An In-Silico Sequence-Structure-Function Analysis of the N-Terminal Lobe in CT Group Bacterial ADP-Ribosyltransferase Toxins

Abstract

The C3-like toxins are single-domain proteins that represent a minimal mono-ADP-ribosyl transferase (mART) enzyme with a simple model scaffold for the entire cholera toxin (CT)-group. These proteins possess a single (A-domain) that modifies Rho proteins. In contrast, C2-like toxins require a binding/translocation partner (B-component) for intoxication. These are A-only toxins that contain the E-x-E motif, modify G-actin, but are two-domains with a C-domain possessing enzymatic activity. The N-domain of the C2-like toxins is unstructured, and its function is currently unknown. A sequence-structure-function comparison was performed on the N-terminal region of the mART domain of the enzymatic component of the CT toxin group in the CATCH fold (3.90.210.10). Special consideration was given to the N-domain distal segment, the α-lobe (α1–α4), and its different roles in these toxin sub-groups. These results show that the role of the N-terminal α-lobe is to provide a suitable configuration (i) of the α2–α3 helices to feature the α3-motif that has a role in NAD+ substrate binding and possibly in the interaction with the protein target; (ii) the α3–α4 helices to provide the α3/4-loop with protein-protein interaction capability; and (iii) the α1-Ntail that features specialized motif(s) according to the toxin type (A-only or A-B toxins) exhibiting an effect on the catalytic activity via the ARTT-loop, with a role in the inter-domain stability, and with a function in the binding and/or translocation steps during the internalization process.

The N-terminus ofPaenibacillus larvaeC3larvinA modulates catalytic efficiency

“…The characterization of N-terminal variants showed decreased protein stability and enzymatic activity in relation to the WT toxin. These findings support the importance of residues previously identified through computational studies, while further identifying novel interactions between the α 1 -helix and the ARTT-loop, which houses the catalytic Q-X-E motif (42). Lastly, C3larvinA was shown to have improved RhoA-targeting capabilities within macrophage cells compared to C3larvin trunc .…”

“…C3larvinA that was missing from C3larvin trunc , one that was shared amongst both C2-and C3toxins. These conserved residues belong to the Structure-motif (S-motif), and were predicted to contribute to protein stability through interactions made with the PN-and ARTT-loops (42).…”

Characterization of C3larvinA, a novel RhoA-targeting ADP-ribosyltransferase toxin produced by the honey bee pathogen,Paenibacillus larvae

“…Interestingly, the intervening 'X' residue in the Gln-X-Glu motif in both C3cer and C3larvin is a Tyr residue further cementing their identical catalytic signatures and detailed mechanism for RhoA modification. As observed for C3cer, the Tyr 184 in C3larvinA interacts with a patch on RhoA composed of Val 43 , Ala 56 , and Trp 58 . The hydroxyl group of Tyr 184 forms an H-bond with the main-chain carbonyl group of Leu 57 in RhoA.…”

“…This is interesting, since the N-terminal adaptor domain of the iota I a -subunit (PDB: 4GY2) is required to bind to the translocating I b -domain (B-domain) via its N-terminal Ca 2+ -binding motif [54,55]. This may point to a divergent evolution of C3-like toxins from an ancestral C2-like toxin where the binding machinery evolved to accommodate a single-domain enzyme [56].…”

A Structural Study on the Listeria Monocytogenes Internalin A—Human E-cadherin Interaction: A Molecular Tool to Investigate the Effects of Missense Mutations

“…To note, in silico analysis already proved to be a reliable analytical tool to investigate the interaction of macromolecules with either small molecules (e.g., [20]) or other macromolecules, as shown already for InlA-Ecad [21]. Additionally, the computational assessment of inter-molecules interaction may eventually result in a reliable estimate of biological outcomes (e.g., [22][23][24][25]). Specifically, in the present work, a structure-based molecular modeling approach was developed and validated as a proof of concept using a set of previously characterized engineered mutations [9].…”