SspH/IpaH bacterial effector E3 ubiquitin (Ub) ligases, unrelated in sequence or structure to eukaryotic E3s, are utilized by a wide variety of Gram-negative bacteria during pathogenesis. These E3s function in a eukaryotic environment, utilize host cell E2 ubiquitin-conjugating enzymes of the Ube2D family, and target host proteins for ubiquitylation. Despite several crystal structures, details of Ube2D~Ub binding and the mechanism of ubiquitin transfer are poorly understood. Here, we show that the catalytic E3 ligase domain of SspH1 can be divided into two subdomains: an N-terminal subdomain that harbors the active-site cysteine and a C-terminal subdomain containing the Ube2D~Ub-binding site. SspH1 mutations designed to restrict subdomain motions show rapid formation of an E3~Ub intermediate, but impaired Ub transfer to substrate. NMR experiments using paramagnetic spin labels reveal how SspH1 binds Ube2D~Ub and targets the E2~Ub active site. Unexpectedly, hydrogen/deuterium exchange MS shows that the E2~Ub-binding region is dynamic but stabilized in the E3~Ub intermediate. Our results support a model in which both subunits of an Ube2D~Ub clamp onto a dynamic region of SspH1, promoting an E3 conformation poised for transthiolation. A conformational change is then required for Ub transfer from E3~Ub to substrate.