RNA Binding by the KTS Splice Variants of Wilms' Tumor Suppressor Protein WT1.

Wilms' tumor suppressor protein WT1 regulates the expression of multiple genes through binding of the Cys2-His2 zinc finger domain to promoter sites. WT1 has also been proposed to be involved in post-transcriptional regulation, by binding to RNA using the same set of zinc fingers. WT1 has two major splice variants, where the Lys-Thr-Ser (KTS) tripeptide is inserted into the linker between the third and fourth zinc fingers. To obtain insights into the mechanism by which the different WT1 splice variants recognize both DNA and RNA, we have determined the solution structure of the WT1 (-KTS) zinc finger domain in complex with a 29mer stem-loop RNA. Zinc fingers 1-3 bind in a widened major groove favored by the presence of a bulge nucleotide in the double-stranded helical stem. Fingers 2 and 3 make specific contacts with the nucleobases in a conserved AUGG sequence in the helical stem. Nuclear magnetic resonance chemical shift mapping and relaxation analysis show that fingers 1-3 of the two splice variants (-KTS and +KTS) of WT1 form similar complexes with RNA. Finger 4 of the -KTS isoform interacts weakly with the RNA loop, an interaction that is abrogated in the +KTS isoform, and both isoforms bind with similar affinity to the RNA. In contrast, finger 4 is required for high-affinity binding to DNA and insertion of KTS into the linker of fingers 3 and 4 abrogates DNA binding. While finger 1 is required for RNA binding, it is dispensable for binding to consensus DNA sites.

Coordination of a Single Calcium Ion in the EF-hand Maintains the Off State of the Stromal Interaction Molecule Luminal Domain.

Store operated calcium (Ca2+) entry (SOCE) is the process whereby endoplasmic reticulum (ER) Ca2+ store depletion causes Orai1-composed Ca2+ channels on the plasma membrane (PM) to open, mediating a rise in cytosolic Ca2+ levels. Stromal interaction molecules (STIMs) are the proteins that directly sense ER Ca2+ content and gate Orai1 channels due to store depletion. The trigger for STIM activation is Ca2+ unbinding from the ER lumen-oriented domains, which consist of a nonconserved amino (N) terminal region and EF-hand and sterile α motif (SAM) domains (EF-SAM), highly conserved from humans to Caenorhabditis elegans. Solution NMR structures of the human EF-SAM domains have been determined at high Ca2+ concentrations; however, no direct structural view of the Ca2+ binding mode has been elucidated. Further, no atomic resolution data currently exists on EF-SAM at low Ca2+ levels. Here, we determined the X-ray crystal structure of the C. elegans STIM luminal domain, revealing that EF-SAM binds a single Ca2+ ion with pentagonal bipyramidal geometry and an ancillary α-helix formed by the N-terminal region acts as a brace to stabilize EF-SAM. Using solution NMR, we observed EF-hand domain unfolding and a conformational exchange between folded and unfolded states involving the ancillary α-helix and the canonical EF-hand in low Ca2+. Remarkably, we also detected an α-helix (+Ca2+) to ß-strand (-Ca2+) transition at the terminal SAM domain α-helix. Collectively, our analyses indicate that one canonically bound Ca2+ ion is sufficient to stabilize the quiescent luminal domain structure, precluding unfolding, conformational exchange, and secondary structure transformation.

Force-dependent allostery of the α-catenin actin-binding domain controls adherens junction dynamics and functions.

α-catenin is a key mechanosensor that forms force-dependent interactions with F-actin, thereby coupling the cadherin-catenin complex to the actin cytoskeleton at adherens junctions (AJs). However, the molecular mechanisms by which α-catenin engages F-actin under tension remained elusive. Here we show that the α1-helix of the α-catenin actin-binding domain (αcat-ABD) is a mechanosensing motif that regulates tension-dependent F-actin binding and bundling. αcat-ABD containing an α1-helix-unfolding mutation (H1) shows enhanced binding to F-actin in vitro. Although full-length α-catenin-H1 can generate epithelial monolayers that resist mechanical disruption, it fails to support normal AJ regulation in vivo. Structural and simulation analyses suggest that α1-helix allosterically controls the actin-binding residue V796 dynamics. Crystal structures of αcat-ABD-H1 homodimer suggest that α-catenin can facilitate actin bundling while it remains bound to E-cadherin. We propose that force-dependent allosteric regulation of αcat-ABD promotes dynamic interactions with F-actin involved in actin bundling, cadherin clustering, and AJ remodeling during tissue morphogenesis.

Inhibition of K-RAS4B by a Unique Mechanism of Action: Stabilizing Membrane-Dependent Occlusion of the Effector-Binding Site.

KRAS is frequently mutated in several of the most lethal types of cancer; however, the KRAS protein has proven a challenging drug target. K-RAS4B must be localized to the plasma membrane by prenylation to activate oncogenic signaling, thus we endeavored to target the protein-membrane interface with small-molecule compounds. While all reported lead compounds have low affinity for KRAS in solution, the potency of Cmpd2 was strongly enhanced when prenylated K-RAS4B is associated with a lipid bilayer. We have elucidated a unique mechanism of action of Cmpd2, which simultaneously engages a shallow pocket on KRAS and associates with the lipid bilayer, thereby stabilizing KRAS in an orientation in which the membrane occludes its effector-binding site, reducing RAF binding and impairing activation of RAF. Furthermore, enrichment of Cmpd2 on the bilayer enhances potency by promoting interaction with KRAS. This insight reveals a novel approach to developing inhibitors of membrane-associated proteins.

Multiplexed Real-Time NMR GTPase Assay for Simultaneous Monitoring of Multiple Guanine Nucleotide Exchange Factor Activities from Human Cancer Cells and Organoids.

Small GTPases (sGTPases) are critical switch-like regulators that mediate several important cellular functions and are often mutated in human cancers. They are activated by guanine nucleotide exchange factors (GEFs), which specifically catalyze the exchange of GTP for GDP. GEFs coordinate signaling networks in normal cells, and are frequently deregulated in cancers. sGTPase signaling pathways are complex and interconnected; however, most GEF assays do not reveal such complexity. In this Communication, we describe the development of a unique real-time NMR-based multiplexed GEF assay that employs distinct isotopic labeling schemes for each sGTPase protein to enable simultaneous observation of six proteins of interest. We monitor nucleotide exchange of KRas, Rheb, RalB, RhoA, Cdc42 and Rac1 in a single system, and assayed the activities of GEFs in lysates of cultured human cells and 3D organoids derived from pancreatic cancer patients. We observed potent activation of RhoA by lysates of HEK293a cells transfected with GEF-H1, along with weak stimulation of Rac1, which we showed is indirect. Our functional analyses of pancreatic cancer-derived organoids revealed higher GEF activity for RhoA than other sGTPases, in line with RNA-seq data indicating high expression of RhoA-specific GEFs.

From Stores to Sinks: Structural Mechanisms of Cytosolic Calcium Regulation.

All eukaryotic cells have adapted the use of the calcium ion (Ca2+) as a universal signaling element through the evolution of a toolkit of Ca2+ sensor, buffer and effector proteins. Among these toolkit components, integral and peripheral proteins decorate biomembranes and coordinate the movement of Ca2+ between compartments, sense these concentration changes and elicit physiological signals. These changes in compartmentalized Ca2+ levels are not mutually exclusive as signals propagate between compartments. For example, agonist induced surface receptor stimulation can lead to transient increases in cytosolic Ca2+ sourced from endoplasmic reticulum (ER) stores; the decrease in ER luminal Ca2+ can subsequently signal the opening surface channels which permit the movement of Ca2+ from the extracellular space to the cytosol. Remarkably, the minuscule compartments of mitochondria can function as significant cytosolic Ca2+ sinks by taking up Ca2+ in a coordinated manner. In non-excitable cells, inositol 1,4,5 trisphosphate receptors (IP3Rs) on the ER respond to surface receptor stimulation; stromal interaction molecules (STIMs) sense the ER luminal Ca2+ depletion and activate surface Orai1 channels; surface Orai1 channels selectively permit the movement of Ca2+ from the extracellular space to the cytosol; uptake of Ca2+ into the matrix through the mitochondrial Ca2+ uniporter (MCU) further shapes the cytosolic Ca2+ levels. Recent structural elucidations of these key Ca2+ toolkit components have improved our understanding of how they function to orchestrate precise cytosolic Ca2+ levels for specific physiological responses. This chapter reviews the atomic-resolution structures of IP3R, STIM1, Orai1 and MCU elucidated by X-ray crystallography, electron microscopy and NMR and discusses the mechanisms underlying their biological functions in their respective compartments within the cell.

Backbone resonance assignments of the F-actin binding domain of mouse αN-catenin.

α-Catenin is a filamentous actin (F-actin) binding protein that links the classical cadherin-catenin complex to the actin cytoskeleton at adherens junctions (AJs). Its C-terminal F-actin binding domain is required for regulating the dynamic interaction between AJs and the actin cytoskeleton during tissue development. Thus, obtaining the molecular details of this interaction is a crucial step towards understanding how α-catenin plays critical roles in biological processes, such as morphogenesis, cell polarity, wound healing and tissue maintenance. Here we report the backbone atom (1HN, 15N, 13Cα, 13Cß and 13C') resonance assignments of the C-terminal F-actin binding domain of αN-catenin.

Biochemical Classification of Disease-associated Mutants of RAS-like Protein Expressed in Many Tissues (RIT1).

RAS-like protein expressed in many tissues 1 (RIT1) is a disease-associated RAS subfamily small guanosine triphosphatase (GTPase). Recent studies revealed that germ-line and somatic RIT1 mutations can cause Noonan syndrome (NS), and drive proliferation of lung adenocarcinomas, respectively, akin to RAS mutations in these diseases. However, the locations of these RIT1 mutations differ significantly from those found in RAS, and do not affect the three mutational "hot spots" of RAS. Moreover, few studies have characterized the GTPase cycle of RIT1 and its disease-associated mutants. Here we developed a real-time NMR-based GTPase assay for RIT1 and investigated the effect of disease-associated mutations on GTPase cycle. RIT1 exhibits an intrinsic GTP hydrolysis rate similar to that of H-RAS, but its intrinsic nucleotide exchange rate is ∼4-fold faster, likely as a result of divergent residues near the nucleotide binding site. All of the disease-associated mutations investigated increased the GTP-loaded, activated state of RIT1 in vitro, but they could be classified into two groups with different intrinsic GTPase properties. The S35T, A57G, and Y89H mutants exhibited more rapid nucleotide exchange, whereas F82V and T83P impaired GTP hydrolysis. A RAS-binding domain pulldown assay indicated that RIT1 A57G and Y89H were highly activated in HEK293T cells, whereas T83P and F82V exhibited more modest activation. All five mutations are associated with NS, whereas two (A57G and F82V) have also been identified in urinary tract cancers and myeloid malignancies. Characterization of the effects on the GTPase cycle of RIT1 disease-associated mutations should enable better understanding of their role in disease processes.

Calmodulin and STIM proteins: Two major calcium sensors in the cytoplasm and endoplasmic reticulum.

The calcium (Ca(2+)) ion is a universal signalling messenger which plays vital physiological roles in all eukaryotes. To decode highly regulated intracellular Ca(2+) signals, cells have evolved a number of sensor proteins that are ideally adapted to respond to a specific range of Ca(2+) levels. Among many such proteins, calmodulin (CaM) is a multi-functional cytoplasmic Ca(2+) sensor with a remarkable ability to interact with and regulate a plethora of structurally diverse target proteins. CaM achieves this 'multi-talented' functionality through two EF-hand domains, each with an independent capacity to bind targets, and an adaptable flexible linker. By contrast, stromal interaction molecule-1 and -2 (STIMs) have evolved for a specific role in endoplasmic reticulum (ER) Ca(2+) sensing using EF-hand machinery analogous to CaM; however, whereas CaM structurally adjusts to dissimilar binding partners, STIMs use the EF-hand machinery to self-regulate the stability of the Ca(2+) sensing domain. The molecular mechanisms underlying the Ca(2+)-dependent signal transduction by CaM and STIMs have revealed a remarkable repertoire of actions and underscore the flexibility of nature in molecular evolution and adaption to discrete Ca(2+) levels. Recent genomic sequencing efforts have uncovered a number of disease-associated mutations in both CaM and STIM1. This article aims to highlight the most recent key structural and functional findings in the CaM and STIM fields, and discusses how these two Ca(2+) sensor proteins execute their biological functions.

Siladenoserinols A-L: new sulfonated serinol derivatives from a tunicate as inhibitors of p53-Hdm2 interaction.

Siladenoserinols A-L were isolated from a tunicate as inhibitors of p53-Hdm2 interaction, a promising target for cancer chemotherapy. Their structures including the absolute configurations were elucidated to be new sulfonated serinol derivatives, each of which contains a 6,8-dioxabicyclo[3.2.1]octane unit and either glycerophosphocholine or glycerophosphoethanolamine moiety. They inhibited p53-Hdm2 interaction with IC(50) values of 2.0-55 µM. Among them, siladenoserinol A and B exhibited the strongest inhibition with an IC(50) value of 2.0 µM.