Chain-Selective Isotopic Labeling of the Heterodimeric Type III Secretion Chaperone, Scc4:Scc1, Reveals the Total Structural Rearrangement of the Chlamydia trachomatis Bi-Functional Protein, Scc4.

Thilini O Ukwaththage,Li Shen,Samantha M Keane,Megan A Macnaughtan

doi:10.3390/biom10111480

Thilini O Ukwaththage, Li Shen + Show 2 more

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https://doi.org/10.3390/biom10111480

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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 σ66-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 15N-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. 1H,15N-heteronuclear single quantum coherence spectra of the 15N-Scc4:Scc1 and Scc4:15N-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.

Highlights

Chlamydia trachomatis (CT) is a gram-negative, obligate intracellular bacterial pathogen with distinct biovars that cause the most common sexually transmitted bacterial diseases or infectious blindness due to trachoma, a neglected infectious disease [1,2]
We showed that Scc4 and Scc1 associate in vivo when co-expressed in E. coli [16]
The interaction was strong allowing for the complex to be purified by Nickel immobilized affinity chromatography (Ni-IMAC) when either Scc4 or Scc1 had a His6-tag

Summary

Introduction

Chlamydia trachomatis (CT) is a gram-negative, obligate intracellular bacterial pathogen with distinct biovars that cause the most common sexually transmitted bacterial diseases or infectious blindness due to trachoma, a neglected infectious disease [1,2]. CT utilizes a type three secretion system (T3SS) to establish and maintain infection by delivering over 100 effector proteins into the host epithelial cell [3,4,5,6]. Once the bacteria enter the host cell, they localize in a specialized membrane-surrounded vacuole, termed an inclusion, where the effector proteins defend against the host’s immune response and provide critical nutrients from the host cell. CT undergoes a unique, biphasic developmental cycle within the inclusions from infectious elemental bodies (EBs) to noninfectious, vegetative reticulate bodies (RBs). RBs transit back to EBs prior to release of EBs from the host cell to reinitiate new infections. The T3SS and transcriptional control of the developmental cycle are essential for infection, and Scc

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