Structure prediction and validation of an affibody engineered for cell-specific nucleic acid targeting

Abstract

Cell-penetrating peptides comprising cloned epitopes that contribute to membrane transduction, DNA-binding and cell targeting functions are known to facilitate nucleic acid delivery. Using the ITASSER software, we predicted the 3-D structure of a well characterized and efficient transfecting cell-penetrating peptide, namely TAT-Mu and its derivative TAT-Mu-AF protein that harbors a targeting ligand, the HER2-binding affibody. Our model predicts TAT-Mu-AF fusion protein as primarily comprising α-helices. The affibody in TAT-Mu-AF is predicted as a 3-helical domain that is distinct from the TAT-Mu domain. Its positioning in three-dimensional structure is oriented in a manner that possibly favors interactions with receptor and facilitates transport to the target site. The linker region between TAT-Mu and the affibody is also predicted as a helix that is likely to stabilize the overall fold of the TAT-Mu-AF complex. Further, the evaluation of secondary structure of the designed TAT-Mu-AF fusion protein by circular dichroism is in support of our predictions.

Keywords: 3-D modelling; Affibody; CPPs; Cell-targeting; Nucleic acid delivery; TAT fusions; iTASSER.

Fig. 1

a Schematic representation of designed TMAF with cell-targeting ligand (AF) with the vector backbone depicted as thin rectangles. The linker domain depicted in L separates the TAT and Mu domains from the AF domain (Not drawn to scale). b. Pairwise alignment of the amino acid sequences of TM and TMAF using ClustalW program (Thompson et al. 1994). Both TM and TMAF are N-terminal His-tag chimeras followed by a 11-amino acid TAT epitope (YGRKKRRQRRR), 19-amino acid Mu epitope (MRRAHHRRRRASHRRMRG), linker region (TYLSE....NPT) and AF (VDNK......QAPK) followed by the Stop signal. The vector sequences are depicted in thin. Asterisk below the sequence represents identical domains of the designed constructs

Fig. 2

a Structure of recombinant TMAF protein predicted by I-TASSER program. Models were generated using PyMOL software (DeLano 2002). TAT epitope (red), Mu epitope (blue) and AF (pale cyan). The linker regions (yellow) separate the TAT-Mu domains from the AF. b. CD profile of TMAF protein in the far-UV spectral region (195–250 nm). Mean residue mass ellipticity was obtained using a Jasco J-815 spectropolarimeter (Jasco, MD). Five scans were averaged and the calculated MRE values were plotted as a function of the far-UV-spectrum. The characteristic shape and magnitude of the CD spectral trace is indicative of α-helix conformation