Abstract
Scc4 is an unusual bi-functional protein from Chlamydia trachomatis (CT) that functions as a type III secretion system (T3SS) chaperone and an RNA polymerase (RNAP)-binding protein. Both functions require interactions with protein partners during specific stages of the CT developmental cycle. As a T3SS chaperone, Scc4 binds Scc1 during the late stage of development to form a heterodimer complex, which chaperones the essential virulence effector, CopN. During the early-middle stage of development, Scc4 regulates T3SS gene expression by binding the σ(66)-containing RNAP holoenzyme. In order to study the structure and association mechanism of the Scc4:Scc1 T3SS chaperone complex using nuclear magnetic resonance (NMR) spectroscopy, we developed an approach to selectively label each chain of the Scc4:Scc1 complex with the (15)N-isotope. The approach allowed one protein to be visible in the NMR spectrum at a time, which greatly reduced resonance overlap and permitted comparison of the backbone structures of free and bound Scc4. (1)H,(15)N-heteronuclear single quantum coherence spectra of the (15)N-Scc4:Scc1 and Scc4:(15)N-Scc1 complexes showed a total structural rearrangement of Scc4 upon binding Scc1 and a dynamic region isolated to Scc1, respectively. Development of the chain-selective labeling approach revealed that the association of Scc4 and Scc1 requires partial denaturation of Scc1 to form the high affinity complex, while low affinity interactions occurred between the isolated proteins under non-denaturing conditions. These results provide new models for Scc4's functional switching mechanism and Scc4:Scc1 association in CT.