Rearranging and concatenating a native RTX domain to understand sequence modularity

Abstract

The use of repetitive peptide sequences forming predictable secondary structures has been a key paradigm in recent efforts to engineer biomolecular recognition. The modularity and predictability of these scaffolds enables precise identification and mutation of the active interface, providing a level of control which non-repetitive scaffolds often lack. However, the majority of these scaffolds are well-folded stable structures. If the structures had a stimulus-responsive character, this would enable the allosteric regulation of their function. The calcium-responsive beta roll-forming repeats in toxin (RTX) domain potentially offer both of these properties. To further develop this scaffold, we synthesized a set of RTX peptides ranging in size from 5 to 17 repeats, with and without C-terminal capping. We found that while the number of repeats can be altered to tune the size of the RTX face, repeat ordering and C-terminal capping are critical for successful folding. Comparing all of the constructs, we also observed that native configuration with nine repeats exhibited the highest affinity for calcium. In addition, we performed a comparison on a set of known RTX-containing proteins and find that C-terminal repeats often possess deviations from the consensus RTX sequence which may be essential for proper folding. We further find that there seems to be a narrow size range in which RTX domains exist. These results demonstrate that the deviations from the consensus RTX sequence that are observed in natural proteins are important for high-affinity calcium binding and folding. Therefore, the RTX scaffolds will be less modular as compared with other, non-responsive scaffolds, and the sequence-dependent interactions between different repeats will need to be retained in these scaffolds as they are developed in future protein-engineering efforts.

Structural and functional dissection reveals distinct roles of Ca2+-binding sites in the giant adhesin SiiE of Salmonella enterica

“…In the bacterial cytosol, the RTX domain is intrinsically disordered. After exit of the T1SS duct, the β-rolls sequentially bind Ca 2+ at the outside of the cell envelope, initiate folding of the C-terminal domain and thereby provide directionality and secretion support for the rest of the protein [28, 29]. The proposed model is in line with earlier findings, namely that ATP-hydrolysis and membrane potential are only required for the initiation of secretion, while further secretion is driven by the folding of portions of the substrate protein that have left the T1SS [11, 14].…”

Block V RTX Domain of Adenylate Cyclase from Bordetella pertussis: A Conformationally Dynamic Scaffold for Protein Engineering Applications

“…Thus, the binding of each calcium ion leads to an increase in the affinity of the adjacent binding site, producing a polarized folding mechanism that propagates from the C-terminus towards the N-terminus. In addition, a C-terminal capping group was found to be necessary for proper folding and function [ 13 , 18 , 61 , 62 , 63 , 64 , 65 ].…”

Negatively charged residues of the segment linking the enzyme and cytolysin moieties restrict the membrane-permeabilizing capacity of adenylate cyclase toxin

“…The 3D structure of the AC domain in complex with calmodulin was solved30 and the structure-function relationships of the other domains of the RTX moiety are currently under intense exploration1720232426313233343536373839. However, little is known about the structure and function of the ~100 residue-long segment that is located between residues 400 to 500 of CyaA and links its AC domain to the pore-forming domain of the Hly moiety.…”