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Yersinia pathogenicity is dependent on polarized translocation of effectorproteins via the type III secretion system (T3SS). The tip complex situatedon the needle structure of the T3SS is required for contact with the eukaryotichost membrane and is to an extent composed of pentameric LcrV. LcrVis a multifunctional protein that also acts as a regulator of the T3SS by virtueof forming a high-affinity complex in the cytoplasm with its chaperone, LcrG.By employing a structure-based approach centered on mass spectrometry,FRET and NMR spectroscopy, we demonstrated that the LcrV-LcrG complexis best described as a multivalent complex, and that the N-terminaldomain of LcrV contributes by negatively affecting the LcrG binding affinity.The N-terminal domain of LcrV is dynamic and undergoes a conformationalchange to accommodate LcrG binding. 19F NMR spectroscopy analysissuggests that the conformational change is an intrinsic property of the protein,which agrees with a conformational selection model. An analysis ofeffector secretion into a culture supernatant demonstrated that the low synthesisand low secretion phenotypes of a LcrV mutant where the N-terminaldomain has been removed are linked to the structure, interactions and stabilityof the LcrV N-terminal domain. In summary, our results add insightsinto the dynamics of LcrV and its complex with LcrG.