# Bacterial Type I Toxins: Folding and Membrane Interactions
## 5 Protein Folding of the _S. aureus sprG1_-Encoded Type I Toxins  
We will present here original results concerning the first solution structure of the _Staphylococcus aureus_ SprG1-encoded toxin peptide, determined by NMR. The SprG1-encoded type I toxins belong to the _sprG1_/SprF1 TA system located within a pathogenicity island (PI) in _S. aureus_ and identified by homology with the _B. subtilis txpA_/RatA type I TA system [81; 83]. The _sprG1_ mRNA encodes two membrane peptides from a single internal reading frame: a long (44 amino acids, SprG144 also named PepG144) and a short version (31 amino acids, SprG131 also named PepG131). The SprG144 peptide has 13 extra amino acids in the N-terminus compared to SprG131 (Table 1). Ectopic overexpression of both SprG1-encoded peptides causes _S. aureus_ cell death accompanied by disruption of membrane integrity within 1 h upon induction (Figure 2) [81]. The dual-function RNA antitoxin SprF1 promotes _sprG1_ mRNA degradation and prevents _sprG1_ mRNA translation by interacting in _cis_ with its overlapping 3'-end [81]. Moreover, thanks to a purine-rich sequence located at its 5'-end, SprF1 also interacts with a subset of polysomes and ribosomes that could promote translation attenuation and persister cell formation [82]. The extracellular addition of chemically-synthesized peptides SprG144 and SprG131 or of membrane extracts prepared from _S. aureus_ cells overexpressing SprG1-encoded peptides trigger the lysis of both competing bacteria (Gram-negative and positive bacteria) and human erythrocytes [81]. To better understand the mechanism of action of the SprG1-encoded peptides responsible for membrane permeabilization, we decided to solve their structure by NMR (Section S1, S2) and focused on the short SprG131 peptide (Figure 1). Chemically-synthesized SprG131 peptide is hydrophobic and thus not soluble in water. It dissolves upon addition of deuterated isopropanol. The addition of 50% v/v \(d\)8-isopropanol avoids the presence of undissolved peptide in the NMR tube and yields acceptable NMR resonances line broadenings. In such conditions, SprG131 peptide adopts a single well-folded conformation based on the TOCSY and NOESY spectra (Figure 4, PDB id: 7 NS1). The 3D structure using NMR restraints displays an almost perfect \(\alpha\)-helix about 39A long, ranging from I4 to S28. Most hydrophilic residues are at the N- and C-termini. The three N-terminal MIT residues and the three hydrophilic C-terminal NKK residues are unstructured, with enhanced flexibility for the C-terminus. Albeit not stacked, the two phenylalanine residues (F10 and F13) in the first half of the peptide are about 7 A from each other. They also share the same strongly hydrophobic side with L14, L17, I18, L20, and V21 (Figure S1). The NMR spectra recorded in the same conditions on the SprG144 peptide disclose large resonances especially in the N-terminal region, indicating that this part of the peptide is not well structured and/or very flexible (Figure S2). Despite a poorer resolution, a good superposition is observed for the amino acids corresponding to the medium part and the C-terminus part of SprG131 peptide (Figure S2). From the similarity of chemical shift and connectivities, we can conclude that the helical structure ranging from L8 to S28 in the SprG131 peptide is retained in SprG144 (i.e., from L21 to S41) (Table 1). Inparticular, the well-defined part of the \(\alpha\)-helix comprising the two phenylalanine residues is conserved. On the opposite, the cross-peaks of the amino acids I15-M20 corresponding to SprG1\({}_{31}\) I2-M7 are shifted and/or broadened (sometimes broadened out to baseline), and many connections are lost. This indicates that the extra N-terminal amino acids in SprG1\({}_{44}\) peptide destabilizes the region corresponding to the N-terminus of the SprG1\({}_{31}\) peptide. In our experimental conditions, there is no evidence that SprG1\({}_{31}\) peptide undergoes conformational exchange or multimerizes.