Part:BBa_K5185028
HNP1_G12A_Y22A
The antibacterial activity of the defensive element relies on dimerization. Through modeling, we replaced the 12th amino acid G and the 22th amino acid Y of HNP1Ala with A and A respectively, to further enhance the stability of HNP1 dimers.
Usage and Biology
α-defensins typically have a triple-stranded beta-sheet structure based on internal disulfide bonds (Lehrer et al. 2012) and further stabilized by other immutable structures. Apart from these residues, point mutations on alpha-defensins tend to have limited effect on the general conformation. Alpha-defensin dimers are formed when amino acids on the second beta-strand interact with each other. Specifically, carboxyl and nitric groups in the amino acid backbone of the two monomers formed 4 hydrogen bonds during dimerization. In addition, cationicity and hydrophobicity are both key determinants in dimerization rates, with the latter showing more significant impact than the former (Rajabi et al. 2012). Thus, our general goal is to create a stable dimer structure while increasing cationicity and hydrophobicity.
Modeling
Our models can mainly be categorized in active site amino acid mutations and other amino acid mutations. We sought to stabilize the active site by introducing additional interactions between the two monomers. Upon considerations, we decided attempt creating pi-pi, pi-cation, and hydrogen bonding interactions between amino acid side chains. Aromatic rings composes of conjugate pi-bonds, which causes the pi-electrons to be delocalized and shared across multiple molecules. A pi-pi interaction is when the delocalized electrons are shared between two aromatic rings when placed adjacently, while pi-cation interaction is characterized by an attraction between a positively group and the delocalized pi-electrons. It should be noted that mutagenesis in our models might not accurately reflect the actual configuration of side chain of the modified HNP-1 that are produced in experimentation. Therefore, we would prioritize pi-pi interactions (increase hydrophobicity) and pi-cation (increase hydrophobicity and cationicity) over hydrogen bonding under the same predicted affinity. The tetramer of HNP-1 was obtained from the Protein Data Bank (PDB id: 3GNY), and the dimer molecule was extracted. The amino acids of the active site are T-18, C-19, and I-20 (Amino acid - index), and as C-19 is immutable (as discussed later), we will focus on mutating T-18 and I-20. Apart from active site amino acids, other amino acids were also mutated to increase hydrophobicity. It is important to note that there are several immutable that constructs the basic structure of alpha-defensin, namely the cytosine residues (C-2, C-4, C-9, C-19, C-29, C-30) that constitute disulfide bonds, arginine and glutamic acid residues (R-5, E-13) that constitute an essential salt bridge, and an invariant glycine (G-17). Of the remaining amino acids, we selected all non-positive hydrophilic amino acids and mutated them to alanine to increase hydrophobicity without drastically changing the general conformation. Therefore, in this mutation, we first mutated G12 and Y22 to Ala to enhance the hydrophobicity of defensins(Fig. 1A), thus generating the HNP1 mutant HNP1_G12A_Y22A (HNP1Ala).After the alanine substitution of amino acids, the alpha-fold predicted model shows high similarity with the original HNP-1 when aligned in PyMOL (rmsd = 0.482). Hdock result of the final structure HNP-1_Ala gives a confidence score of 0.8058. We then performed point mutations on amino acids G18 and L20 of the active site to obtain three other HNP1 mutants：HNP1ANF，HNP1AWW(BBa_K5185030)和HNP1AWK(BBa_K5185031) Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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