Immunoglobulin-alpha (IgA)-bound antigens induce immune effector responses by activating the IgA-specific receptor FcalphaRI (CD89) on immune cells. Here we present crystal structures of human FcalphaRI alone and in a complex with the Fc region of IgA1 (Fcalpha). FcalphaRI has two immunoglobulin-like domains that are oriented at approximately right angles to each other. Fcalpha resembles the Fcs of immunoglobulins IgG and IgE, but has differently located interchain disulphide bonds and external rather than interdomain N-linked carbohydrates. Unlike 1:1 FcgammaRIII:IgG and Fc epsilon RI:IgE complexes, two FcalphaRI molecules bind each Fcalpha dimer, one at each Calpha2-Calpha3 junction. The FcalphaRI-binding site on IgA1 overlaps the reported polymeric immunoglobulin receptor (pIgR)-binding site, which might explain why secretory IgA cannot initiate phagocytosis or bind to FcalphaRI-expressing cells in the absence of an integrin co-receptor.

We demonstrate a method for generating discretely structured protein nanotubes from the simple ring-shaped building block, homohexameric Hcp1 from Pseudomonas aeruginosa. Our design exploited the observation that the crystal lattice of Hcp1 contains rings stacked in a repeating head-to-tail pattern. High-resolution detail of the ring-ring interface allowed the selection of sites for specific cysteine mutations capable of engaging in disulfide bond formation across rings, thereby generating stable Hcp1 nanotubes. Protein nanotubes containing up to 25 subunits ( approximately 100 nm in length) were self-assembled under simple conditions. Furthermore, we demonstrate that the tube ends and interior can be independently and specifically functionalized to generate nanocapsules.

Regulated protein localization is critical for many cellular processes. Several techniques have been developed for experimental control over protein localization, including chemically induced and light-induced dimerization, which both provide temporal control. Light-induced dimerization offers the distinct advantage of spatial precision within subcellular length scales. A number of elegant systems have been reported that utilize natural light-sensitive proteins to induce dimerization via direct protein–protein binding interactions, but the application of these systems at cellular locations beyond the plasma membrane has been limited. Here we present a new technique to rapidly and reversibly control protein localization in living cells with subcellular spatial resolution using a cell-permeable, photoactivatable chemical inducer of dimerization. We demonstrate light-induced recruitment of a cytosolic protein to individual centromeres, kinetochores, mitochondria and centrosomes in human cells, indicating that our system is widely applicable to many cellular locations.

Aurora B kinase is concentrated and activated at centromeres before release and diffusion to reach spatially distributed substrates necessary for cell division.

Maintenance of genome stability during cell division depends on establishing correct attachments between chromosomes and spindle microtubules. Correct, bi-oriented attachments are stabilized, while incorrect attachments are selectively destabilized. This process relies largely on increased phosphorylation of kinetochore substrates of Aurora B kinase at misaligned versus aligned kinetochores. Current models explain this differential phosphorylation by spatial changes in the position of substrates relative to a constant pool of kinase at the inner centromere. However, these models are based on studies in aneuploid cells. We show that normal diploid cells have a more robust error correction machinery. Aurora B is enriched at misaligned centromeres in these cells, and the dynamic range of Aurora B substrate phosphorylation at misaligned versus aligned kinetochores is increased. These findings indicate that in addition to Aurora B regulating kinetochore-microtubule binding, the kinetochore also controls Aurora B recruitment to the inner centromere. We show that this recruitment depends on both activity of Plk1, a kinetochore-localized kinase, and activity of Aurora B itself. Our results suggest a feedback mechanism in which Aurora B both regulates and is regulated by chromosome attachment to the spindle, which amplifies the differential phosphorylation of kinetochore substrates and increases the efficiency of error correction.

SUMMARY The spindle assembly checkpoint (SAC) kinase Mps1 not only inhibits anaphase but also corrects erroneous attachments that could lead to missegregation and aneuploidy. However, Mps1’s error correction-relevant substrates are unknown. Using a chemically tuned kinetochore-targeting assay, we show that Mps1 destabilizes microtubule attachments (K-fibers) epistatically to Aurora B, the other major error-correcting kinase. Through quantitative proteomics, we identify multiple sites of Mps1-regulated phosphorylation at the outer kinetochore. Substrate modification was microtubule-sensitive and opposed by PP2A-B56 phosphatases that stabilize chromosome-spindle attachment. Consistently, Mps1 inhibition rescued K-fiber stability after depleting PP2A-B56. We also identify the Ska complex as a key effector of Mps1 at the kinetochore-microtubule interface, as mutations that mimic constitutive phosphorylation destabilized K-fibers in vivo and reduced the efficiency of the Ska complex’s conversion from lattice diffusion to end-coupled microtubule binding in vitro. Our results reveal how Mps1 dynamically modifies kinetochores to correct improper attachments and ensure faithful chromosome segregation.

Type I sulfatases require an unusual co-or post-translational modification for their activity in hydrolyzing sulfate esters. In eukaryotic sulfatases, an active site cysteine residue is oxidized to the aldehyde-containing C ␣ -formylglycine residue by the formylglycine-generating enzyme (FGE). The machinery responsible for sulfatase activation is poorly understood in prokaryotes. Here we describe the identification of a prokaryotic FGE from Mycobacterium tuberculosis. In addition, we solved the crystal structure of the Streptomyces coelicolor FGE homolog to 2.1 Å resolution. The prokaryotic homolog exhibits remarkable structural similarity to human FGE, including the position of catalytic cysteine residues. Both biochemical and structural data indicate the presence of an oxidized cysteine modification in the active site that may be relevant to catalysis. In addition, we generated a mutant M. tuberculosis strain lacking FGE. Although global sulfatase activity was reduced in the mutant, a significant amount of residual sulfatase activity suggests the presence of FGE-independent sulfatases in this organism.Type I sulfatases are members of an expanding family of enzymes that employ novel co-or post-translationally derived cofactors to facilitate catalysis (1, 2). The formylglycine (Fgly) 4 residue positioned within the active site of type I sulfatases is thought to undergo hydration to a gem-diol, after which one of the hydroxyl groups acts as a catalytic nucleophile to initiate sulfate ester cleavage (Fig. 1a) (3). The FGly residue is located within a ϳ13-residue consensus sequence termed the sulfatase motif (4) that defines this family of enzymes and is highly conserved throughout all domains of life (Fig. 1b). Although FGly is formed from cysteine residues in eukaryotic sulfatases, either cysteine (within the core motif CX[P/A]XR) or serine (SXPXR) can be oxidized to FGly in prokaryotic type I sulfatases. The coor post-translational machineries necessary for these respective modifications appear to be different; FGE is able to activate CXPXR-type sulfatases (5-7) and anaerobic sulfatase-maturating enzyme is responsible for modifying SXPXR-type sulfatases and CXAXR-type sulfatases (8, 9). Some prokaryotes, such as Mycobacterium tuberculosis, have only CXPXR-type sulfatases (10), whereas other species have only SXPXR-type sulfatases or a combination of type I sulfatases (11). Some prokaryotes also contain FGly-independent sulfatases. These sulfatases function by distinct enzymatic mechanisms and are divided into two categories, Fe(II) ␣-ketoglutarate-dependent dioxygenase sulfatases (type II sulfatases) and metallo-␤-lactamase sulfatases (type III sulfatases) (12-14). Unlike type I sulfatases, which share a high degree of sequence similarity, type II and III sulfatases have highly divergent sequences, complicating the discovery of these proteins by genomic search algorithms.In higher eukaryotes, sulfatases are involved in a variety of essential tasks, including extracellular matrix remodeling and steroid titer regu...

Aurora B kinase, a key regulator of cell division, localizes to specific cellular locations, but the regulatory mechanisms responsible for phosphorylation of substrates located remotely from kinase enrichment sites are unclear. Here, we provide evidence that this activity at a distance depends on both sites of high kinase concentration and the bistability of a coupled kinase-phosphatase system. We reconstitute this bistable behavior and hysteresis using purified components to reveal co-existence of distinct high and low Aurora B activity states, sustained by a two-component kinase autoactivation mechanism. Furthermore, we demonstrate these non-linear regimes in live cells using a FRET-based phosphorylation sensor, and provide a mechanistic theoretical model for spatial regulation of Aurora B phosphorylation. We propose that bistability of an Aurora B-phosphatase system underlies formation of spatial phosphorylation patterns, which are generated and spread from sites of kinase autoactivation, thereby regulating cell division.DOI: http://dx.doi.org/10.7554/eLife.10644.001