Regions of intrinsic disorder help identify a novel nuclear localization signal in Toxoplasma gondii histone acetyltransferase TgGCN5-B

Abstract

We have previously shown that protozoan parasites, such as Toxoplasma gondii, contain a high prevalence of intrinsically disordered regions in their predicted proteins. Here, we determine that both TgGCN5-family histone acetyltransferases (HATs) contain unusually high levels of intrinsic disorder. A previously identified basic-rich nuclear localization signal (NLS) in the N-terminus of TgGCN5-A is located within such a region of predicted disorder, but this NLS is not conserved in TgGCN5-B. We therefore analyzed the intrinsically disordered regions of TgGCN5-B for basic-rich sequences that could be indicative of a functional NLS, and this led to the identification of a novel NLS for TgGCN5-B, RPAENKKRGR. The functionality of the GCN5-B NLS was validated experimentally and has predictive value. These studies demonstrate that basic-rich sequences within regions predicted to be intrinsically disordered constitute criteria for a candidate NLS.

Figure 1. Intrinsic disorder predictions and domain structures of TgGCN5-A (A) and TgGCN5-B (B)

The per-residue propensity for intrinsic disorder was evaluated using a set of PONDR algorithms (VL-XT – red lines; VSL2 – purple lines; VL3 – blue lines). In PONDR plots (top graphs of each plot), segments with scores above 0.5 correspond to the disordered regions, whereas those below 0.5 correspond to the ordered regions/binding sites. Long regions of predicted disorder are highlighted in gray. Position of the NLS is shown in purple. Below each plot a cartoon showing the key domains of each TgGCN5 protein is shown: HAT domain in blue and bromodomain (bromo) in gold, separated by the ADA2-interacting domain (ADA2). NLS is shown in purple.

Figure 2. Mapping of the NLS for TgGCN5-B

IFAs using antibody to FLAG tag were used to detect various forms of TgGCN5-B or β-gal fusion proteins. Diagram of each protein is shown to the right with FLAG epitope tag and proteins domains indicated. The blue box of each TgGCN5-B protein diagram represents the HAT catalytic domain and the orange box depicts the bromodomain. β-gal protein cartoon is represented in green. A. Localization of TgGCN5-B lacking the first 313 (Δ313) or B. 310 (Δ310) amino acid residues. C. Localization of TgGCN5-B after an internal deletion of the ten residue NLS (ΔNLS). D. Localization of β-gal_FLAG. E. Localization of β-gal-NLS_FLAG. F. Localization of GCN5-BΔ310 containing alanine substitutions for R311 and P312. hN, host cell nucleus; TgN, Toxoplasma nucleus; green = anti-FLAG; red = DAPI.

Figure 3. Intrinsic disorder analysis and localization of AT-hook 054600

A. Intrinsic disorder predictions for the full-length AT-hook protein TGGT1_056400. B. Distribution of the PONDR scores over the 2000–3000 amino acid fragment of the AT-hook protein TGGT1_056400. The per-residue propensity for intrinsic disorder was evaluated using a set of PONDR algorithms (VL-XT – red lines; VSL2 – purple lines; VL3 – blue lines). Segments with scores above 0.5 correspond to the disordered regions, whereas those below 0.5 correspond to the ordered regions/binding sites. Long regions of predicted disorder are highlighted in gray. Approximate area of predicted NLS (amino acids 2,515 – 2,522) is indicated in purple. C. AT-hook 056400 (TGGT1_056400) was endogenously tagged with a C-terminal 3xHA epitope tag and localized by IFA using anti-HA (green). Diagram at the right is a schematic of AT-hook 056400 with AT-hook domains as red boxes and predicted NLS as a green box.