S-Sulfhydration of the Catalytic Cysteine in the Rhodanese Domain of YgaP is Complex Dynamic Process

ISSN:2297-824

Detergent/Nanodisc Screening for High-Resolution NMR Studies of an Integral Membrane Protein Containing a Cytoplasmic Domain

<div><p>Because membrane proteins need to be extracted from their natural environment and reconstituted in artificial milieus for the 3D structure determination by X-ray crystallography or NMR, the search for membrane mimetic that conserve the native structure and functional activities remains challenging. We demonstrate here a detergent/nanodisc screening study by NMR of the bacterial α-helical membrane protein YgaP containing a cytoplasmic rhodanese domain. The analysis of 2D [¹⁵N,¹H]-TROSY spectra shows that only a careful usage of low amounts of mixed detergents did not perturb the cytoplasmic domain while solubilizing in parallel the transmembrane segments with good spectral quality. In contrast, the incorporation of YgaP into nanodiscs appeared to be straightforward and yielded a surprisingly high quality [¹⁵N,¹H]-TROSY spectrum opening an avenue for the structural studies of a helical membrane protein in a bilayer system by solution state NMR.</p> </div

YgaP⁻ incorporation into DMPC nanodiscs.

<p>(<b>A</b>) Size exclusion chromatography (Superdex 200 10/300GL) of YgaP⁻ in 20 mM bis-Tris-HCl pH 7, 150 mM NaCl, 3 mM DHPC-7, 1 mM LMPG, and 5 mM TCEP. (upper panel) and YgaP⁻ in MSP1/DMPC nanodiscs. (<b>B</b>) SDS-PAGE of YgaP⁻ in DMPC nanodiscs. 12% NuPAGE Bis-Tris gel (Invitrogen, Carslbad). Lanes: (MW) SeeBlue plus2 prestained (Invitrogen, Carslbad), (1)-(3) Different dilutions of the YgaP/DMPC nanodisc reaction mixture in the SDS sample buffer after the removal of detergents by Biobeads to resolve the partial overlap due to apparent over-staining in lane 1 for the individual identification of YgaP and MSP1 as indicated. (<b>C</b>)–(<b>D</b>) 2D [¹⁵N,¹H]-TROSY spectra of ²H,¹⁵N-labeled YgaP purified in 6 mM DHPC-7 and 1 mM LMPG and (<b>C</b>) incorporated in DMPC nanodiscs or (<b>D</b>) in nanodiscs with deuterated d-54 DMPC. (<b>E</b>) 2D [¹⁵N,¹H]-TROSY spectrum of ²H,¹⁵N-labeled YgaP purified in 3 mM FC12. The sample of (E) was used for a DMPC nanodisc preparation as shown in (<b>F</b>): 2D [¹⁵N,¹H]-TROSY spectrum of ²H,¹⁵N-labeled YgaP.</p

NMR spectra of YgaP in various micellar systems as indicated.

<p>2D [¹⁵N,¹H]-TROSY spectra of ²H,¹⁵N-labeled YgaP⁻ in (<b>A</b>) FC12, (<b>B</b>) DHPC-7, (<b>C</b>) DHPC-7 and FC12, (<b>D, F</b>) DHPC-7 and LMPG. (<b>E</b>) 2D [¹⁵N,¹H]-TROSY of the N-terminal rhodanese domain of YgaP. The individual cross peaks are labeled according to the sequential assignment. (<b>G</b>) ¹H and ¹⁵N chemical shift differences (labeled Δδ¹H^N and Δδ¹⁵N) between the N-terminal rhodanese domain in solution and the N-terminal rhodanese domain of full length YgaP⁻ in the optimized mixed micellar conditions (i.e. 6 mM DHPC, 1 mM LMPG). The lack of profound up- or down-filed <b>Δ</b>δ ¹H^N and <b>Δ</b>δ¹⁵N chemical shift differences indicates the same tertiary structure of the rhodanese domain in solution and in presence of mixed micelles.</p

Effects of DHPC-7/LMPG mixed micelles on the N-terminal rhodanese domain and full length YgaP

<p>⁻<b>.</b> (<b>A</b>) 2D [¹⁵N,¹H]-TROSY spectra of the N-terminal rhodanese domain in absence (Black) and in presence of 9 mM DHPC-7, 2 mM LMPG (Red). For better clarity a portion of the spectrum is magnified as indicated. (<b>B</b>) 2D [¹⁵N,¹H]-TROSY spectra of ²H,¹⁵N-labeled of YgaP with optimum detergent concentration (i.e. 6 mM DHPC, 1 mM LMPG) (Black) and in presence of 9 mM DHPC-7, and 2 mM LMPG, respectively (Red). Black arrows indicate regions of the red spectrum where resonances are missing, indicating the effect of detergent excess in the quality of the spectrum. (<b>C</b>) SDS-PAGE of the nickel affinity purification of YgaP⁻ in DHPC-7/LMPG. 4–12% NuPAGE Bis-Tris gel (Invitrogen, Carslbad). Lanes: (MW) SeeBlue plus2 prestained (Invitrogen, Carslbad), (1) YgaP⁻ after membrane extraction in DHPC-7/LMPG micelles, (2) Loading flow-through fraction of Nickel resin, (3) Washing of Nickel resin, (4) Elution of YgaP⁻ with buffer containing 500 mM imidazole (details of the buffer used are given in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054378#s2" target="_blank">Material and Methods</a> section).</p

S-Nitrosylation Induces Structural and Dynamical Changes in a Rhodanese Family Protein.

S-Nitrosylation is well established as an important post-translational regulator in protein function and signaling. However, relatively little is known about its structural and dynamical consequences. We have investigated the effects of S-nitrosylation on the rhodanese domain of the Escherichia coli integral membrane protein YgaP by NMR, X-ray crystallography, and mass spectrometry. The results show that the active cysteine in the rhodanese domain of YgaP is subjected to two competing modifications: S-nitrosylation and S-sulfhydration, which are naturally occurring in vivo. It has been observed that in addition to inhibition of the sulfur transfer activity, S-nitrosylation of the active site residue Cys63 causes an increase in slow motion and a displacement of helix 5 due to a weakening of the interaction between the active site and the helix dipole. These findings provide an example of how nitrosative stress can exert action at the atomic level

The Solution Structure of a Domain from the Neisseria meningitidis Lipoprotein PilP Reveals a New β-Sandwich Fold

Type IV pili are long, thin fibres, which extend from the surface of the bacterial pathogen Neisseria meningitidis; they play a key role in adhesion and colonisation of host cells. PilP is a lipoprotein, suggested to be involved in the assembly and stabilization of an outer membrane protein, PilQ, which is required for pilus formation. Here we describe the expression of a recombinant fragment of PilP, spanning residues 20 to 181, and determination of the solution structure of a folded domain, spanning residues 85 to 163, by NMR. The N-terminal third of the protein, from residues 20 to 84, is apparently unfolded. Protease digestion yielded a 113 residue fragment that contained the folded domain. The domain adopts a simple beta-sandwich type fold, consisting of a three-stranded beta-sheet packed against a four-stranded beta-sheet. There is also a short segment of 3(10) helix at the N-terminal part of the folded domain. We were unable to identify any other proteins that are closely related in structure to the PilP domain, although the fold appears to be distantly related to the lipocalin family. Over 40 homologues of PilP have been identified in Gram-negative bacteria and the majority of conserved residues lie within the folded domain. The fourth beta-strand and adjacent loop regions contain a high proportion of conserved residues, including three glycine residues, which seem to play a role in linking the two beta-sheets. The two beta-sheets pack together to form a crevice, lined with conserved hydrophobic residues: we suggest that this feature could act as a binding site for a small ligand. The results show that PilP and its homologues have a conserved, folded domain at the C-terminal end of the protein that may be involved in mediating binding to hydrophobic ligands