Molecular Insights into CLD Domain Dynamics and Toxin Recruitment of the HlyA E. coli T1SS

Gentile R, Schott-Verdugo S, Khosa S, Günes C, Bonus M, Reiners J, Smits S, Schmitt L, Gohlke H, Journal of Molecular Biology :169485 (2025) DOI

SASDXZ4 – Hemolysin A Type 1 Secretion System (Hly T1SS)

Alpha-hemolysin translocation ATP-binding protein HlyB
HlyA (GFP-Hemolysin with Flag-Tag insertion)
Membrane fusion protein (MFP) family protein (R14C; L229V)
Outer membrane protein TolC
MWI(0) 1043 kDa
MWexpected 1112 kDa
VPorod 2837 nm3
log I(s) 1.04×103 1.04×102 1.04×101 1.04×100
Alpha-hemolysin translocation ATP-binding protein HlyB HlyA (GFP-Hemolysin with Flag-Tag insertion) Membrane fusion protein (MFP) family protein (R14C; L229V) Outer membrane protein TolC small angle scattering data  s, nm-1
ln I(s)
Alpha-hemolysin translocation ATP-binding protein HlyB HlyA (GFP-Hemolysin with Flag-Tag insertion) Membrane fusion protein (MFP) family protein (R14C; L229V) Outer membrane protein TolC Guinier plot ln 1.04×103 Rg: 11.4 nm 0 (11.4 nm)-2 s2
(sRg)2I(s)/I(0)
Alpha-hemolysin translocation ATP-binding protein HlyB HlyA (GFP-Hemolysin with Flag-Tag insertion) Membrane fusion protein (MFP) family protein (R14C; L229V) Outer membrane protein TolC Kratky plot 1.104 0 √3 sRg
p(r)
Alpha-hemolysin translocation ATP-binding protein HlyB HlyA (GFP-Hemolysin with Flag-Tag insertion) Membrane fusion protein (MFP) family protein (R14C; L229V) Outer membrane protein TolC pair distance distribution function Rg: 12.2 nm 0 Dmax: 46.2 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Alpha-hemolysin translocation ATP-binding protein HlyB HlyA (GFP-Hemolysin with Flag-Tag insertion) Membrane fusion protein (MFP) family protein (R14C; L229V) Outer membrane protein TolC DAMMIF model
Synchrotron SAXS data from solutions of Hemolysin A Type 1 Secretion System (Hly T1SS) in 50 mM Tris, 150 mM NaCl, 10 mM CaCl2, 0.0063% glyco-diosgenin (GDN), pH 7.5 were collected on the BM29 beam line at the ESRF (Grenoble, France) using a Pilatus3 2M detector at a sample-detector distance of 2.8 m and at a wavelength of λ = 0.099 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). One solute concentration of 0.50 mg/ml was measured at 10°C. 10 successive 1 second frames were collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

Glyco-diosgenin (GDN): https://pubchem.ncbi.nlm.nih.gov/compound/162642462

Alpha-hemolysin translocation ATP-binding protein HlyB (HlyB)
Mol. type   Protein
Organism   Escherichia coli (strain UTI89 / UPEC)
Olig. state   Hexamer
Mon. MW   80.1 kDa
 
UniProt   Q1R2T6 (2-707)
Sequence   FASTA
 
HlyA (GFP-Hemolysin with Flag-Tag insertion) (HlyA)
Mol. type   Protein
Organism   Escherichia coli
Olig. state   Monomer
Mon. MW   143.6 kDa
 
UniProt   A0A236M9V9 (1-1024)
Sequence   FASTA
 
Membrane fusion protein (MFP) family protein (R14C; L229V) (HlyD)
Mol. type   Protein
Organism   Escherichia coli (strain UTI89 / UPEC)
Olig. state   Hexamer
Mon. MW   54.5 kDa
 
UniProt   Q1R2T7 (1-478)
Sequence   FASTA
 
Outer membrane protein TolC (TolC)
Mol. type   Protein
Organism   Escherichia coli (strain K12)
Olig. state   Trimer
Mon. MW   53.7 kDa
 
UniProt   P02930 (1-493)
Sequence   FASTA