NSRRC Activity Report 2023

Neutron Science 083 Scattering data revealed a distinct upward shift in the dimensionless Kratky plot, signifying inherent flexibility. Rigid body modeling using the ensemble optimization method BILBOMD, which combines high-resolution structures with flexible linker regions, was employed ( Fig. 1 ). Structural ensembles were selected based on combinations of conformers that best matched experimental data. Results indicated that SANS data ( Fig. 2 ) were consistent with a predominant compact state (~60%), where the terminal regions of both proteins interact, alongside a more extended form (~10%) with reduced contacts. The remaining ~30% represented free NADase, in agreement with previous binding studies. The compact state showcased contacts between SLO domain 4 and the NADase N-terminal translocation region, which is supported by mutagenesis data. Conversely, the extended form depicts separated terminal segments, likely corresponding to NADase dissociation from cell-bound SLO during delivery into host cells. Validation of the conformational ensemble was sought through SANS measurements on the same samples in varying D 2 O percentages. The resulting contrast variation series quantitatively aligned with the distribution of compact, extended, and free populations from initial dataset modeling. Fig. 2 : SANS results: (a) SANS data (offset for clarity) collected from the D NADase/SLO complex with the multistate model scattering curves overlaid (solid black line); (b) Stuhrmann plot for the NADase/SLO complex A plot I(0) normalized by concentration as a function of D 2 O content of the supporting solvent. (c) A plot I(0) normalized by concentration as a function of D 2 O content of the supporting solvent. [Reproduced from Ref. 3] In summary, neutron scattering experiments uncovered functionally relevant flexibility in the virulence factor complex (NADase/SLO), challenging assumptions derived from rigid crystal structures. These observations contribute to an integrated model that elucidates NADase delivery into host cells through dynamic association with SLO pores. Thus, SANS offers unique structural insights that are crucial for understanding the synergistic toxicity of these proteins in GAS infection. (Reported by Chun-Ming Wu) This report features the work of Shuying Wang and her collaborators published in Commun. Biol. 6 , 124 (2023). ANSTO QUOKKA – Small-angle Neutron Scattering • SANS • Protein Structure, Polymer, Biomaterial, Drug Delivery, Magnetism, Soft Matter References 1. A. L. Bricker, V. J. Carey, M. R. Wessels, Infect. Immun. 73 , 6562 (2005). 2. B. Limbago, V. Penumalli, B. Weinrick, J. R. Scott, Infect. Immun. 68 , 6384 (2000). 3. W.-J. Tsai, Y.-H. Lai, Y.-A. Shi, M Hammel, A. P. Duff, A. E. Whitten, K. L. Wilde, C.-M. Wu, R. Knott, U.-S. Jeng, C.-Y. Kang, C.-Y. Hsu, J.-L. Wu, P.-J. Tsai, C.-C. Ni, J.-J. Wu, Y.-S. Lin, C.-C. Liu, T. Senda, S.-Y. Wang, Commun. Biol. 6 , 124 (2023).