Entropy of stapled peptide inhibitors in free state is the major contributor to the improvement of binding affinity with the GK domain.

Stapled peptides are promising protein-protein interaction (PPI) inhibitors that can increase the binding potency. Different from small-molecule inhibitors in which the binding mainly depends on energetic interactions with their protein targets, the binding of stapled peptides has long been suggested to be benefited from entropy. However, it remains challenging to reveal the molecular features that lead to this entropy gain, which could originate from the stabilization of the stapled peptide in solution or from the increased flexibility of the complex upon binding. This hinders the rational design of stapled peptides as PPI inhibitors. Using the guanylate kinase (GK) domain of the postsynaptic density protein 95 (PSD-95) as the target, we quantified the enthalpic and entropic contributions by combining isothermal titration calorimetry (ITC), X-ray crystallography, and free energy calculations based on all-atom molecular dynamics (MD) simulations. We successfully designed a stapled peptide inhibitor (staple 1) of the PSD-95 GK domain that led to a 25-fold increase in the binding affinity (from tens of µMs to 1.36 µM) with high cell permeability. We showed that entropy indeed greatly enhanced the binding affinity and the entropy gain was mainly due to the constrained-helix structure of the stapled peptide in solution (free state). Based on staple 1, we further designed two other stapled peptides (staple 2 and 3), which exerted even larger entropy gains compared to staple 1 because of their more flexible bound complexes (bound state). However, for staple 2 and 3, the overall binding affinities were not improved, as the loose binding in their bound states led to an enthalpic loss that largely compensated the excess entropy gain. Our work suggests that increasing the stability of the stapled peptide in free solution is an effective strategy for the rational design of stapled peptides as PPI inhibitors.

Structural basis of GABARAP-mediated GABAA receptor trafficking and functions on GABAergic synaptic transmission.

GABAA receptors (GABAARs) are the primary fast inhibitory ion channels in the central nervous system. Dysfunction of trafficking and localization of GABAARs to cell membranes is clinically associated with severe psychiatric disorders in humans. The GABARAP protein is known to support the stability of GABAARs in synapses, but the underlying molecular mechanisms remain to be elucidated. Here, we show that GABARAP/GABARAPL1 directly binds to a previously unappreciated region in the γ2 subunit of GABAAR. We demonstrate that GABARAP functions to stabilize GABAARs via promoting its trafficking pathway instead of blocking receptor endocytosis. The GABARAPL1-γ2-GABAAR crystal structure reveals the mechanisms underlying the complex formation. We provide evidence showing that phosphorylation of γ2-GABAAR differentially modulate the receptor's binding to GABARAP and the clathrin adaptor protein AP2. Finally, we demonstrate that GABAergic synaptic currents are reduced upon specific blockage of the GABARAP-GABAAR complex formation. Collectively, our results reveal that GABARAP/GABARAPL1, but not other members of the Atg8 family proteins, specifically regulates synaptic localization of GABAARs via modulating the trafficking of the receptor.

Structural Basis Underlying Strong Interactions between Ankyrins and Spectrins.

Ankyrins (encoded by ANK1/2/3 corresponding to Ankyrin-R/B/G or AnkR/B/G), via binding to spectrins, connect plasma membranes with actin cytoskeleton to maintain mechanical strengths and to modulate excitabilities of diverse cells such as neurons, muscle cells, and erythrocytes. Cellular and genetic evidences suggest that each isoform of ankyrins pairs with a specific ß-spectrin in discrete subcellular membrane microdomains for distinct functions, although the molecular mechanisms underlying such ankyrin/ß-spectrin pairings are unknown. In this study, we discover that a conserved and short extension N-terminal to the ZU5N-ZU5C-UPA tandem (exZZU) is critical for each ankyrin to bind to ß-spectrins with high affinities. Structures of AnkB/G exZZU in complex with spectrin repeats13-15 of ß2/ß4-spectrins solved here reveal that the extension sequence of exZZU forms an additional ß-strand contributing to the structural stability and enhanced affinity of each ZU5N/spectrin repeat interaction. The complex structures further reveal that the UPA domain of exZZU directly participates in spectrin binding. Formation of the exZZU supramodule juxtaposes the ZU5N and UPA domains for simultaneous interacting with spectrin repeats 14 and 15. However, our biochemical and structural investigations indicate that the direct and strong interactions between ankyrins and ß-spectrins do not appear to determine their pairing specificities. Therefore, there likely exists additional mechanism(s) for modulating functional pairings between ankyrins and ß-spectrins in cells.

Potent and specific Atg8-targeting autophagy inhibitory peptides from giant ankyrins.

The mammalian Atg8 family proteins are central drivers of autophagy and contain six members, classified into the LC3 and GABARAP subfamilies. Due to their high sequence similarity and consequent functional overlaps, it is difficult to delineate specific functions of Atg8 proteins in autophagy. Here we discover a super-strong GABARAP-selective inhibitory peptide harbored in 270/480 kDa ankyrin-G and a super-potent pan-Atg8 inhibitory peptide from 440 kDa ankyrin-B. Structural studies elucidate the mechanism governing the Atg8 binding potency and selectivity of the peptides, reveal a general Atg8-binding sequence motif, and allow development of a more GABARAP-selective inhibitory peptide. These peptides effectively blocked autophagy when expressed in cultured cells. Expression of these ankyrin-derived peptides in Caenorhabditis elegans also inhibited autophagy, causing accumulation of the p62 homolog SQST-1, delayed development and shortened life span. Thus, these genetically encodable autophagy inhibitory peptides can be used to occlude autophagy spatiotemporally in living animals.

miR-23a/b promote tumor growth and suppress apoptosis by targeting PDCD4 in gastric cancer.

MicroRNAs (miRNAs) are short non-coding RNAs of 21-23 nucleotides that play important roles in virtually all biological pathways in mammals and in other multicellular organisms. miR-23a and miR-23b (miR-23a/b) are critical oncomiRs (miRNAs that are associated with human cancers) of gastric cancer, but their detailed roles in the initiation and progression of gastric cancer remain to be elucidated. In this study, we found that miR-23a/b were consistently upregulated in gastric cancer tissues. We then investigated the molecular mechanisms through which miR-23a/b contribute to gastric cancer and identified programmed cell death 4 (PDCD4) as a direct target gene of miR-23a/b. In contrast to the upregulated expression levels of miR-23a/b, PDCD4 protein levels were dramatically downregulated and inversely correlated with miR-23a/b in gastric cancer tissues. Moreover, we observed that cell apoptosis was increased by miR-23a/b inhibitors and decreased by miR-23a/b mimics in gastric cancer cells and that the restoration of PDCD4 expression attenuated the anti-apoptotic effects of miR-23a/b in gastric cancer cells, indicating that PDCD4 is a direct mediator of miR-23a/b functions. Finally, we showed that miR-23a/b significantly suppressed PDCD4 expression and enhanced tumor growth in a gastric cancer xenograft mouse model. Taken together, this study highlights an important role for miR-23a/b as oncomiRs in gastric cancer through the inhibition of PDCD4 translation. These findings may shed new light on the molecular mechanism of gastric carcinogenesis and provide a new avenue for gastric cancer treatment.