Production and structural studies of lung surfactant protein B (SP-B) peptides

Lung surfactant is a mixture of lipids and proteins which is critical for normal breathing by lining the air-water interface to reduce the surface tension. Lung surfactant protein B (SP-B) is the only essential protein component of lung surfactant complex due to the lethality of any SP-B deficiency. It is thought that SP-B functions by enhancing lipid rearrangements at various phases of the breathing cycle. However, the high-resolution structure and mechanism of SP-B are not yet understood. In the first part of this research, SP-B and a 7-residue deletion mutant of SP-B were produced recombinantly and partially characterized. In the second part of this research, circular dichroism, solution and solid-state nuclear magnetic resonance (NMR) methods were used to assess the structure of two SP-B-based peptides, Super-Mini-B and N-terminal insertion sequence (SP-B₁₋₇). Super-Mini-B is composed of the N-terminal 7-residue insertion sequence and the N-and C-terminal helices of SP-B. Interestingly, it was observed that Super-Mini-B produces greater lipid membrane perturbation than the peptide which lacks the N-terminal insertion sequence (i.e. Mini-B). Comparing the results of structural studies on Mini-B, SP-B₁₋₇ and Super-Mini-B helps unveil the contribution of the 7-residue insertion sequence to the function of SP-B

Structural and Biochemical Characterization of VirB8 Protein in Type IV Secretion Systems

Secretion is the passage of macromolecules across cellular membranes. In bacteria, secretion is essential for virulence and survival. Gram-negative bacteria use specialized envelope-spanning multiprotein complexes to secrete macromolecules called type IV secretion system (T4SS). T4SSs mediate the secretion of monomeric proteins, multisubunit protein toxins and nucleoprotein complexes. Also, they contribute to the horizontal spread of plasmid-encoded antibiotic resistance genes. Consequently, they are potential targets for antivirulence drugs. Gram- negative bacteria have two membranes that the secretion complex spans. As a result, the T4SS consists of proteins inserted in the membranes and of soluble proteins that face into or out of the bacterial cell. The details of channel assembly and structure are not known, although recent advances have revealed the structure of the core secretion channel. VirB8 is an inner membrane protein of the complex that interacts with many other T4SS subunits and works as nucleation factor for T4SS channel assembly. Biophysical studies and NMR experiments in particular were conducted to characterize the structural aspects of VirB8 interactions. Dynamic regions of VirB8 during monomer-to-dimer transition were identified by NMR spectroscopy. X-ray crystal and NMR analyses revealed structural differences at the helical regions (α-1 and α-4) of wild-type VirB8 and its monomeric variant VirB8M102R. Fragment screening identified small molecules binding to the wild-type and monomeric variant. In silico docking analyses suggested that the surface groove in the VirB8 structure is important for effective binding of the small molecules. NMR experiments and biochemical assays demonstrated that the β-sheet domain (β1 in particular) is the binding interface of VirB8 for the interaction with VirB10. The identified interface has functional importance for T4SS-mediated conjugation. In addition, I used NMR spectroscopy to identify changes in the structure of VirB8 upon interaction with VirB5. Altogether, structural and biochemical studies on periplasmic and full length VirB8 enabled us to characterize the sequence of interactions between VirB8 and other VirB proteins during T4SS complex assembly and function. The results of this research may lead to an innovative strategy for the development of novel antimicrobial drugs.La sécrétion est le passage de macromolécules à travers les membranes cellulaires. Chez les bactéries, la sécrétion est essentielle pour la virulence et la survie. Les bactéries à Gramnégatif utilisent le système de sécrétion de type IV (SST4) pour la sécrétion de toxines et de nucléoprotéines. Les SST4 contribuent notamment à la propagation des gènes de résistance aux antibiotiques. Pour cette raison, les composants du SST4 sont des cibles potentielles pour le développement de médicaments antivirulence. Le SST4 est un complexe protéique qui s’étend entre la double membrane de la bactérie à Gram-négatif. Les protéines qui le composent sont insérées dans les membranes cellulaires ou solubles. Bien que la structure du pore central du SST4 ait été résolue récemment, les détails de l'assemblage et la structure de ce complexe ne sont pas connus. VirB8 est une protéine de la membrane interne qui interagit avec de nombreuses autres sous-unités du SST4. Il s’agit d’un acteur central de l'assemblage du SST4. Des études biophysiques, et notamment des expériences de RMN ont ainsi été réalisées pour caractériser les aspects structuraux des interactions avec VirB8. Des regions dynamiques dans la structure de VirB8 ont été identifiées par spectroscopie RMN lors de la transition entre la forme monomérique et dimérique. Les analyses de cristallographie et de RMN ont révélé des différences structurales dans les régions hélicoïdales (α1 et α4) de VirB8 wild-type et du variant monomérique VirB8M102R. Le criblage de fragments a permis d’identifier de petites molécules capables de se lier à VirB8 ainsi qu’au variant monomérique. Les analyses d’arrimage moléculaire in silico suggèrent que la rainure de surface dans la structure VirB8 est importante pour laliaison de ces petites molécules. Les expériences de RMN et les essais biochimiques révèlent que le feuillet β (β1 en particulier) constitue l'interface d’interaction entre VirB8 et VirB10. Cette interface d’interaction est d’ailleurs importante pour la conjugaison du SST4. De plus, j'ai identifié des changements dans la structure de VirB8 lors de l'interaction avec VirB5. Les études sur la protéine VirB8 nous ont permis de caractériser la séquence d'événements entre VirB8 et d'autres protéines VirB, régulant l'assemblage et la fonction du SST4

²H spectra of mechanically oriented 7∶3 POPC-<i>d</i>₃₁:POPG bilayers in the presence (upper panel) and absence (lower panel) of 1 mol% SP-B (1–25,63–78).

<p>The spectra were acquired with 60000 transients at 23°C. Dashed vertical lines indicate the quadrupole splitting of deuterons on the C15 acyl chain segment of POPC-<i>d</i>₃₁ in the absence of SP-B (1–25,63–78).</p

Partial ¹H chemical shift assignments (in ppm) for residues 1–7 of SP-B, as assigned based on 2D TOCSY and NOESY NMR experiments with SP-B (1–7) and SP-B (1–25,63–78) (Super Mini-B) in deuterated SDS at 45°C.

<p>Note that although three sets of proline resonances were identified, none of them could be identified with a particular proline in the sequence.</p

Splittings, Δ ν, and corresponding order parameters, S_CD, for the resolved peaks of the 2H NMR spectra of POPC-<i>d</i>₃₁/POPG (7∶3) (Figure 1) in the absence and presence of SP-B (1–25,63–78) (Super Mini-B).

<p>The splittings quoted derive from a single experiment; however the uncertainty has been estimated based on the standard deviation in splittings derived from 5 separate control experiments with the same lipid composition.</p

Selected regions of 2D NOESY NMR spectra of 1 mM SP-B (1–25,63–78) in 150 mM SDS solution at pH 5 and 45°C (red) and 1 mM SP-B (8–25,63–78) in 150 mM SDS at pH 5 and 37°C (blue).

<p>Selected regions of 2D NOESY NMR spectra of 1 mM SP-B (1–25,63–78) in 150 mM SDS solution at pH 5 and 45°C (red) and 1 mM SP-B (8–25,63–78) in 150 mM SDS at pH 5 and 37°C (blue).</p

HN region of the ¹H NMR spectra of 1 mM SP-B (1–25,63–78) in SDS micelles at 45°C (red) and 1.5 mM SP-B (8–25,63–78) in SDS micelles at 37°C (blue).

<p>The number of transients was 32. The intensity scale is arbitrary but has been normalized to take into account the differences in peptide concentration.</p

A) Far-UV CD spectra of SP-B (8–25,63–78) (triangles), SP-B (1–25,63–78) (circles) and SP-B (1–7) (squares) dissolved in SDS micelles.

<p>Spectra were taken using a 1 mm path-length quartz cuvette from 200 nm to 260 nm at 25°C. Shown are single scans. Three additional spectra of each sample were acquired and the 4 scans averaged together before % secondary structure was extracted (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072821#pone-0072821-t002" target="_blank"><b>Table 2</b></a>).</p

Half ²H NMR spectra of mechanically oriented 7∶3 POPC-<i>d</i>₃₁:POPG bilayers in the absence of peptide (a), in comparison to (b) with 1 mol% SP-B (8–25,63–78) or (c) SP-B (1–25,63–78).

<p>The spectra were all acquired at 23°C. Vertical lines indicate the quadrupole splitting of POPC-<i>d</i>₃₁ deuterons in the absence of peptide.</p

DOSY NMR analysis of SuperMini-B in SDS micelles at 37°C.

<p>DOSY NMR analysis of SuperMini-B in SDS micelles at 37°C.</p