The rare disease challenge and how to promote a productive rare disease community: case study of Birt-Hogg-Dubé symposia.

Resources for rare diseases are lacking. Patients do not have the information and support that they need, and researchers struggle to make progress due to a shortage of skills and collaborations within the field. One way to overcome these hurdles is to host annual Symposia, focused on a specific rare disease. Here, we use the example of Birt-Hogg-Dubé Symposia to discuss the practical issues of such meetings, including the importance of timing and the choice of invited speakers. We highlight the ways in which rare disease symposia can create a single community, removing barriers between patients, clinicians and researchers.

Structure and organisation of SinR, the master regulator of biofilm formation in Bacillus subtilis.

sinR encodes a tetrameric repressor of genes required for biofilm formation in Bacillus subtilis. sinI, which is transcribed under Spo0A control, encodes a dimeric protein that binds to SinR to form a SinR-SinI heterodimer in which the DNA-binding functions of SinR are abrogated and repression of biofilm genes is relieved. The heterodimer-forming surface comprises residues conserved between SinR and SinI. Each forms a pair of α-helices that hook together to form an intermolecular four-helix bundle. Here, we are interested in the assembly of the SinR tetramer and its binding to DNA. Size-exclusion chromatography with multi-angle laser light scattering and crystallographic analysis reveal that a DNA-binding fragment of SinR (residues 1-69) is a monomer, while a SinI-binding fragment (residues 74-111) is a tetramer arranged as a dimer of dimers. The SinR(74-111) chain forms two α-helices with the organisation of the dimer similar to that observed in the SinR-SinI complex. The tetramer is formed through interactions of residues at the C-termini of the four chains. A model of the intact SinR tetramer in which the DNA binding domains surround the tetramerisation core was built. Fluorescence anisotropy and surface plasmon resonance experiments showed that SinR binds to an oligonucleotide duplex, 5'-TTTGTTCTCTAAAGAGAACTTA-3', containing a pair of SinR consensus sequences in inverted orientation with a K(d) of 300 nM. The implications of these data for promoter binding and the curious quaternary structural transitions of SinR upon binding to (i) SinI and (ii) the SinR-like protein SlrR, which "repurposes" SinR as a repressor of autolysin and motility genes, are discussed.

Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen.

SpoIIE is a dual function protein that plays important roles during sporulation in Bacillus subtilis. It binds to the tubulin-like protein FtsZ causing the cell division septum to relocate from mid-cell to the cell pole, and it dephosphorylates SpoIIAA phosphate leading to establishment of differential gene expression in the two compartments following the asymmetric septation. Its 872 residue polypeptide contains a multiple-membrane spanning sequence at the N-terminus and a PP2C phosphatase domain at the C-terminus. The central segment that binds to FtsZ is unlike domains of known structure or function, moreover the domain boundaries are poorly defined and this has hampered the expression of soluble fragments of SpoIIE at the levels required for structural studies. Here we have screened over 9000 genetic constructs of spoIIE using a random incremental truncation library approach, ESPRIT, to identify a number of soluble C-terminal fragments of SpoIIE that were aligned with the protein sequence to map putative domains and domain boundaries. The expression and purification of three fragments were optimised, yielding multimilligram quantities of the PP2C phosphatase domain, the putative FtsZ-binding domain and a larger fragment encompassing both these domains. All three fragments are monomeric and the PP2C domain-containing fragments have phosphatase activity.

Structural rearrangement accompanying ligand binding in the GAF domain of CodY from Bacillus subtilis.

The GAF domain is a simple module widespread in proteins of diverse function, including cell signalling proteins and transcription factors. Its structure, typically spanning 150 residues, has three tiers: a basal layer of two or more alpha-helices, a middle layer of beta-pleated sheet and a top layer formed by segments of the polypeptide that connect strands of the beta-sheet. In structures of GAF domains in complex with their effectors, these polypeptide segments envelop the ligand, enclosing it in a cavity whose base is formed by the beta-sheet, such that ligand binding and release must be accompanied by conformational rearrangements of the distal portion of the structure. Descriptions of binding are presently limited by the absence of a GAF domain for which both liganded and unliganded structures are known. Earlier, we solved the crystal structure of the GAF domain of CodY, a branched-chain amino acid and GTP-responsive regulator of the transcription of stationary-phase and virulence genes in Bacillus, in complexes with isoleucine and valine. Here, we report the structure of this domain in its unliganded form, allowing definition of the structural changes accompanying ligand binding. The core of the protein and its dimerisation interface are essentially unchanged, in agreement with circular dichroism spectroscopy experiments that show that the secondary structure composition is unperturbed by ligand binding. There is however extensive refolding of the binding site loops, with up to 15-A movements of the coiled segment linking beta3 and beta4, such that the binding pocket is not formed in the absence of the ligand. The implications of these structural rearrangements for ligand affinity and specificity are discussed. Finally, saturation-transfer-difference NMR spectroscopy showed binding of isoleucine but not that of GTP to the GAF domain, suggesting that the two cofactors do not have a common binding site.