Exceptionally high-affinity Ras binders that remodel its effector domain

The Ras proteins are aberrantly activated in a wide range of human cancers, often endowing tumors with aggressive properties and resistance to therapy. Decades of effort to develop direct Ras inhibitors for clinical use have thus far failed, largely because of a lack of adequate small-molecule–binding pockets on the Ras surface. Here, we report the discovery of Ras-binding miniproteins from a naïve library and their evolution to afford versions with midpicomolar affinity to Ras. A series of biochemical experiments indicated that these miniproteins bind to the Ras effector domain as dimers, and high-resolution crystal structures revealed that these miniprotein dimers bind Ras in an unprecedented mode in which the Ras effector domain is remodeled to expose an extended pocket that connects two isolated pockets previously found to engage small-molecule ligands. We also report a Ras point mutant that stabilizes the protein in the open conformation trapped by these miniproteins. These findings provide new tools for studying Ras structure and function and present opportunities for the development of both miniprotein and small-molecule inhibitors that directly target the Ras proteins

The Autophagy-Related Beclin‑1 Protein Requires the Coiled-Coil and BARA Domains To Form a Homodimer with Submicromolar Affinity

Beclin-1 (BECN1) is an essential component of macroautophagy. This process is a highly conserved survival mechanism that recycles damaged cellular components or pathogens by encasing them in a bilayer vesicle that fuses with a lysosome to allow degradation of the vesicular contents. Mutations or altered expression profiles of BECN1 have been linked to various cancers and neurodegenerative diseases. Viruses, including HIV and herpes simplex virus 1 (HSV-1), are also known to specifically target BECN1 as a means of evading host defense mechanisms. Autophagy is regulated by the interaction between BECN1 and Bcl-2, a pro-survival protein in the apoptotic pathway that stabilizes the BECN1 homodimer. Disruption of the homodimer by phosphorylation or competitive binding promotes autophagy through an unknown mechanism. We report here the first recombinant synthesis (3–5 mg/L in an <i>Escherichia coli</i> culture) and characterization of full-length, human BECN1. Our analysis reveals that full-length BECN1 exists as a soluble homodimer (<i>K</i>_D ∼ 0.45 μM) that interacts with Bcl-2 (<i>K</i>_D = 4.3 ± 1.2 μM) and binds to lipid membranes. Dimerization is proposed to be mediated by a coiled-coil region of BECN1. A construct lacking the C-terminal BARA domain but including the coiled-coil region exhibits a homodimer <i>K</i>_D 3.5-fold weaker than that of full-length BECN1, indicating that both the BARA domain and the coiled-coil region of BECN1 contribute to dimer formation. Using site-directed mutagenesis, we show that residues at the C-terminus of the coiled-coil region previously shown to interact with the BARA domain play a key role in dimerization and mutations weaken the interface by ∼5-fold

THE EFFECT OF AUDIENCE SIZE ON MANAGERS' PRIVATE DISCLOSURES

I use experimental and survey evidence to investigate how and why audience size affects managers’ disclosure content in private meetings with investors. The experiment uses a 2x2 between-subjects design with 328 experienced managers. I predict and find that larger audiences decrease bad news disclosure because of increases in managers’ self-focus. I provide evidence of self-focus as the causal mechanism using both experimental manipulation and measurement. The survey elicits responses from 114 investor relations officers on actual private disclosure experiences and provides (a) support for my experimental assumption about the variability of audience size in practice and (b) evidence consistent with the experiment suggesting managers are unaware of the effect of audience size. I contribute by (1) providing evidence on the determinants of content in managers’ private disclosures, (2) demonstrating the effects of managers’ self-focus as a previously unexamined determinant of voluntary disclosure, and (3) providing additional descriptive evidence on managers’ private disclosures. My results may also provide increased understanding of the differences between private and public disclosures