upstream of the purF gene that codes for the enzyme amidophosphoribosyltransferase. This enzyme synthesizes the compound 5-phospho-D-ribosylamine (PRA), which is the first committed step in purine biosynthesis. The consensus model for Fibro-purF motif RNAs includes a predicted three-stem junction that carries numerous conserved nucleotide positions within the regions joining the stems. This architecture appears to be of sufficient size and complexity for the formation of the ligand-binding aptamer portion of a riboswitch. In this study, we conducted biochemical analyses of a representative Fibro-purF motif RNA to confirm that the RNA generally folds according to the predicted consensus model. However, due to the instability of PRA, binding of this ligand candidate by the RNA could not be directly assessed. Genetic analyses were used to demonstrate that Fibro-purF motif RNAs regulate gene expression in accordance with predicted PRA concentrations. These findings indicate that Fibro-purF motif RNAs are genetic regulation elements that likely suppress PRA biosynthesis when sufficient levels of this purine precursor are present.
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