From Gatekeeper to Effector: A Structural and Functional Analysis of the Multifaceted Chlamydial protein, CopN.
Archuleta, Tara Lakshimi
Chlamydiae species are obligate intracellular pathogens that utilize a type three-secretion system (T3SS) to deliver effector proteins and manipulate host cell processes. T3SSs are composed of cytosolic components, a needle complex, which forms the conduit for secreted proteins, and translocators, which form the pore in the target cell. A hierarchy of secretion in which needle components are secreted, followed by translocators, and finally effectors, is necessary for this system to be effective. While the effectors are diverse, the components that form the T3SS are conserved. Despite this conservation, these proteins can acquire additional functions while maintaining their roles in the T3SS. One such multifunctional protein, CopN, is both T3SS effector and a regulator of secretion. In this study, we describe the Chlamydial protein, CopN, which is a multidomain protein with similarity to a family of T3SS regulators known as “gatekeeper” proteins that control effector secretion in other organisms. In its effector role, CopN binds free αβ tubulin and inhibits microtubule polymerization. I also present the structure of CopN bound to Scc3, a translocator specific chaperone. The CopN-Scc3 structure reveals a secretion scaffold formed by CopN and Scc3 that likely recruits translocators to the secretion pore and ensures that translocators are secreted immediately following plug secretion. Structure based mutagenesis of the homologous proteins in Shigella flexneri confirms that translocator secretion requires the plug-chaperone secretion scaffold. The CopN structure reveals a conserved basic surface shown by mutagenesis to be essential for tubulin binding. Competition binding experiments indicate that CopN binds a conserved acidic face on tubulin, mimicking a tubulin binding strategy used by host proteins. The tubulin-binding interface is adjacent to the Scc3 binding site, revealing a strategy used by Chlamydia to encode effector activity on a conserved molecular structure.