Toxin-antitoxin (TA) systems are present in many bacteria and play important roles in bacterial growth, physiology, and pathogenicity. Those that are best studied are the type II TA systems, in which both toxins and antitoxins are proteins. The HicAB system is one of the prototypic TA systems, found in many bacterial species. Complex interactions between the protein toxin (HicA), the protein antitoxin (HicB), and the DNA upstream of the encoding genes regulate the activity of this system, but few structural details are available about how HicA destabilizes the HicB-DNA complex. Here, we determined the X-ray structures of HicB and the HicAB complex to 1.8 and 2.5 Å resolution, respectively, and characterized their DNA interactions. This revealed that HicB forms a tetramer and HicA and HicB form a heterooctameric complex that involves structural reorganization of the C-terminal (DNA-binding) region of HicB. Our observations indicated that HicA has a profound impact on binding of HicB to DNA sequences upstream of hicAB in a stoichiometric-dependent way. At low ratios of HicA:HicB, there was no effect on DNA binding, but at higher ratios, the affinity for DNA declined cooperatively, driving dissociation of the HicA:HicB:DNA complex. These results reveal the structural mechanisms by which HicA de-represses the HicB-DNA complex. This work was supported by the Biotechnology and Biological Sciences Research Council-funded South West Biosciences Doctoral Training Partnership Training Grant BB/J014400/1. The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1-S10 and Tables S1-S12. The atomic coordinates and structure factors (codes 6G1C, 6G1N, and 6G26) have been deposited in the Protein Data Bank (http://wwpdb.org/). Experimental SAXS data and derived models of both HicB 4 and HicAB 4 have been deposited in the Small Angle Scattering Biological Data Bank (SASBDB) under the accession codes SASDD45 and SASDD55.
Mupirocin, a commercially available antibiotic produced by Pseudomonas fluorescens and thiomarinol, isolated from the marine bacterium Pseudoalteromonas sp. SANK 73390, both consist of a polyketide-derived monic acid homologue esterified with either 9-hydroxynonanoic acid (mupirocin, 9HN) or 8-hydroxyoctanoic acid (thiomarinol, 8HO). The mechanisms of formation of these deceptively simple 9HN and 8HO fatty acid moieties in mup and tml respectively remain unresolved. To define starter unit generation the purified mupirocin proteins MupQ, MupS and MacpD and their thiomarinol equivalents (TmlQ, TmlS and TacpD) have been expressed and shown to convert malonylcoenzyme A (CoA) and succinyl-CoA to 3-hydroxypropionoyl (3-HP) or 4-hydroxybutyryl (4-HB) fatty acid starter units respectively via the MupQ/TmlQ catalysed generation of an unusual bis-CoA/acyl carrier protein (ACP) thioester, followed by MupS/TmlS catalysed reduction. Mix and match experiments show MupQ/TmlQ to be highly selective for the correct CoA, MacpD/TacpD were interchangeable but an alternate transacting ACPs from the mupirocin pathway (MacpA/TacpA) or a heterologous ACP (BatA) were non-functional. MupS and TmlS selectivity was more varied and these reductases differed in their substrate and ACP selectivity. The solution structure of MacpD determined by NMR revealed a C-terminal extension with partial helical character that has been shown to be important for maintaining high titres of mupirocin. We generated a truncated MacpD construct, MacpD_T, which lacks this C-terminal extension but retains an ability to generate 3-HP with MupS and MupQ, suggesting further downstream roles in protein-protein interactions for this region of the ACP.
Patients with diabetes mellitus (DM) often have alterations of the autonomic nervous system (ANS), even early in their disease course. Previous research has not evaluated whether these changes may have consequences on adaptation mechanisms in DM, e.g. to mental stress. We therefore evaluated whether patients with DM who already had early alterations of the ANS reacted with an abnormal regulatory pattern to mental stress. We used the spectral analysis technique, known to be valuable and reliable in the investigation of disturbances of the ANS. We investigated 34 patients with DM without clinical evidence of ANS dysfunction (e.g. orthostatic hypotension) and 44 normal control subjects (NC group). No patients on medication known to alter ANS responses were accepted. The investigation consisted of a resting state evaluation and a mental stress task (BonnDet). In basal values, only the 21 patients with type 2 DM were different in respect to body mass index and systolic blood pressure. In the study parameters we found significantly lower values in resting and mental stress spectral power of mid-frequency band (known to represent predominantly sympathetic influences) and of high-frequency and respiration bands (known to represent parasympathetic influences) in patients with DM (types 1 and 2) compared with NC group (5.3 +/- 1.2 ms2 vs. 6.1 +/- 1.3 ms2, and 5.5 +/- 1.6 ms2 vs. 6.2 +/- 1.5 ms2, and 4.6 +/- 1.7 ms2 vs. 6.2 +/- 1.5 ms2, for resting values respectively; 4.7 +/- 1.4 ms2 vs. 5.9 +/- 1.2 ms2, and 4.6 +/- 1.9 ms2 vs. 5.6 +/- 1.7 ms2, and 3.7 +/- 2.1 ms2 vs. 5.6 +/- 1.7 ms2, for stress values respectively; M/F ratio 6/26 vs. 30/14). These differences remained significant even when controlled for age, sex, and body weight. However, patients with DM type 2 (and significantly higher body weight) showed only significant values in mental stress modulus values. There were no specific group effects in the patients with DM in adaptation mechanisms to mental stress compared with the NC group. These findings demonstrate that power spectral examinations at rest are sufficiently reliable to diagnose early alterations in ANS in patients with DM. The spectral analysis technique is sensitive and reliable in investigation of ANS in patients with DM without clinically symptomatic autonomic dysfunction.
Menisporopsin A is a fungal bioactive macrocyclic polylactone, the biosynthesis of which requires only reducing (R) and nonreducing (NR) polyketide synthases (PKSs) to guide a series of esterification and cyclolactonization reactions. There is no structural information pertaining to these PKSs. Here, we report the solution characterization of singlet and doublet acyl carrier protein (ACP 2 and ACP 1 -ACP 2 )-thioesterase (TE) domains from NR-PKS involved in menisporopsin A biosynthesis. Small-angle X-ray scattering (SAXS) studies in combination with homology modelling reveal that these polypeptides adopt a distinctive beads-on-a-string configuration, characterized by the presence of highly flexible interdomain linkers. These models provide a platform for studying domain organization and interdomain interactions in fungal NR-PKSs, which may be of value in directing the design of functionally optimized polyketide scaffolds.
The cation-independent mannose 6-phosphate (M6P)/ Insulin-like growth factor-2 receptor (CI-MPR/ IGF2R) is a ~300 kDa transmembrane protein responsible for trafficking M6P-tagged lysosomal hydrolases and the internalisation of IGF2. Insulin-like growth factor 2 (IGF2). The extracellular region of the CI-MPR is composed of 15 has fifteen homologous domains including M6P binding domains (D) D3, D5, D9 and D15 and IGF2 binding D11. but how it interacts with extracellular ligands at neutral pH is poorly understood. We have focused structural work on key CI-MPR domains of human CI-MPR and report the first structures of human D7, D8, D9 and D10 within two multi-domain constructs, D9-10 and D7-11. Together These structures provide the first high-resolution description of the high-affinity M6P binding domain D9. Domain 9 stabilises a well-defined hub formed by D7-11 in which whereby two penta-domains intertwine to form a dimeric helical-type coil. Remarkably the D7-11 ligand free structure of this penta-domain closely matches the IGF2 bound state suggesting this may be an intrinsically stable conformation at neutral pH. An Interdomain clusters of histidine and proline residues identified between several pairs of domains impart rigidity and may impart receptor rigidity and play a role in structural transitions of the receptor at low pH.
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