Crystal structure of the P2 C-repressor: a binder of non-palindromic direct DNA repeats

As opposed to the vast majority of prokaryotic repressors, the immunity repressor of temperate Escherichia coli phage P2 (C) recognizes non-palindromic direct repeats of DNA rather than inverted repeats. We have determined the crystal structure of P2 C at 1.8 Å. This constitutes the first structure solved from the family of C proteins from P2-like bacteriophages. The structure reveals that the P2 C protein forms a symmetric dimer oriented to bind the major groove of two consecutive turns of the DNA. Surprisingly, P2 C has great similarities to binders of palindromic sequences. Nevertheless, the two identical DNA-binding helixes of the symmetric P2 C dimer have to bind different DNA sequences. Helix 3 is identified as the DNA-recognition motif in P2 C by alanine scanning and the importance for the individual residues in DNA recognition is defined. A truncation mutant shows that the disordered C-terminus is dispensable for repressor function. The short distance between the DNA-binding helices together with a possible interaction between two P2 C dimers are proposed to be responsible for extensive bending of the DNA. The structure provides insight into the mechanisms behind the mutants of P2 C causing dimer disruption, temperature sensitivity and insensitivity to the P4 antirepressor

Structural and biochemical studies of phage P2 DNA-binding proteins and human tetraspanins

Biochemical studies of proteins are crucial for a more detailed view of the world around us. The focus of biochemical studies can vary, from a complex mammalian system to a more simple viral entity, but the same methods and principles apply. In biochemistry one rely on both in vitro and in vivo analyses to understand biological processes. Protein crystallography has since the late 1950s been an additional important tool. By visualizing the structures of molecules involved in a biological process one can truly comprehend the molecular mechanisms of an organism or cell at the chemical level. This thesis includes structural biochemical work in combination with mutational and functional studies of proteins from both human and virus. Human tetraspanins are integral membrane proteins grouped by their conserved structural features. Many of them have been shown to regulate cell migration, fusion, and signalling in the cell by functioning as organizers of multi-molecular membrane complexes. Several tetraspanins are also implicated in different forms of human cancers. How tetraspanins perform their function is still not known at the molecular level and today very little structural data exist on complete tetraspanin proteins. Structural biochemical studies require mg quantities of purified protein, something that is not easily obtained for membrane proteins. This thesis includes a family-wide approach to achieve full-length tetraspanins for biochemical studies. To facilitate this process a GFP-based optimization scheme for production and purification of membrane proteins in E. coli and S. cerevisiae has been applied. By utilizing this approach, we identified 8 human tetraspanins that can be produced and isolated from either E. coli or S. cerevisiae, and in one case using either system. The temperate bacteriophage P2 is a virus, which can enter both the lytic and the lysogenic cycle upon infection of its host. The outcome of the infection is regulated by and dependent on several proteins encoded by the viral genome. The immunity repressor P2 and the Cox repressor direct the phage into either cycle. Integration and excision of the virus DNA requires the enzyme P2 integrase. The work in this thesis presents high-resolution crystal structures of these key proteins from the regulation of lysogeny in bacteriophage P2. By using a crystallographic approach in combination with mutational studies, key characteristics of these three proteins are presented. At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p

Structural and biochemical studies of phage P2 DNA-binding proteins and human tetraspanins

Biochemical studies of proteins are crucial for a more detailed view of the world around us. The focus of biochemical studies can vary, from a complex mammalian system to a more simple viral entity, but the same methods and principles apply. In biochemistry one rely on both in vitro and in vivo analyses to understand biological processes. Protein crystallography has since the late 1950s been an additional important tool. By visualizing the structures of molecules involved in a biological process one can truly comprehend the molecular mechanisms of an organism or cell at the chemical level. This thesis includes structural biochemical work in combination with mutational and functional studies of proteins from both human and virus. Human tetraspanins are integral membrane proteins grouped by their conserved structural features. Many of them have been shown to regulate cell migration, fusion, and signalling in the cell by functioning as organizers of multi-molecular membrane complexes. Several tetraspanins are also implicated in different forms of human cancers. How tetraspanins perform their function is still not known at the molecular level and today very little structural data exist on complete tetraspanin proteins. Structural biochemical studies require mg quantities of purified protein, something that is not easily obtained for membrane proteins. This thesis includes a family-wide approach to achieve full-length tetraspanins for biochemical studies. To facilitate this process a GFP-based optimization scheme for production and purification of membrane proteins in E. coli and S. cerevisiae has been applied. By utilizing this approach, we identified 8 human tetraspanins that can be produced and isolated from either E. coli or S. cerevisiae, and in one case using either system. The temperate bacteriophage P2 is a virus, which can enter both the lytic and the lysogenic cycle upon infection of its host. The outcome of the infection is regulated by and dependent on several proteins encoded by the viral genome. The immunity repressor P2 and the Cox repressor direct the phage into either cycle. Integration and excision of the virus DNA requires the enzyme P2 integrase. The work in this thesis presents high-resolution crystal structures of these key proteins from the regulation of lysogeny in bacteriophage P2. By using a crystallographic approach in combination with mutational studies, key characteristics of these three proteins are presented. At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p

Impact of Paranasal Sinus Surgery in Granulomatosis With Polyangiitis: A Longitudinal Computed Tomography Study

Objectives/Hypothesis Severe chronic rhinosinusitis (CRS) in patients with granulomatosis with polyangiitis (GPA) failing medical therapies can be treated with paranasal sinus surgery. Whether this surgery protects from progressive sinonasal damage remains unknown. Here, we aimed to analyze time‐dependent relations between sinus surgeries and computed tomography (CT) imaging features in the CRS of GPA. Study Design Longitudinal observational study. Methods We assessed CRS features including bone thickening by global osteitis scoring scale, bone erosions, and mucosal thickening by Lund‐Mackay scores in serial paranasal sinus CT scans (742 CT scans in total) from a cohort of 127 well‐characterized GPA patients. Data on sinonasal surgical procedures were from a mandatory national registry and from chart review. We defined the time from baseline CT to last CT as the study observation period in each patient. Datasets were analyzed by linear mixed models. Results We found that 23/127 cohort patients had one or more paranasal sinus surgical procedures, and 96% of these (22/23) had osteitis by CT after surgery. In patients with nasal surgery alone or no surgery, we identified osteitis in 7/11 (64%) and 45/93 (48%), respectively. During the observation period of a median of 5 years, 38 patients had progression of their sinus osteitis, with the highest annual osteitis progression rates observed around the time of surgery. Conclusions In this cohort, paranasal sinus surgery was associated with prevalence, severity, and progression rate of sinus osteitis, indicating that sinus surgery does not reduce the bone damage development in the CRS of GPA

Development of CT-based methods for longitudinal analyses of paranasal sinus osteitis in granulomatosis with polyangiitis

Background Even though progressive rhinosinusitis with osteitis is a major clinical problem in granulomatosis with polyangiitis (GPA), there are no studies on how GPA-related osteitis develops over time, and no quantitative methods for longitudinal assessment. Here, we aimed to identify simple and robust CT-based methods for capture and quantification of time-dependent changes in GPA-related paranasal sinus osteitis and compare performance of the methods under study in a largely unselected GPA cohort. Methods GPA patients (n = 121) with ≥3 paranasal CT scans obtained ≥12 months apart and control patients not having GPA or rhinosinusitis (n = 15) were analysed by: (i) Global osteitis scoring scale (GOSS), originally developed for chronic rhinosinusitis; (ii) Paranasal sinus volume by manual segmentation; (iii) Mean maxillary and sphenoid diameter normalised to landmark distances (i.e. diameter ratio measurement, DRM). Results Time-dependent changes in GPA-related osteitis were equally well measured by the simple DRM and the labour-intensive volume method while GOSS missed ongoing changes in cases with extensive osteitis. GOSS at last CT combined with DRM identified three distinct patient groups: (i) The no osteitis group, who had no osteitis and no change in DRM from baseline CT to last CT (45/121 GPA patients and 15/15 disease controls); (ii) Stable osteitis group, with presence of osteitis, but no change in DRM across time (31 GPA); (iii) Progressive osteitis, defined by declining DRM (45 GPA). Conclusions We suggest DRM and GOSS as complementary methods for capturing, classifying and quantifying time-dependent changes in GPA-related osteitis

Sequence alignment of selected tetraspanins.

<p>Predicted localization of transmembrane helices with TOPCONS server [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134041#pone.0134041.ref078" target="_blank">78</a>] are indicated with a gray background. Residues conserved across the family–indicated in yellow and predicted glycosylation sites [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134041#pone.0134041.ref079" target="_blank">79</a>] are indicated with a red background. Sequence alignments were generated with the ClustalW server [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134041#pone.0134041.ref080" target="_blank">80</a>] by feeding 219 tetraspanin sequences from diverge spices to improve alignment statistics.</p

Tetraspanin topology scheme.

<p>Transmembrane helices are numbered 1–4, conserved helices in the large extracellular domain indicated with letters A,B,E according to nomenclature by Seigneuret et al. <b>[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134041#pone.0134041.ref032" target="_blank">32</a>]</b>. Conserved residues are shown in circles, where x stands for any amino acid. Possible post-translational modifications are indicated as palmitoylation sites shown as waves close to the intracellular side of the protein and available N-linked glycosylation sites shown as forks on the extracellular domains.</p

Size exclusion chromatogram of purified CO-029.

<p>Size exclusion of the purified CO-029 protein after proteolytic cleavage and removal of the GFP-tag. The elution was monitored with the absorbance at 280 nm.</p

Experimental studies reporting tetraspanin production or isolation.

<p>^a A tetraspanin superfamily member lacking the CCG motif [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134041#pone.0134041.ref044" target="_blank">44</a>].</p><p>MDCK, Madin-Darby Canine Kidney Epithelial Cells; HEK293, Human Embryonic Kidney 293 cells</p

Solubilization efficiency of TSPAN-GFP fusion proteins.

<p>The solubilization degree of five tetraspanin-containing yeast membranes with the help of detergents and SMA polymers. The solubilization ratio was determined by comparing the fluorescence counts of solubilized material after ultracentrifugation to the mix before separation of solubilized and non-solubilized material.</p