Cooperative binding ensures the obligatory melibiose/Na(+) cotransport in MelB

MelB catalyzes the obligatory cotransport of melibiose with Na(+), Li(+), or H(+). Crystal structure determination of the Salmonella typhimurium MelB (MelB(St)) has revealed a typical major facilitator superfamily (MFS) fold at a periplasmic open conformation. Cooperative binding of Na(+) and melibiose has been previously established. To determine why cotranslocation of sugar solute and cation is obligatory, we analyzed each binding in the thermodynamic cycle using three independent methods, including the determination of melting temperature by circular dichroism spectroscopy, heat capacity change (ΔC(p)), and regulatory phosphotransferase EIIA(Glc) binding with isothermal titration calorimetry (ITC). We found that MelB(St) thermostability is increased by either substrate (Na(+) or melibiose) and observed a cooperative effect of both substrates. ITC measurements showed that either binary formation yields a positive sign in the ΔC(p), suggesting MelB(St) hydration and a likely widening of the periplasmic cavity. Conversely, formation of a ternary complex yields negative values in ΔCp, suggesting MelB(St) dehydration and cavity closure. Lastly, we observed that EIIA(Glc), which has been suggested to trap MelB(St) at an outward-open state, readily binds to the MelB(St) apo state at an affinity similar to MelB(St)/Na(+). However, it has a suboptimal binding to the ternary state, implying that MelB(St) in the ternary complex may be conformationally distant from the EIIA(Glc)-preferred outward-facing conformation. Our results consistently support the notion that binding of one substrate (Na(+) or melibiose) favors MelB(St) at open states, whereas the cooperative binding of both substrates triggers the alternating-access process, thus suggesting this conformational regulation could ensure the obligatory cotransport.