Structural and functional characterisation of human RNA helicase DHX8 provides insights into the mechanism of RNA-stimulated ADP release.

DHX8 is a crucial DEAH-box RNA helicase involved in splicing and required for the release of mature mRNA from the spliceosome. Here, we report the biochemical characterisation of full-length human DHX8 and the catalytically active helicase core DHX8Δ547, alongside crystal structures of DHX8Δ547 bound to ADP and a structure of DHX8Δ547 bound to poly(A)6 single-strand RNA. Our results reveal that DHX8 has an in vitro binding preference for adenine-rich RNA and that RNA binding triggers the release of ADP through significant conformational flexibility in the conserved DEAH-, P-loop and hook-turn motifs. We demonstrate the importance of R620 and both the hook-turn and hook-loop regions for DHX8 helicase activity and propose that the hook-turn acts as a gatekeeper to regulate the directional movement of the 3' end of RNA through the RNA-binding channel. This study provides an in-depth understanding of the activity of DHX8 and contributes insights into the RNA-unwinding mechanisms of the DEAH-box helicase family.

Inhibition of Ral GTPases Using a Stapled Peptide Approach.

Aberrant Ras signaling drives numerous cancers, and drugs to inhibit this are urgently required. This compelling clinical need combined with recent innovations in drug discovery including the advent of biologic therapeutic agents, has propelled Ras back to the forefront of targeting efforts. Activated Ras has proved extremely difficult to target directly, and the focus has moved to the main downstream Ras-signaling pathways. In particular, the Ras-Raf and Ras-PI3K pathways have provided conspicuous enzyme therapeutic targets that were more accessible to conventional drug-discovery strategies. The Ras-RalGEF-Ral pathway is a more difficult challenge for traditional medicinal development, and there have, therefore, been few inhibitors reported that disrupt this axis. We have used our structure of a Ral-effector complex as a basis for the design and characterization of α-helical-stapled peptides that bind selectively to active, GTP-bound Ral proteins and that compete with downstream effector proteins. The peptides have been thoroughly characterized biophysically. Crucially, the lead peptide enters cells and is biologically active, inhibiting isoform-specific RalB-driven cellular processes. This, therefore, provides a starting point for therapeutic inhibition of the Ras-RalGEF-Ral pathway.

Crystal structures of stapled and hydrogen bond surrogate peptides targeting a fully buried protein-helix interaction.

Constrained α-helical peptides are an exciting class of molecule designed to disrupt protein-protein interactions (PPIs) at a surface-exposed helix binding site. Complexes that engage more than one helical face account for over a third of structurally characterized helix PPIs, including several examples where the helix is fully buried. However, no constrained peptides have been reported that have targeted this class of interaction. We report the design of stapled and hydrogen bond surrogate (HBS) peptides mimicking the helical tail of the malaria parasite invasion motor myosin (myoA), which presents polar and hydrophobic functionality on all three faces in binding its partner, myoA tail interacting protein (MTIP), with high affinity. The first structures of these different constrained peptides bound to the same target are reported, enabling a direct comparison between these constraints and between staples based on monosubstituted pentenyl glycine (pGly) and disubstituted pentenyl alanine (pAla). Importantly, installation of these constraints does not disrupt native interactions in the buried site, so the affinity of the wild-type peptide is maintained.

Multimeric complexes among ankyrin-repeat and SOCS-box protein 9 (ASB9), ElonginBC, and Cullin 5: insights into the structure and assembly of ECS-type Cullin-RING E3 ubiquitin ligases.

Proteins of the ankyrin-repeat and SOCS-box (ASB) family act as the substrate-recognition subunits of ECS-type (ElonginBC-Cullin-SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that catalyze the specific polyubiquitination of cellular proteins to target them for degradation by the proteasome. Therefore, ASB multimeric complexes are involved in numerous cell processes and pathways; however, their interactions, assembly, and biological roles remain poorly understood. To enhance our understanding of ASB CRL systems, we investigated the structure, affinity, and assembly of the quaternary multisubunit complex formed by ASB9, Elongin B, Elongin C (EloBC), and Cullin 5. Here, we describe the application of several biophysical techniques including differential scanning fluorimetry, isothermal titration calorimetry (ITC), nanoelectrospray ionization, and ion-mobility mass spectrometry (IM-MS) to provide structural and thermodynamic information for a quaternary ASB CRL complex. We find that ASB9 is unstable alone but forms a stable ternary complex with EloBC that binds with high affinity to the Cullin 5 N-terminal domain (Cul5NTD) but not to Cul2NTD. The structure of the monomeric ASB9-EloBC-Cul5NTD quaternary complex is revealed by molecular modeling and is consistent with IM-MS and temperature-dependent ITC data. This is the first experimental study to validate structural information for the assembly of the quaternary N-terminal region of an ASB CRL complex. The results suggest that ASB E3 ligase complexes function and assemble in an analogous manner to that of other CRL systems and provide a platform for further molecular investigation of this important protein family. The data reported here will also be of use for the future development of chemical probes to examine the biological function and modulation of other ECS-type CRL systems.

Regulation of the Plasmodium motor complex: phosphorylation of myosin A tail-interacting protein (MTIP) loosens its grip on MyoA.

The interaction between the C-terminal tail of myosin A (MyoA) and its light chain, myosin A tail domain interacting protein (MTIP), is an essential feature of the conserved molecular machinery required for gliding motility and cell invasion by apicomplexan parasites. Recent data indicate that MTIP Ser-107 and/or Ser-108 are targeted for intracellular phosphorylation. Using an optimized MyoA tail peptide to reconstitute the complex, we show that this region of MTIP is an interaction hotspot using x-ray crystallography and NMR, and S107E and S108E mutants were generated to mimic the effect of phosphorylation. NMR relaxation experiments and other biophysical measurements indicate that the S108E mutation serves to break the tight clamp around the MyoA tail, whereas S107E has a smaller but measurable impact. These data are consistent with physical interactions observed between recombinant MTIP and native MyoA from Plasmodium falciparum lysates. Taken together these data support the notion that the conserved interactions between MTIP and MyoA may be specifically modulated by this post-translational modification.

Interaction and dynamics of the Plasmodium falciparum MTIP-MyoA complex, a key component of the invasion motor in the malaria parasite.

The myosin tail domain interacting protein-myosin A (MTIP-MyoA) protein complex is an essential element of the motor driving invasion of red blood cells by the Plasmodium species that cause malaria. Here we report the key determinants of binding at the MTIP/MyoA interface, and the first structural study on the complex in solution using protein NMR.