A highly potent and specific MET therapeutic protein antagonist with both ligand-dependent and ligand-independent activity.

Activation of the MET oncogenic pathway has been implicated in the development of aggressive cancers that are difficult to treat with current chemotherapies. This has led to an increased interest in developing novel therapies that target the MET pathway. However, most existing drug modalities are confounded by their inability to specifically target and/or antagonize this pathway. Anticalins, a novel class of monovalent small biologics, are hypothesized to be "fit for purpose" for developing highly specific and potent antagonists of cancer pathways. Here, we describe a monovalent full MET antagonist, PRS-110, displaying efficacy in both ligand-dependent and ligand-independent cancer models. PRS-110 specifically binds to MET with high affinity and blocks hepatocyte growth factor (HGF) interaction. Phosphorylation assays show that PRS-110 efficiently inhibits HGF-mediated signaling of MET receptor and has no agonistic activity. Confocal microscopy shows that PRS-110 results in the trafficking of MET to late endosomal/lysosomal compartments in the absence of HGF. In vivo administration of PRS-110 resulted in significant, dose-dependent tumor growth inhibition in ligand-dependent (U87-MG) and ligand-independent (Caki-1) xenograft models. Analysis of MET protein levels on xenograft biopsy samples show a significant reduction in total MET following therapy with PRS-110 supporting its ligand-independent mechanism of action. Taken together, these data indicate that the MET inhibitor PRS-110 has potentially broad anticancer activity that warrants evaluation in patients.

Crystal structures of the Cid1 poly (U) polymerase reveal the mechanism for UTP selectivity.

Polyuridylation is emerging as a ubiquitous post-translational modification with important roles in multiple aspects of RNA metabolism. These poly (U) tails are added by poly (U) polymerases with homology to poly (A) polymerases; nevertheless, the selection for UTP over ATP remains enigmatic. We report the structures of poly (U) polymerase Cid1 from Schizoscaccharomyces pombe alone and in complex with UTP, CTP, GTP and 3'-dATP. These structures reveal that each of the 4 nt can be accommodated at the active site; however, differences exist that suggest how the polymerase selects UTP over the other nucleotides. Furthermore, we find that Cid1 shares a number of common UTP recognition features with the kinetoplastid terminal uridyltransferases. Kinetic analysis of Cid1's activity for its preferred substrates, UTP and ATP, reveal a clear preference for UTP over ATP. Ultimately, we show that a single histidine in the active site plays a pivotal role for poly (U) activity. Notably, this residue is typically replaced by an asparagine residue in Cid1-family poly (A) polymerases. By mutating this histidine to an asparagine residue in Cid1, we diminished Cid1's activity for UTP addition and improved ATP incorporation, supporting that this residue is important for UTP selectivity.

Structural insights into cis element recognition of non-polyadenylated RNAs by the Nab3-RRM.

Transcription termination of non-polyadenylated RNAs in Saccharomyces cerevisiae occurs through the action of the Nrd1-Nab3-Sen1 complex. Part of the decision to terminate via this pathway occurs via direct recognition of sequences within the nascent transcript by RNA recognition motifs (RRMs) within Nrd1 and Nab3. Here we present the 1.6 Å structure of Nab3-RRM bound to its UCUU recognition sequence. The crystal structure reveals clear density for a UCU trinucleotide and a fourth putative U binding site. Nab3-RRM establishes a clear preference for the central cytidine of the UCUU motif, which forms pseudo-base pairing interactions primarily through hydrogen bonds to main chain atoms and one serine hydroxyl group. Specificity for the flanking uridines is less defined; however, binding experiments confirm that these residues are also important for high affinity binding. Comparison of the Nab3-RRM to other structures of RRMs bound to polypyrimidine RNAs showed that this mode of recognition is similar to what is observed for the polypyrimidine-tract binding RRMs, and that the serine residue involved in pseudo-base pairing is only found in RRMs that bind to polypyrimidine RNAs that contain a cytosine base, suggesting a possible mechanism for discriminating between cytosine and uracil bases in RRMs that bind to polypyrimidine-containing RNA.

Cooperative interaction of transcription termination factors with the RNA polymerase II C-terminal domain.

Phosphorylation of the C-terminal domain (CTD) of RNA polymerase II controls the co-transcriptional assembly of RNA processing and transcription factors. Recruitment relies on conserved CTD-interacting domains (CIDs) that recognize different CTD phosphoisoforms during the transcription cycle, but the molecular basis for their specificity remains unclear. We show that the CIDs of two transcription termination factors, Rtt103 and Pcf11, achieve high affinity and specificity both by specifically recognizing the phosphorylated CTD and by cooperatively binding to neighboring CTD repeats. Single-residue mutations at the protein-protein interface abolish cooperativity and affect recruitment at the 3' end processing site in vivo. We suggest that this cooperativity provides a signal-response mechanism to ensure that its action is confined only to proper polyadenylation sites where Ser2 phosphorylation density is highest.

RNA-binding proteins: modular design for efficient function.

Many RNA-binding proteins have modular structures and are composed of multiple repeats of just a few basic domains that are arranged in various ways to satisfy their diverse functional requirements. Recent studies have investigated how different modules cooperate in regulating the RNA-binding specificity and the biological activity of these proteins. They have also investigated how multiple modules cooperate with enzymatic domains to regulate the catalytic activity of enzymes that act on RNA. These studies have shown how, for many RNA-binding proteins, multiple modules define the fundamental structural unit that is responsible for biological function.

Mis-translation of a computationally designed protein yields an exceptionally stable homodimer: implications for protein engineering and evolution.

We recently used computational protein design to create an extremely stable, globular protein, Top7, with a sequence and fold not observed previously in nature. Since Top7 was created in the absence of genetic selection, it provides a rare opportunity to investigate aspects of the cellular protein production and surveillance machinery that are subject to natural selection. Here we show that a portion of the Top7 protein corresponding to the final 49 C-terminal residues is efficiently mis-translated and accumulates at high levels in Escherichia coli. We used circular dichroism, size-exclusion chromatography, small-angle X-ray scattering, analytical ultra-centrifugation, and NMR spectroscopy to show that the resulting C-terminal fragment (CFr) protein adopts a compact, extremely stable, homo-dimeric structure. Based on the solution structure, we engineered an even more stable variant of CFr by disulfide-induced covalent circularisation that should be an excellent platform for design of novel functions. The accumulation of high levels of CFr exposes the high error rate of the protein translation machinery. The rarity of correspondingly stable fragments in natural proteins coupled with the observation that high quality ribosome binding sites are found to occur within E. coli protein-coding regions significantly less often than expected by random chance implies a stringent evolutionary pressure against protein sub-fragments that can independently fold into stable structures. The symmetric self-association between two identical mis-translated CFr sub-domains to generate an extremely stable structure parallels a mechanism for natural protein-fold evolution by modular recombination of protein sub-structures.