NSRRC Activity Report 2023

082 NSRRC ACTIVITY REPORT 2023 W e often deal with the hassle of infections caused by Group A Streptococcus in our daily lives. This germ releases two powerful secret weapons, known as NAD+ glycohydrolase (NADase) and Streptolysin O (SLO). The challenge stems from the interplay between these two factors, creating a complication that brings complexity to our day-to-day existence. 1,2 To unveil its solution structure and its conformational flexibility, small-angle neutron scattering (SANS) experiments were conducted on the deuterated-NADase/SLO complex. This was imperative as its crystal structure lacked functionally crucial terminal regions. Chun-Ming Wu from the NSRRC and Shuying Wang’s research group at National Cheng Kung University have Fig. 1 : Crystal structure of the NADase 193-451,G330D /SLO 106-574 complex. (a) Ribbon diagram of the crystal structure of the NADase 193-451,G330D /SLO 106-574 complex in two perspectives related by a rotation of 90° about the horizontal axis. where yellow is NADase 193-451,G330D and magenta is SLO 106-574 . (b) Schematic diagrams of NADase and SLO, where gray is the secretory signal (SS). [Reproduced from Ref. 3] Neutron-Enabled Probing of Virulence Factor Dynamics and Functional Insights for Infection Mechanisms Understanding the molecular basis of streptococcal infection may lead to targeted therapies or vaccines against the bacteria’s unique pathogenic mechanisms. recently initiated a collaborative project focusing on SANS measurements using the QUOKKA instrument at Open-pool Australian lightwater reactor in Australian Nuclear Science and Technology Organisation (OPAL, ANSTO) with deuterated protein technology. The goal is to emphasize the dominance of NADase through contrast matching with SLO in a 42% D 2 O solution. Deuterated- NADase/SLO aliquots were acquired through size-exclusion chromatography (SEC) utilizing a Superdex 200 Increase 10/300 column. However, efforts to examine the structural details of the SLO component faced challenges due to aggregation in the 100% D 2 O sample, leading to difficulties in obtaining a clear interpretation. Despite this, valuable data were obtained, enabling the modeling of the complex’s overall structure and dynamics.