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Type III secretion systems are complex nanomachines used for injection of proteins from Gram-negative bacteria into eukaryotic cells. While they are assembled when the environmental conditions are appropriate, they only start secreting upon contact with a host cell. Secretion is hierarchical: first, the pore-forming translocators are released, next, effector proteins are injected. Hierarchy between these protein classes is mediated by a conserved gate-keeper protein, MxiC in Shigella. As its molecular mechanism of action is still poorly understood, we used its structure to guide site-directed mutagenesis and dissect its function. We identified mutants predominantly affecting all known features of MxiC regulation: secretion of translocators, MxiC and/or effectors. Using molecular genetics we then mapped at which point in the regulatory cascade the mutants were affected. Analysis of some of these mutants led us to a set of electron paramagnetic resonance experiments that provide evidence that MxiC interacts directly with IpaD. We suggest how this interaction regulates a switch in its conformation that is key to its functions.