Crystal Structure Of An Eiic Trapped In An Inward-Facing Conformation

Enzyme IIC (EIIC) is a membrane embedded sugar transporter and is part of the phosphoenolpyruvate-dependent phosphotransferase system. Crystal structures of two EIICs, bcChbC and bcMalT, have been reported. Both transporters have a substrate binding domain, and the substrate binding domain assumes an inward facing conformation in bcChbC and an outward-facing conformation in bcMalT. This has led to the hypothesis that substrate translocation can be achieved by rigid-body motions of the substrate binding domain. However, to understand the transport mechanism, it is necessary to obtain different conformations of the same transporter. Here we present a crystal structure of bcMalT whose substrate binding domain is trapped in an inward-facing conformation by a mercury ion that bridges two strategically placed cysteine residues. To find out if the crosslinked structure is distorted significantly from its native state, we did the follow two experiments. First, all-atom molecular dynamics simulations were performed for the inward facing conformation with or without the cysteine crosslink, and both structures remain stable after 1 microsecond. Second, single-molecule Foster resonance energy transfer (smFRET) showed that large distance changes predicted by the crosslinked structure indeed occur on bcMalT without the crosslinking. The structure of the inward facing bcMalT therefore supports the hypothesis that the substrate binding domain moves as a rigid body to shuttle the substrate across the membrane, and provides details of the conformational changes.