Figure 3. A 2.6 Å resolution crystal structure of the SagB-SpdC complex establishes that two interfaces are critical for its function.

a, A cartoon representation of the SagB-SpdC crystal structure. The extracellular domains of both proteins interact (blue box); a helix at the bottom of the active site cleft of SagB (violet) contacts an extracellular loop between TM3 and TM4 of SpdC (green). The approximate location of the membrane is denoted in gray. b, Several hydrogen bonds and a salt-bridge form at the interface between the SagB helix and the SpdC loop. c, A view from the extracellular face of the transmembrane helices shows that SagB closely contacts TM3 of SpdC. SagB lacking its TM helix does not co-purify with SpdC (Extended Data 8). d and e, Radiolabeled peptidoglycan oligomers were incubated with SagB-SpdC or with constructs containing mutations designed to either disrupt or stabilize the extracellular interface between SagB and SpdC. d, SagBinterface* denotes SagBN115S, K118N, R119Q, V122D, D123G, L127E, in which SagB residues at the interface were switched to the corresponding SagA residues. e, A variant of SagB-SpdC with two cysteinesubstituted residues, SagB^K118C - SpdC^D106C, was purified as the disulfide-linked complex (Supplementary Fig 12). Activity of the oxidized complex (lane 3) was compared to the activity of SagB^K118C-SpdC^D106C incubated with reducing agent (lane 4), and to wild-type SagB-SpdC without and with reducing agent (lanes 2 and 5). Unlabeled cleavage products were also treated with ColM and analyzed using LC-MS analysis. Extracted ion chromatogram (EIC) traces are shown for both wild-type SagB-SpdC and SagB^K118C-SpdC^D106C reactions, and further confirm that longer oligosaccharide products are preferred for a disulfide-restricted complex. Notably, short oligosaccharides ionize better relative to longer oligosaccharides. Experiments were performed at least three times.