Cryo-EM structures of ClpB complexes reveal a pathway for substrate transfer and translocation during protein disaggregation.

Summary AAA+ proteins form asymmetric hexameric rings that hydrolyze ATP and thread substrate proteins through a central channel via mobile substrate-binding pore loops. Understanding how ATPase and threading activities are regulated and intertwined is key to understanding the AAA+ protein mechanism. We studied the disaggregase ClpB, which contains tandem ATPase domains (AAA1, AAA2) and shifts between low and high ATPase and threading activities. Coiled-coil M-domains repress ClpB activity by encircling the AAA1 ring. Here, we determine the mechanism of ClpB activation by comparing ATPase mechanisms and cryo-EM structures of ClpB wild-type and a constitutively active ClpB M-domain mutant. We show that ClpB activation reduces ATPase cooperativity and induces a sequential mode of ATP hydrolysis in the AAA2 ring, the main ATPase motor. AAA1 and AAA2 rings do not work synchronously but in alternating cycles. This ensures high grip, enabling substrate threading via a processive, rope-climbing mechanism.

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Several bacterial pathogens produce nucleotidyl cyclase toxins to manipulate eukaryotic host cells. Inside host cells they are activated by endogenous cofactors to produce high levels of cyclic nucleotides (cNMPs). The ExoY toxin from Pseudomonas aeruginosa (PaExoY) and the ExoY-like module (VnExoY) found in the MARTX (Multifunctional-Autoprocessing Repeats-in-ToXin) toxin of Vibrio nigripulchritudo share modest sequence similarity (~38%) but were both recently shown to be activated by actin after their delivery to the eukaryotic host cell. Here, we further characterized the ExoY-like cyclase of V. nigripulchritudo. We show that, in contrast to PaExoY that requires polymerized actin (F-actin) for maximum activation, VnExoY is selectively activated by monomeric actin (G-actin). These two enzymes also display different nucleotide substrate and divalent cation specificities. In vitro in presence of the cation Mg2+, the F-actin activated PaExoY exhibits a promiscuous nucleotidyl cyclase activity with the substrate preference GTP>ATP≥UTP>CTP, while the G-actin activated VnExoY shows a strong preference for ATP as substrate, as it is the case for the well-known calmodulin-activated adenylate cyclase toxins from Bordetella pertussis or Bacillus anthracis. These results suggest that the actin-activated nucleotidyl cyclase virulence factors despite sharing a common activator may actually display a greater variability of biological effects in infected cells than initially anticipated.

Many actin-binding proteins (ABPs) use complex multidomain architectures to integrate and coordinate multiple signals and interactions with the dynamic remodeling of actin cytoskeleton. In these proteins, small segments that are intrinsically disordered in their unbound native state can be functionally as important as identifiable folded units. These functional intrinsically disordered regions (IDRs) are however difficult to identify and characterize in vitro. Here, we try to summarize the state of the art in understanding the structural features and interfacial properties of IDRs involved in actin self-assembly dynamics. Recent structural and functional insights into the regulation of widespread, multifunctional WH2/b-thymosin domains, and of other IDRs such as those associated with WASP/ WAVE, formin or capping proteins are examined. Understanding the functional versatility of IDRs in actin assembly requires apprehending by multiple structural and functional approaches their large conformational plasticity and dynamics in their interactions. In many modular ABPs, IDRs relay labile interactions with multiple partners and act as interaction hubs in interdomain and protein-protein interfaces. They thus control multiple conformational transitions between the inactive and active states or between various active states of multidomain ABPs, and play an important role to coordinate the high turnover of interactions in actin self-assembly dynamics. V C 2013 Wiley Periodicals, Inc.

Because of their large conformational heterogeneity, structural characterization of intrinsically disordered proteins (IDPs) is very challenging using classical experimental methods alone. In this study, we use NMR and small-angle x-ray scattering (SAXS) data with multiple molecular dynamics (MD) simulations to describe the conformational ensemble of the fully disordered verprolin homology domain of the neural Aldrich syndrome protein involved in the regulation of actin polymerization. First, we studied several back-calculation software of SAXS scattering intensity and optimized the adjustable parameters to accurately calculate the SAXS intensity from an atomic structure. We also identified the most appropriate force fields for MD simulations of this IDP. Then, we analyzed four conformational ensembles of neural Aldrich syndrome protein verprolin homology domain, two generated with the program flexible-meccano with or without NMR-derived information as input and two others generated by MD simulations with two different force fields. These four conformational ensembles were compared to available NMR and SAXS data for validation. We found that MD simulations with the AMBER-03w force field and the TIP4P/ 2005s water model are able to correctly describe the conformational ensemble of this 67-residue IDP at both local and global level.

Immunotoxins are emerging candidates for cancer therapeutics. These biomolecules consist of a cell targeting protein combined to a polypeptide toxin.Associations of both entities can be achieved either chemically by covalent bonds or genetically creating fusion proteins. However, chemical agents can affect activity and/or stability of the conjugate proteins and additional purification steps are often required to isolate the final conjugate from unwanted by-products. As for fusion proteins, they often suffer from low solubility and yield.In this report, we describe a straightforward conjugation process to generate an immunotoxin using co-associating peptides (named K3 and E3), originating from the tetramerization domain of p53. To that end, a nanobody targeting the human epidermal growth factor receptor 2 (nano-HER2) and a protein toxin fragment from Pseudomonas aeruginosa Exotoxin A (TOX) were genetically fused to the E3 and K3 peptides. Entities were produced separately in E. coli in soluble forms and at high yields. The nano-HER2 fused to the E3 or K3 helixes (nano-HER2-E3 and nano-HER2-K3) and the co-assembled immunotoxins (nano-HER2-K3E3-TOX and nano-HER2-E3K3-TOX) presented binding specificity on HER2 overexpressing cells with relative binding constants in the low nanomolar to picomolar range. Both toxin modules (E3-TOX and K3-TOX) and the combined immunotoxins exhibited similar cytotoxicity levels compared to the toxin alone (TOX). Finally, nano-HER2-K3E3-TOX and nano-HER2-E3K3-TOX evaluated on various breast cancer cells were highly potent and specific to kill HER2-overexpressing breast cancer cells with IC 50 values in the picomolar range. Altogether, we demonstrate that this non-covalent conjugation method using two co-assembling peptides can be easily implemented for modular engineering of immunotoxins targeting different types of cancers.

ErbB2 (or HER2) is a receptor tyrosine kinase overexpressed in some breast cancers, associated with poor prognosis. Treatments targeting the receptor extracellular and kinase domains have greatly improved disease outcome in the last twenty years. In parallel, the structures of these domains have been described, enabling better mechanistic understanding of the receptor function and targeted inhibition. However, ErbB2 disordered C-terminal cytoplasmic tail (CtErbB2) remains very poorly characterized in terms of structure, dynamics and detailed functional mechanism. Yet, it is where signal transduction is triggered, via phosphorylation of tyrosine residues, and carried out, via interaction with adaptor proteins. Here we report the first description of ErbB2 disordered tail at atomic resolution, using NMR and SAXS. We show that although no part of CtErbB2 has any stable secondary or tertiary structure, it has around 20% propensity for a N-terminal helix that is suspected to interact with the kinase domain, and many PPII stretches distributed all along the sequence, forming potential SH3 and WW domains binding sites. Moreover, we identified a long-range transient contact involving CtErbB2 termini. These characteristics suggest new potential mechanisms of auto-regulation and protein-protein interaction. SIGNIFICANCE We report here the first description of the receptor tyrosine kinase ErbB2 disordered tail (CtErbB2) at atomic resolution, using NMR and SAXS. We show that although CtErbB2 exhibits no stable structure, it does exhibit partial secondary and tertiary structures likely important for its function. These structural elements are consistent with an active role of the C-terminal tail in the regulation of the receptor's activity, thanks to the presence of preformed structures for intramolecular interactions, as well as long-range contacts modulating accessibility of those sites and proline interaction sites distinct from the main tyrosine sites. Together, those results reinforce the view that disordered tails of receptors are more than random anchors for partners.