An octameric PqiC toroid stabilises the outer-membrane interaction of the PqiABC transport system.

The E. coli Paraquat Inducible (Pqi) Pathway is a putative Gram-negative phospholipid transport system. The pathway comprises three components: an integral inner membrane protein (PqiA), a periplasmic spanning MCE family protein (PqiB) and an outer membrane lipoprotein (PqiC). Interactions between all complex components, including stoichiometry, remain uncharacterised; nevertheless, once assembled into their quaternary complex, the trio of Pqi proteins are anticipated to provide a continuous channel between the inner and outer membranes of diderms. Here, we present X-ray structures of both the native and a truncated, soluble construct of the PqiC lipoprotein, providing insight into its biological assembly, and utilise neutron reflectometry to characterise the nature of the PqiB-PqiC-membrane interaction. Finally, we employ phenotypic complementation assays to probe specific PqiC residues, which imply the interaction between PqiB and PqiC is less intimate than previously anticipated.

Ubiquitin-specific protease 11 structure in complex with an engineered substrate mimetic reveals a molecular feature for deubiquitination selectivity.

Ubiquitin-specific proteases (USPs) are crucial for controlling cellular proteostasis and signaling pathways but how deubiquitination is selective remains poorly understood, in particular between paralogues. Here, we developed a fusion tag method by mining the Protein Data Bank and trapped USP11, a key regulator of DNA double-strand break repair, in complex with a novel engineered substrate mimetic. Together, this enabled structure determination of USP11 as a Michaelis-like complex that revealed key S1 and S1' binding site interactions with a substrate. Combined mutational, enzymatic, and binding experiments identified Met77 in linear diubiquitin as a significant residue that leads to substrate discrimination. We identified an aspartate "gatekeeper" residue in the S1' site of USP11 as a contributing feature for discriminating against linear diubiquitin. When mutated to a glycine, the corresponding residue in paralog USP15, USP11 acquired elevated activity toward linear diubiquitin in-gel shift assays, but not controls. The reverse mutation in USP15 confirmed that this position confers paralog-specific differences impacting diubiquitin cleavage rates. The results advance our understanding of the molecular basis for the higher selectivity of USP11 compared to USP15 and may aid targeted inhibitor development. Moreover, the reported carrier-based crystallization strategy may be applicable to other challenging targets.

Protein target highlights in CASP15: Analysis of models by structure providers.

We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology.

Moving toward a better understanding of the model bacterial predator Bdellovibrio bacteriovorus.

The bacterial predator Bdellovibrio bacteriovorus is a model for the wider phenomenon of bacteria:bacteria predation, and the specialization required to achieve a lifestyle dependent on prey consumption. Bdellovibrio bacteriovorus is able to recognize, enter and ultimately consume fellow Gram-negative bacteria, killing these prey from within their periplasmic space, and lysing the host at the end of the cycle. The classic phenotype-driven characterization (and observation of predation) has benefitted from an increased focus on molecular mechanisms and fluorescence microscopy and tomography, revealing new features of several of the lifecycle stages. Herein we summarize a selection of these advances and describe likely areas for exploration that will push the field toward a more complete understanding of this fascinating 'two-cell' system.

Discovery of peptide ligands targeting a specific ubiquitin-like domain-binding site in the deubiquitinase USP11.

Ubiquitin-specific proteases (USPs) reverse ubiquitination and regulate virtually all cellular processes. Defined noncatalytic domains in USP4 and USP15 are known to interact with E3 ligases and substrate recruitment factors. No such interactions have been reported for these domains in the paralog USP11, a key regulator of DNA double-strand break repair by homologous recombination. We hypothesized that USP11 domains adjacent to its protease domain harbor unique peptide-binding sites. Here, using a next-generation phage display (NGPD) strategy, combining phage display library screening with next-generation sequencing, we discovered unique USP11-interacting peptide motifs. Isothermal titration calorimetry disclosed that the highest affinity peptides (KD of ∼10 µm) exhibit exclusive selectivity for USP11 over USP4 and USP15 in vitro Furthermore, a crystal structure of a USP11-peptide complex revealed a previously unknown binding site in USP11's noncatalytic ubiquitin-like (UBL) region. This site interacted with a helical motif and is absent in USP4 and USP15. Reporter assays using USP11-WT versus a binding pocket-deficient double mutant disclosed that this binding site modulates USP11's function in homologous recombination-mediated DNA repair. The highest affinity USP11 peptide binder fused to a cellular delivery sequence induced significant nuclear localization and cell cycle arrest in S phase, affecting the viability of different mammalian cell lines. The USP11 peptide ligands and the paralog-specific functional site in USP11 identified here provide a framework for the development of new biochemical tools and therapeutic agents. We propose that an NGPD-based strategy for identifying interacting peptides may be applied also to other cellular targets.

The structure of the deubiquitinase USP15 reveals a misaligned catalytic triad and an open ubiquitin-binding channel.

Ubiquitin-specific protease 15 (USP15) regulates important cellular processes, including transforming growth factor ß (TGF-ß) signaling, mitophagy, mRNA processing, and innate immune responses; however, structural information on USP15's catalytic domain is currently unavailable. Here, we determined crystal structures of the USP15 catalytic core domain, revealing a canonical USP fold, including a finger, palm, and thumb region. Unlike for the structure of paralog USP4, the catalytic triad is in an inactive configuration with the catalytic cysteine ∼10 Å apart from the catalytic histidine. This conformation is atypical, and a similar misaligned catalytic triad has so far been observed only for USP7, although USP15 and USP7 are differently regulated. Moreover, we found that the active-site loops are flexible, resulting in a largely open ubiquitin tail-binding channel. Comparison of the USP15 and USP4 structures points to a possible activation mechanism. Sequence differences between these two USPs mainly map to the S1' region likely to confer specificity, whereas the S1 ubiquitin-binding pocket is highly conserved. Isothermal titration calorimetry monoubiquitin- and linear diubiquitin-binding experiments showed significant differences in their thermodynamic profiles, with USP15 displaying a lower affinity for monoubiquitin than USP4. Moreover, we report that USP15 is weakly inhibited by the antineoplastic agent mitoxantrone in vitro A USP15-mitoxantrone complex structure disclosed that the anthracenedione interacts with the S1' binding site. Our results reveal first insights into USP15's catalytic domain structure, conformational changes, differences between paralogs, and small-molecule interactions and establish a framework for cellular probe and inhibitor development.

Bdellovibrio bacteriovorus uses chimeric fibre proteins to recognize and invade a broad range of bacterial hosts.

Predatory bacteria, like the model endoperiplasmic bacterium Bdellovibrio bacteriovorus, show several adaptations relevant to their requirements for locating, entering and killing other bacteria. The mechanisms underlying prey recognition and handling remain obscure. Here we use complementary genetic, microscopic and structural methods to address this deficit. During invasion, the B. bacteriovorus protein CpoB concentrates into a vesicular compartment that is deposited into the prey periplasm. Proteomic and structural analyses of vesicle contents reveal several fibre-like proteins, which we name the mosaic adhesive trimer (MAT) superfamily, and show localization on the predator surface before prey encounter. These dynamic proteins indicate a variety of binding capabilities, and we confirm that one MAT member shows specificity for surface glycans from a particular prey. Our study shows that the B. bacteriovorus MAT protein repertoire enables a broad means for the recognition and handling of diverse prey epitopes encountered during bacterial predation and invasion.

Bacterial invasion and killing by predatory Bdellovibrio primed by predator prey cell recognition and self protection.

Bacterial predation, as exemplified by the periplasm-invading model predator Bdellovibrio bacteriovorus, is a fascinating multistage process facilitated by several adaptations to 'regular' bacterial lifestyles. We are beginning to understand more about such adaptations at the molecular level, particularly those concerning processes near the beginning of the predatory lifecycle (recognition, invasion, prey cell wall manipulation). In this review we highlight recent advances in investigating predation and tie these into some of the classical observations and phenotypes that make this two-cell system such an interesting case study in evolution.

Structural basis of the leukocyte integrin Mac-1 I-domain interactions with the platelet glycoprotein Ib.

Cell-surface receptor interactions between leukocyte integrin macrophage-1 antigen (Mac-1, also known as CR3, αMß2, CD11b/CD18) and platelet glycoprotein Ibα (GPIbα) are critical to vascular inflammation. To define the key residues at the binding interface, we used nuclear magnetic resonance (NMR) to assign the spectra of the mouse Mac-1 I-domain and mapped the residues contacting the mouse GPIbα N-terminal domain (GPIbαN) to the locality of the integrin metal ion-dependant adhesion site (MIDAS) surface. We next determined the crystal structures of the mouse GPIbαN and Mac-1 I-domain to 2 Å and 2.5 Å resolution, respectively. The mouse Mac-1 I-domain crystal structure reveals an active conformation that is stabilized by a crystal contact from the α7-helix with a glutamate side chain completing the octahedral coordination sphere of the MIDAS Mg2+ ion. The amino acid sequence of the α7-helix and disposition of the glutamic acid matches the C-terminal capping region α-helix of GPIbα effectively acting as a ligand mimetic. Using these crystal structures in combination with NMR measurements and docking analysis, we developed a model whereby an acidic residue from the GPIbα leucine-rich repeat (LRR) capping α-helix coordinates directly to the Mac-1 MIDAS Mg2+ ion. The Mac-1:GPIbαN complex involves additional interactions consolidated by an elongated pocket flanking the GPIbαN LRR capping α-helix. The GPIbαN α-helix has an HxxxE motif, which is equivalent by homology to RxxxD from the human GPIbαN. Subsequent mutagenesis of residues at this interface, coupled with surface plasmon resonance studies, confirmed the importance of GPIbαN residues H218, E222, and the Mac-1 MIDAS residue T209 to formation of the complex.