X-ray structure of a human cardiac muscle troponin C/troponin I chimera in two crystal forms

The X-ray crystal structure of a human cardiac muscle troponin C/troponin I chimera has been determined in two different crystal forms and shows a conformation of the complex that differs from that previously observed by NMR. The chimera consists of the N-terminal domain of troponin C (cTnC; residues 1–80) fused to the switch region of troponin I (cTnI; residues 138–162). In both crystal forms, the cTnI residues form a six-turn α-helix that lays across the hydrophobic groove of an adjacent cTnC molecule in the crystal structure. In contrast to previous models, the cTnI helix runs in a parallel direction relative to the cTnC groove and completely blocks the calcium desensitizer binding site of the cTnC–cTnI interface.

Mechanisms of CP190 Interaction with Architectural Proteins in Drosophila Melanogaster

Most of the known Drosophila architectural proteins interact with an important cofactor, CP190, that contains three domains (BTB, M, and D) that are involved in protein–protein interactions. The highly conserved N-terminal CP190 BTB domain forms a stable homodimer that interacts with unstructured regions in the three best-characterized architectural proteins: dCTCF, Su(Hw), and Pita. Here, we identified two new CP190 partners, CG4730 and CG31365, that interact with the BTB domain. The CP190 BTB resembles the previously characterized human BCL6 BTB domain, which uses its hydrophobic groove to specifically associate with unstructured regions of several transcriptional repressors. Using GST pull-down and yeast two-hybrid assays, we demonstrated that mutations in the hydrophobic groove strongly affect the affinity of CP190 BTB for the architectural proteins. In the yeast two-hybrid assay, we found that architectural proteins use various mechanisms to improve the efficiency of interaction with CP190. Pita and Su(Hw) have two unstructured regions that appear to simultaneously interact with hydrophobic grooves in the BTB dimer. In dCTCF and CG31365, two adjacent regions interact simultaneously with the hydrophobic groove of the BTB and the M domain of CP190. Finally, CG4730 interacts with the BTB, M, and D domains of CP190 simultaneously. These results suggest that architectural proteins use different mechanisms to increase the efficiency of interaction with CP190.

Selective binding of small molecules to Vibrio cholerae DsbA offers a starting point for the design of novel antibacterials

DsbA enzymes catalyze oxidative folding of proteins that are secreted into the periplasm of Gram-negative bacteria, and they are indispensable for the virulence of human pathogens such as Vibrio cholerae and Escherichia coli. Therefore, targeting DsbA represents an attractive approach to control bacterial virulence. X-ray crystal structures reveal that DsbA enzymes share a similar fold, however, the hydrophobic groove adjacent to the active site, which is implicated in substrate binding, is shorter and flatter in the structure of V. cholerae DsbA (VcDsbA) compared to E. coli DsbA (EcDsbA). The flat and largely featureless nature of this hydrophobic groove is challenging for the development of small molecule inhibitors. Using fragment-based screening approaches, we have identified a novel small molecule, based on the benzimidazole scaffold, that binds to the hydrophobic groove of oxidized VcDsbA with a KD of 446 ± 10 µM. The same benzimidazole compound has ~8-fold selectivity for VcDsbA over EcDsbA and binds to oxidized EcDsbA, with KD > 3.5 mM. We generated a model of the benzimidazole complex with VcDsbA using NMR data but were unable to determine the structure of the benzimidazole bound EcDsbA using either NMR or X-ray crystallography. Therefore, a structural basis for the observed selectivity is unclear. To better understand ligand binding to these two enzymes we crystallized each of them in complex with a known ligand, the bile salt sodium taurocholate. The crystal structures show that taurocholate adopts different binding poses in complex with VcDsbA and EcDsbA, and reveals the protein-ligand interactions that stabilize the different modes of binding. This work highlights the capacity of fragment-based drug discovery to identify inhibitors of challenging protein targets. In addition, it provides a starting point for development of more potent and specific VcDsbA inhibitors that act through a novel anti-virulence mechanism.

Structural basis for membrane recruitment of ATG16L1 by WIPI2 in Autophagy

Autophagy is a cellular process that degrades cytoplasmic cargo by engulfing it in a double membrane vesicle, known as the autophagosome, and delivering it to the lysosome. The ATG12-5-16L1 complex is responsible for conjugating members of the ubiquitin-like ATG8 protein family to phosphatidylethanolamine in the growing autophagosomal membrane, known as the phagophore. ATG12-5-16L1 is recruited to the phagophore by a subset of the phosphatidylinositol 3-phosphate-binding seven bladed â-propeller WIPI proteins. We determined the crystal structure of WIPI2d in complex with the WIPI2 interacting region (W2IR) of ATG16L1 comprising residues 207-230 at 1.85 Å resolution. The structure shows that the ATG16L1 W2IR adopts an alpha helical conformation and binds in an electropositive and hydrophobic groove between WIPI2 â-propeller blades 2 and 3. Mutation of residues at the interface reduces or blocks the recruitment of ATG12-5-16L1 and the conjugation of the ATG8 protein LC3B to synthetic membranes. Interface mutants show a decrease in starvation-induced autophagy. Comparisons across the four human WIPIs suggest that WIPI1 and 2 belong to a W2IR-binding subclass responsible for localizing ATG12-5-16L1 and driving ATG8 lipidation, whilst WIPI3 and 4 belong to a second W34IR-binding subclass responsible for localizing ATG2, and so directing lipid supply to the nascent phagophore. The structure provides a framework for understanding the regulatory node connecting two central events in autophagy initiation, the action of the autophagic PI 3-kinase complex on the one hand, and ATG8 lipidation on the other.

Identification of ubiquitin variants that inhibit the E2 ubiquitin conjugating enzyme, Ube2k

Transfer of ubiquitin to substrate proteins regulates most processes in eukaryotic cells. E2 enzymes are a central component of the ubiquitin machinery, and generally determine the type of ubiquitin signal generated and thus the ultimate fate of substrate proteins. The E2, Ube2k, specifically builds degradative ubiquitin chains on diverse substrates. Here we have identified protein-based reagents, called ubiquitin variants (UbVs), that bind tightly and specifically to Ube2k. Crystal structures reveal that the UbVs bind to the E2 enzyme at a hydrophobic cleft that is distinct from the active site and previously identified ubiquitin binding sites. We demonstrate that the UbVs are potent inhibitors of Ube2k and block both ubiquitin charging of the E2 enzyme, and E3-catalysed ubiquitin transfer. The binding site of the UbVs suggests they directly clash with the ubiquitin activating enzyme, while potentially disrupting interactions with E3 ligases via allosteric effects. Our data reveal the first protein-based inhibitors of Ube2k and unveil a hydrophobic groove that could be an effective target for inhibiting Ube2k and other E2 enzymes.

Structural Basis for Membrane Recruitment of ATG16L1 by WIPI2 in Autophagy

Autophagy is a cellular process that degrades cytoplasmic cargo by engulfing it in a double membrane vesicle, known as the autophagosome, and delivering it to the lysosome. The ATG12-5-16L1 complex is responsible for conjugating members of the ubiquitin-like ATG8 protein family to phosphatidylethanolamine in the growing autophagosomal membrane, known as the phagophore. ATG12-5-16L1 is recruited to the phagophore by a subset of the phosphatidylinositol 3-phosphate-binding seven bladed beta-propeller WIPI proteins. We determined the crystal structure of WIPI2d in complex with the WIPI2 interacting region (W2IR) of ATG16L1 comprising residues 207-230 at 1.85 Angstrom resolution. The structure shows that the ATG16L1 W2IR adopts an alpha helical conformation and binds in an electropositive and hydrophobic groove between WIPI2 beta-propeller blades 2 and 3. Mutation of residues at the interface reduces or blocks the recruitment of ATG12-5-16L1 and the conjugation of the ATG8 protein LC3B to synthetic membranes. Interface mutants show a decrease in starvation-induced autophagy. Comparisons across the four human WIPIs suggest that WIPI1 and 2 belong to a W2IR-binding subclass responsible for localizing ATG12-5-16L1 and driving ATG8 lipidation, whilst WIPI3 and 4 belong to a second W34IR-binding subclass responsible for localizing ATG2, and so directing lipid supply to the nascent phagophore. The structure provides a framework for understanding the regulatory node connecting two central events in autophagy initiation, the action of the autophagic PI 3-kinase complex on the one hand, and ATG8 lipidation on the other.

ERH facilitates microRNA maturation through the interaction with the N-terminus of DGCR8

The microprocessor complex cleaves the primary transcript of microRNA (pri-miRNA) to initiate miRNA maturation. Microprocessor is known to consist of RNase III DROSHA and dsRNA-binding DGCR8. Here, we identify Enhancer of Rudimentary Homolog (ERH) as a new component of Microprocessor. Through a crystal structure and biochemical experiments, we reveal that ERH uses its hydrophobic groove to bind to a conserved region in the N-terminus of DGCR8, in a 2:2 stoichiometry. Knock-down of ERH or deletion of the DGCR8 N-terminus results in a reduced processing of suboptimal pri-miRNAs in polycistronic miRNA clusters. ERH increases the processing of suboptimal pri-miR-451 in a manner dependent on its neighboring pri-miR-144. Thus, the ERH dimer may mediate ‘cluster assistance’ in which Microprocessor is loaded onto a poor substrate with help from a high-affinity substrate in the same cluster. Our study reveals a role of ERH in the miRNA biogenesis pathway.

XPO1E571K Mutation Modifies Exportin 1 Localisation and Interactome in B-Cell Lymphoma

The XPO1 gene encodes exportin 1 (XPO1) that controls the nuclear export of cargo proteins and RNAs. Almost 25% of primary mediastinal B-cell lymphoma (PMBL) and classical Hodgkin lymphoma (cHL) cases harboured a recurrent XPO1 point mutation (NM_003400, chr2:g61718472C>T) resulting in the E571K substitution within the hydrophobic groove of the protein, the site of cargo binding. We investigated the impact of the XPO1E571K mutation using PMBL/cHL cells having various XPO1 statuses and CRISPR–Cas9-edited cells in which the E571K mutation was either introduced or knocked-out. We first confirmed that the mutation was present in both XPO1 mRNA and protein. We observed that the mutation did not modify the export capacity but rather the subcellular localisation of XPO1 itself. In particular, mutant XPO1 bound to importin β1 modified the nuclear export/import dynamics of relevant cargoes.

Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme

PPP-family phosphatases such as PP1 have little intrinsic specificity. Cofactors can target PP1 to substrates or subcellular locations, but it remains unclear how they might confer sequence-specificity on PP1. The cytoskeletal regulator Phactr1 is a neuronally enriched PP1 cofactor that is controlled by G-actin. Structural analysis showed that Phactr1 binding remodels PP1's hydrophobic groove, creating a new composite surface adjacent to the catalytic site. Using phosphoproteomics, we identified mouse fibroblast and neuronal Phactr1/PP1 substrates, which include cytoskeletal components and regulators. We determined high-resolution structures of Phactr1/PP1 bound to the dephosphorylated forms of its substrates IRSp53 and spectrin αII. Inversion of the phosphate in these holoenzyme-product complexes supports the proposed PPP-family catalytic mechanism. Substrate sequences C-terminal to the dephosphorylation site make intimate contacts with the composite Phactr1/PP1 surface, which are required for efficient dephosphorylation. Sequence specificity explains why Phactr1/PP1 exhibits orders-of-magnitude enhanced reactivity towards its substrates, compared to apo-PP1 or other PP1 holoenzymes.