The centrosomal adaptor TACC3 and the microtubule polymerase chTOG interact via defined C-terminal subdomains in an Aurora-A kinase-independent manner.

The cancer-associated, centrosomal adaptor protein TACC3 (transforming acidic coiled-coil 3) and its direct effector, the microtubule polymerase chTOG (colonic and hepatic tumor overexpressed gene), play a crucial function in centrosome-driven mitotic spindle assembly. It is unclear how TACC3 interacts with chTOG. Here, we show that the C-terminal TACC domain of TACC3 and a C-terminal fragment adjacent to the TOG domains of chTOG mediate the interaction between these two proteins. Interestingly, the TACC domain consists of two functionally distinct subdomains, CC1 (amino acids (aa) 414-530) and CC2 (aa 530-630). Whereas CC1 is responsible for the interaction with chTOG, CC2 performs an intradomain interaction with the central repeat region of TACC3, thereby masking the TACC domain before effector binding. Contrary to previous findings, our data clearly demonstrate that Aurora-A kinase does not regulate TACC3-chTOG complex formation, indicating that Aurora-A solely functions as a recruitment factor for the TACC3-chTOG complex to centrosomes and proximal mitotic spindles. We identified with CC1 and CC2, two functionally diverse modules within the TACC domain of TACC3 that modulate and mediate, respectively, TACC3 interaction with chTOG required for spindle assembly and microtubule dynamics during mitotic cell division.

NisC binds the FxLx motif of the nisin leader peptide.

Nisin is a model system for lantibiotics, a class of peptides displaying antimicrobial activity against various Gram-positive bacteria. After ribosomal synthesis, the precursor peptide is modified in two steps, of which the last one involves consecutive cyclization reactions mediated by the cyclase NisC. Here, we present a detailed in vitro study of the interaction between NisC and the nisin precursor peptide. Our results unravel a specific interaction of NisC with the leader peptide independent of the maturation state. Furthermore, mutagenesis studies identified a specific binding sequence within the leader. Two amino acids (F-18 and L-16) within the highly conserved -FNLD- box of class I lantibiotics are essential for binding. They represent a potential general binding motif between leader peptides of a group of lantibiotics with their cyclase family. In summary, these in vitro data provide a new perception on the complexity of the lantibiotic modification machineries.

Role of centrosomal adaptor proteins of the TACC family in the regulation of microtubule dynamics during mitotic cell division.

During the mitotic division cycle, cells pass through an extensive microtubule rearrangement process where microtubules forming the mitotic spindle apparatus are dynamically instable. Several centrosomal- and microtubule-associated proteins are involved in the regulation of microtubule dynamics and stability during mitosis. Here, we focus on members of the transforming acidic coiled coil (TACC) family of centrosomal adaptor proteins, in particular TACC3, in which their subcellular localization at the mitotic spindle apparatus is controlled by Aurora-A kinase-mediated phosphorylation. At the effector level, several TACC-binding partners have been identified and characterized in greater detail, in particular, the microtubule polymerase XMAP215/ch-TOG/CKAP5 and clathrin heavy chain (CHC). We summarize the recent progress in the molecular understanding of these TACC3 protein complexes, which are crucial for proper mitotic spindle assembly and dynamics to prevent faulty cell division and aneuploidy. In this regard, the (patho)biological role of TACC3 in development and cancer will be discussed.

Substrate recognition and specificity of the NisB protein, the lantibiotic dehydratase involved in nisin biosynthesis.

Nisin is a posttranslationally modified antimicrobial peptide containing the cyclic thioether amino acids lanthionine and methyllanthionine. Although much is known about its antimicrobial activity and mode of action, knowledge about the nisin modification process is still rather limited. The dehydratase NisB is believed to be the initial interaction partner in modification. NisB dehydrates specific serine and threonine residues in prenisin, whereas the cyclase NisC catalyzes the (methyl)lanthionine formation. The fully modified prenisin is exported and the leader peptide is cleaved off by the extracellular protease NisP. Light scattering analysis demonstrated that purified NisB is a dimer in solution. Using size exclusion chromatography and surface plasmon resonance, the interaction of NisB and prenisin, including several of its modified derivatives, was studied. Unmodified prenisin binds to NisB with an affinity of 1.05 ± 0.25 µm, whereas the dehydrated and the fully modified derivatives bind with respective affinities of 0.31 ± 0.07 and 10.5 ± 1.7 µm. The much lower affinity for the fully modified prenisin was related to a >20-fold higher off-rate. For all three peptides the stoichiometry of binding was 1:1. Active nisin, which is the equivalent of fully modified prenisin lacking the leader peptide did not bind to NisB, nor did prenisin in which the highly conserved FNLD box within the leader peptide was mutated to AAAA. Taken together our data indicate that the leader peptide is essential for initial recognition and binding of prenisin to NisB.

Stoichiometry and structure of a lantibiotic maturation complex.

Lantibiotics are ribosomally synthesized antimicrobial peptides secreted by mainly Gram-positive bacteria. Class 1 lantibiotics mature via two modification steps introduced by a modification LanBC complex. For the lantibiotic nisin, the dehydratase NisB catalyzes the dehydration of serine and threonine residues in the so-called core peptide. Second, five (methyl)-lanthionine rings are introduced in a regio- and stereospecific manner by the cyclase NisC. Here, we characterized the assembly of the NisBC complex in vitro, which is only formed in the presence of the substrate. The complex is composed of a NisB dimer, a monomer of NisC and one prenisin molecule. Interestingly, the presence of the last lanthionine ring prevented complex formation. This stoichiometry was verified by small-angle X-ray scattering measurements, which revealed the first structural glimpse of a LanBC complex in solution.

Lantibiotic immunity: inhibition of nisin mediated pore formation by NisI.

Nisin, a 3.4 kDa antimicrobial peptide produced by some Lactococcus lactis strains is the most prominent member of the lantibiotic family. Nisin can inhibit cell growth and penetrates the target Gram-positive bacterial membrane by binding to Lipid II, an essential cell wall synthesis precursor. The assembled nisin-Lipid II complex forms pores in the target membrane. To gain immunity against its own-produced nisin, Lactococcus lactis is expressing two immunity protein systems, NisI and NisFEG. Here, we show that the NisI expressing strain displays an IC50 of 73 ± 10 nM, an 8-10-fold increase when compared to the non-expressing sensitive strain. When the nisin concentration is raised above 70 nM, the cells expressing full-length NisI stop growing rather than being killed. NisI is inhibiting nisin mediated pore formation, even at nisin concentrations up to 1 µM. This effect is induced by the C-terminus of NisI that protects Lipid II. Its deletion showed pore formation again. The expression of NisI in combination with externally added nisin mediates an elongation of the chain length of the Lactococcus lactis cocci. While the sensitive strain cell-chains consist mainly of two cells, the NisI expressing cells display a length of up to 20 cells. Both results shed light on the immunity of lantibiotic producer strains, and their survival in high levels of their own lantibiotic in the habitat.

The C-terminus of nisin is important for the ABC transporter NisFEG to confer immunity in Lactococcus lactis.

The lantibiotic nisin is a small 3.4 kDa antimicrobial peptide, which acts against Gram-positive bacteria in the nmol/L range. Nisin is produced and secreted by several Lactococcus lactis strains to ensure advantages against other bacteria in their habitat. Nisin contains five specific lanthionine rings of which the first two are important for Lipid II binding and the last two are crucial for the pore formation in the membrane. To gain immunity against nisin, the producing strain is expressing an ABC transporter called NisFEG, which expels nisin from the membrane. As a result six to eightfold more nisin is needed to affect the cells. The hydrolysis of ATP by NisFEG is required for this immunity as shown by a mutant, where the ATP hydrolysis is disrupted (NisFH181A EG). Furthermore, NisFEG recognizes the C-terminus of nisin, since deletion of the last six amino acids as well as of the last ring lowered the fold of immunity displayed by NisFEG.

Lantibiotics: how do producers become self-protected?

Lantibiotics are small peptides produced by Gram-positive bacteria, which are ribosomally synthesized as a prepeptide. Their genes are highly organized in operons containing all the genes required for maturation, transport, immunity and synthesis. The best-characterized lantibiotic is nisin from Lactococcus lactis. Nisin is active against other Gram-positive bacteria via various modes of actions. To prevent activity against its producer strain, an autoimmunity system has developed consisting of different proteins, the ABC transporter NisFEG and a membrane anchored protein NisI. Together, they circumvent the ability of nisin to fulfill its action and cause cell death of L. lactis. Within this review, the mechanism of regulation, biosynthesis and activity of the immunity machinery will be discussed. Furthermore a short description about the application of these immunity proteins in both medical and industrial fields is highlighted.

Easy and rapid purification of highly active nisin.

Nisin is an antimicrobial peptide produced and secreted by several L. lactis strains and is specifically active against Gram-positive bacteria. In previous studies, nisin was purified via cation exchange chromatography at low pH employing a single-step elution using 1 M NaCl. Here, we describe an optimized purification protocol using a five-step NaCl elution to remove contaminants. The obtained nisin is devoid of impurities and shows high bactericidal activity against the nisin-sensitive L. lactis strain NZ9000. Purified nisin exhibits an IC(50) of ~3 nM, which is a tenfold improvement as compared to nisin obtained via the one-step elution procedure.