Tsubasa Tanaka

Site-2 proteases (S2Ps), conserved intramembrane metalloproteases that maintain cellular homeostasis, are associated with chronic infection and persistence leading to multidrug resistance in bacterial pathogens. A structural model of how S2Ps discriminate and accommodate substrates could help us develop selective antimicrobial agents. We previously proposed that the Escherichia coli S2P RseP unwinds helical substrate segments before cleavage, but the mechanism for accommodating a full-length membrane-spanning substrate remained unclear. Our present cryo-EM analysis of Aquifex aeolicus RseP ( Aa RseP) revealed that a substrate-like membrane protein fragment from the expression host occupied the active site while spanning a transmembrane cavity that is inaccessible via lateral diffusion. Furthermore, in vivo photocrosslinking supported that this substrate accommodation mode is recapitulated on the cell membrane. Our results suggest that the substrate accommodation by threading through a conserved membrane-associated region stabilizes the substrate-complex and contributes to substrate discrimination on the membrane.

Semaphorins constitute a large family of secreted and membrane‐bound proteins that signal through cell‐surface receptors, plexins. Semaphorins generally use low‐affinity protein–protein interactions to bind with their specific plexin(s) and regulate distinct cellular processes such as neurogenesis, immune response, and organogenesis. Sema6D is a membrane‐bound semaphorin that interacts with class A plexins. Sema6D exhibited differential binding affinities to class A plexins in prior cell‐based assays, but the molecular mechanism underlying this selectivity is not well understood. Therefore, we performed hybrid in vitro/in silico analysis to examine the binding mode of Sema6D to class A plexins and to identify residues that give rise to the differential affinities and thus contribute to the selectivity within the same class of semaphorins. Our biophysical binding analysis indeed confirmed that Sema6D has a higher affinity for Plexin‐A1 than for other class A plexins, consistent with the binding selectivity observed in the previous cell‐based assays. Unexpectedly, our present crystallographic analysis of the Sema6D‐Plexin‐A1 complex showed that the pattern of polar interactions is not interaction‐specific because it matches the pattern in the prior structure of the Sema6A‐Plexin‐A2 complex. Thus, we performed in silico alanine scanning analysis and discovered hotspot residues that selectively stabilized the Sema6D‐Plexin‐A1 pair via Van der Waals interactions. We then validated the contribution of these hotspot residues to the variation in binding affinity with biophysical binding analysis and molecular dynamics simulations on the mutants. Ultimately, our present results suggest that shape complementarity in the binding interfaces is a determinant for binding selectivity.