Peter Simpson

Streptococcus pyogenes evades innate immune surveillance in part by deploying the cell-envelope protease ScpA to cleave and inactivate complement anaphylatoxins, thereby suppressing neutrophil recruitment to sites of infection. However, the structural basis of substrate recognition by ScpA has remained unresolved. Here we present cryo-electron microscopy structures of ScpA in complex with C5a and C3a, revealing that ScpA functions as a dual-specificity anaphylatoxinase through a conserved multi-exosite recognition mechanism. The second fibronectin domain of ScpA engages the conserved four-helix bundle core of the anaphylatoxin through complementary electrostatic surfaces, while the PA domain captures the conserved C-terminal arginine of the anaphylatoxin, positioning the flexible substrate tail for cleavage at the protease active site. Solution NMR spectroscopy independently supports this two-site mechanism and shows that intrinsic flexibility within the anaphylatoxin fold is exploited for productive active-site engagement. Rather than recognising a specific linear sequence flanking the scissile bond, ScpA targets conserved structural and electrostatic features shared by C3a and C5a. Although C4a retains the conserved C-terminal arginine and can engage individual ScpA exosites, it fails to form the stable cooperative complex required for productive binding and proteolysis. These findings provide a structural framework for bacterial interference with complement signalling and inform the rational design of improved ScpA-based vaccine antigens and biotherapeutics.

Intrinsically disordered regions (IDRs) are prevalent in RNA-binding proteins (RBPs), yet their roles in RNA interactions remain poorly defined. We examined RNA-binding regulation by structured and disordered regions of LARP6, an RBP with a diverse RNA-binding repertoire. Mass spectrometry-based RNA interaction mapping in living cells identified direct LARP6–RNA contacts within the structured La-module and its flanking IDRs. Mutagenesis and individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP) revealed the La-module, but not the IDRs, as essential for LARP6 RNA binding. Deletion of the N-terminal IDR broadened LARP6 RNA footprints, uncovering a role in RNA-binding selectivity. This is achieved through a composite mechanism of restricting the conformational flexibility of the adjacent La-module, forming auxiliary contacts with the RNA, and modulating RNA access for binding. The IDR-mediated RNA-binding selectivity is critical for LARP6-mediated promotion of cancer cell viability and invasion. Our findings uncover a previously unrecognised critical function for IDRs in promoting selective RBP–RNA recognition, by affecting the binding specificity of their adjacent structured domains.

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays an important role in tumour biology by promoting the stabilisation and activity of oncogenic ‘client’ proteins. Inhibition of Hsp90 by small-molecule drugs, acting via its ATP hydrolysis site, has shown promise as a molecularly targeted cancer therapy. Owing to the importance of Hop and other tetratricopeptide repeat (TPR)-containing cochaperones in regulating Hsp90 activity, the Hsp90-TPR domain interface is an alternative site for inhibitors, which could result in effects distinct from ATP site binders. The TPR binding site of Hsp90 cochaperones includes a shallow, positively charged groove that poses a significant challenge for druggability. Herein, we report the apo, solution-state structure of Hop TPR2A which enables this target for NMR-based screening approaches. We have designed prototype TPR ligands that mimic key native ‘carboxylate clamp’ interactions between Hsp90 and its TPR cochaperones and show that they block binding between Hop TPR2A and the Hsp90 C-terminal MEEVD peptide. We confirm direct TPR-binding of these ligands by mapping ¹H–¹⁵N HSQC chemical shift perturbations to our new NMR structure. Our work provides a novel structure, a thorough assessment of druggability and robust screening approaches that may offer a potential route, albeit difficult, to address the chemically challenging nature of the Hop TPR2A target, with relevance to other TPR domain interactors.

The composition of the mitochondrial membrane is important for its architecture and proper function. Mitochondria depend on a tightly regulated supply of phospholipid via intra‐mitochondrial synthesis and by direct import from the endoplasmic reticulum. The Ups1/PRELI‐like family together with its mitochondrial chaperones (TRIAP1/Mdm35) represent a unique heterodimeric lipid transfer system that is evolutionary conserved from yeast to man. Work presented here provides new atomic resolution insight into the function of a human member of this system. Crystal structures of free TRIAP1 and the TRIAP1–SLMO1 complex reveal how the PRELI domain is chaperoned during import into the intermembrane mitochondrial space. The structural resemblance of PRELI‐like domain of SLMO1 with that of mammalian phoshatidylinositol transfer proteins (PITPs) suggest that they share similar lipid transfer mechanisms, in which access to a buried phospholipid‐binding cavity is regulated by conformationally adaptable loops. See also: [F Yu et al ][1] (July 2015) ![][2] The structures of TRIAP1 and its complex with SLMO1 reveal how the PRELI domain is chaperoned during mitochondrial import. Access to an internal phospholipid binding cavity is likely regulated by conformationally adaptable loops. EMBO Reports (2015) 16: 824–835 [1]: http://dx.doi.org/10.15252/embr.201540137 [2]: pending:yes

Pseudomonas aeruginosa is a Gram‐negative opportunistic bacterium, synonymous with cystic fibrosis patients, which can cause chronic infection of the lungs. This pathogen is a model organism to study biofilms: a bacterial population embedded in an extracellular matrix that provide protection from environmental pressures and lead to persistence. A number of Chaperone‐Usher Pathways, namely CupA‐CupE, play key roles in these processes by assembling adhesive pili on the bacterial surface. One of these, encoded by the cupB operon, is unique as it contains a nonchaperone‐usher gene product, CupB5. Two‐partner secretion (TPS) systems are comprised of a C‐terminal integral membrane β‐barrel pore with tandem N‐terminal POTRA (POlypeptide TRansport Associated) domains located in the periplasm (TpsB) and a secreted substrate (TpsA). Using NMR we show that TpsB4 (LepB) interacts with CupB5 and its predicted cognate partner TpsA4 (LepA), an extracellular protease. Moreover, using cellular studies we confirm that TpsB4 can translocate CupB5 across the P. aeruginosa outer membrane, which contrasts a previous observation that suggested the CupB3 P‐usher secretes CupB5. In support of our findings we also demonstrate that tps4/cupB operons are coregulated by the RocS1 sensor suggesting P. aeruginosa has developed synergy between these systems. Furthermore, we have determined the solution‐structure of the TpsB4‐POTRA1 domain and together with restraints from NMR chemical shift mapping and in vivo mutational analysis we have calculated models for the entire TpsB4 periplasmic region in complex with both TpsA4 and CupB5 secretion motifs. The data highlight specific residues for TpsA4/CupB5 recognition by TpsB4 in the periplasm and suggest distinct roles for each POTRA domain.

The BAG6 complex resides in the cytosol and acts as a sorting point to target diverse hydrophobic protein substrates along their appropriate paths, including proteasomal degradation and ER membrane insertion. Composed of a trimeric complex of BAG6, TRC35 and UBL4A, the BAG6 complex is closely associated with SGTA, a co-chaperone from which it can obtain hydrophobic substrates. SGTA consists of an N-terminal dimerisation domain (SGTA_NT), a central tetratricopeptide repeat (TPR) domain, and a glutamine rich region towards the C-terminus. Here we solve a solution structure of the SGTA dimerisation domain and use biophysical techniques to investigate its interaction with two different UBL domains from the BAG6 complex. The SGTA_NT structure is a dimer with a tight hydrophobic interface connecting two sets of four alpha helices. Using a combination of NMR chemical shift perturbation, isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) experiments we have biochemically characterised the interactions of SGTA with components of the BAG6 complex, the ubiquitin-like domain (UBL) containing proteins UBL4A and BAG6. We demonstrate that the UBL domains from UBL4A and BAG6 directly compete for binding to SGTA at the same site. Using a combination of structural and interaction data we have implemented the HADDOCK protein-protein interaction docking tool to generate models of the SGTA-UBL complexes. This atomic level information contributes to our understanding of the way in which hydrophobic proteins have their fate decided by the collaboration between SGTA and the BAG6 complex.

The small ubiquitin-like modifier (SUMO) can form polymeric chains that are important signals in cellular processes such as meiosis, genome maintenance and stress response. The SUMO-targeted ubiquitin ligase RNF4 engages with SUMO chains on linked substrates and catalyses their ubiquitination, which targets substrates for proteasomal degradation. Here we use a segmental labelling approach combined with solution nuclear magnetic resonance (NMR) spectroscopy and biochemical characterization to reveal how RNF4 manipulates the conformation of the SUMO chain, thereby facilitating optimal delivery of the distal SUMO domain for ubiquitin transfer.

We report the solution structures of the VPg proteins from feline calicivirus (FCV) and murine norovirus (MNV), which have been determined by nuclear magnetic resonance spectroscopy. In both cases, the core of the protein adopts a compact helical structure flanked by flexible N and C termini. Remarkably, while the core of FCV VPg contains a well-defined three-helix bundle, the MNV VPg core has just the first two of these secondary structure elements. In both cases, the VPg cores are stabilized by networks of hydrophobic and salt bridge interactions. The Tyr residue in VPg that is nucleotidylated by the viral NS7 polymerase (Y24 in FCV, Y26 in MNV) occurs in a conserved position within the first helix of the core. Intriguingly, given its structure, VPg would appear to be unable to bind to the viral polymerase so as to place this Tyr in the active site without a major conformation change to VPg or the polymerase. However, mutations that destabilized the VPg core either had no effect on or reduced both the ability of the protein to be nucleotidylated and virus infectivity and did not reveal a clear structure-activity relationship. The precise role of the calicivirus VPg core in virus replication remains to be determined, but knowledge of its structure will facilitate future investigations.

RbpA is a small non–DNA-binding transcription factor that associates with RNA polymerase holoenzyme and stimulates transcription in actinobacteria, including Streptomyces coelicolor and Mycobacterium tuberculosis. RbpA seems to show specificity for the vegetative form of RNA polymerase as opposed to alternative forms of the enzyme. Here, we explain the basis of this specificity by showing that RbpA binds directly to the principal σ subunit in these organisms, but not to more diverged alternative σ factors. Nuclear magnetic resonance spectroscopy revealed that, although differing in their requirement for structural zinc, the RbpA orthologues from S. coelicolor and M. tuberculosis share a common structural core domain, with extensive, apparently disordered, N- and C-terminal regions. The RbpA–σ interaction is mediated by the C-terminal region of RbpA and σ domain 2, and S. coelicolor RbpA mutants that are defective in binding σ are unable to stimulate transcription in vitro and are inactive in vivo . Given that RbpA is essential in M. tuberculosis and critical for growth in S. coelicolor , these data support a model in which RbpA plays a key role in the σ cycle in actinobacteria.