Hypothetical protein CT398 (CdsZ) interacts with σ⁵⁴ (RpoN)‐holoenzyme and the type III secretion export apparatus in Chlamydia trachomatis

Abstract

A significant challenge to bacteriology is the relatively large proportion of proteins that lack sufficient sequence similarity to support functional annotation (i.e. hypothetical proteins). The aim of this study was to apply protein structural homology to gain insights into a candidate protein of unknown function (CT398) within the medically important, obligate intracellular bacterium Chlamydia trachomatis. C. trachomatis is a major human pathogen responsible for numerous infections throughout the world that can lead to blindness and infertility. A 2.12 Å crystal structure of hypothetical protein CT398 was determined that was comprised of N-terminal coiled-coil and C-terminal Zn-ribbon domains. The structure of CT398 displayed a high degree of structural similarity to FlgZ (Flagellarassociated zinc-ribbon domain protein) from Helicobacter pylori. This observation directed analyses of candidate protein partners of CT398, revealing interactions with two paralogous type III secretion system (T3SS) ATPase-regulators (CdsL and FliH) and the alternative sigma factor RpoN (r 54 ). Furthermore, genetic introduction of a conditional expression, affinity-tagged construct into C. trachomatis enabled the purification of a CT398-RpoN-holoenzyme complex, suggesting a potential role for CT398 in modulating transcriptional activity during infection. The interactions reported here, in tandem with previous FlgZ studies in H. pylori, indicate that CT398 functions as a regulator of several key areas of chlamydial biology throughout the developmental cycle. Accordingly, we propose that CT398 be named CdsZ (Contact-dependent secretion-associated zinc-ribbon domain protein).

A working model for the type III secretion mechanism in Chlamydia

“…C. trachomatis CT398 has structural similarity to the Helicobacter flagellar protein FlgZ [6]. This study further revealed interactions of CT398 with the alternative sigma factor RpoN, raising the possibility that CT398 could modulate activity of RNAP.…”

Chlamydia cell biology and pathogenesis

“…Late genes are regulated either by a stage-specific sigma factor (σ 28 ) 19 or through the relief of transcriptional repression of σ 66 -dependent promoters by the repressor early upstream open reading frame (EUO) 19,23 . Transcriptional regulation is also coupled to the T3SS 24–26 . Much of this insight comes from in vitro studies; advances in chlamydial genetics (BOX 2) will enable the analysis of current models in vivo .…”

Crystal structure of theMSMEG_4306gene product fromMycobacterium smegmatis

“…A standard BLAST (Altschul et al, 1990) search against the PDB using the MSMEG_4306 sequence as a query did not result in any significant structural homologue that could be used as a template for solving the structure by the molecularreplacement method. However, a DELTA-BLAST (domain enhanced lookup time accelerated BLAST) search (Boratyn et al, 2012) revealed two crystal structures, namely those of HP0958 from Helicobacter pylori (Caly et al, 2010) and CT398 from Chlamydia trachomatis (Barta et al, 2015), as close homologues of MSMEG_4306, although they share only 15% identity with MSMEG_4306. Since the two homologues are likely to be too distantly related for successful molecular replacement, we decided to solve the structure of MSMEG_ 4306 by experimental phasing.…”

“…The top two hits were CT398 from C. trachomatis (PDB entry 4ilo,r.m.s.d. 3.8 Å , Z-score 15.9, 229 aligned C atoms; Barta et al, 2015) and HP0958 from H. pylori (PDB entry 3na7, r.m.s.d. 7.7 Å , Z-score 15.6, 230 aligned C atoms;Caly et al, 2010), which appeared to be strikingly similar to the structure of MSMEG_4306 (Fig.…”

“…3a and 3b) indicates a remarkable similarity in the zinc-ribbon domain, while the coiled-coil domains show variations in their orientations relative to each other. Although such structural variations may occur owing to different crystal packing (Eyal et al, 2005;Rapp & Pollack, 2005), it is also possible that these variations are organism-specific and that they play a role in proteinprotein interactions (Barta et al, 2015). The search using just the N-terminal domain resulted in 846 hits, which comprised more or less the same proteins as those obtained with the fulllength structure.…”