Lysozyme aptamer

Mar 25, 2024 • Yuxuan Sun, Baowei Huang

Description

In 2013, M. E. Girvin et al. employed the SELEX method to isolate an aptamer with high affinity for hen egg white lysozyme (HEWL). Concurrently, they examined its structure using X-ray diffraction. This aptamer has a sequence length of 59 nucleotides. M. E. Girvin et al. systematically minimized an RNA aptamer (Lys1) selected against HEWL. The resulting 59-nucleotide compact aptamer, Lys1.2minE, retains nanomolar binding affinity and the ability to inhibit lysozyme's catalytic activity. Specifically, Lys1.2minE inhibits the catalysis of large cell wall substrates but not that of small model substrates. Subsequently, M. E. Girvin et al. shortened the terminal stem of Lys1.2minE to four Watson-Crick base pairs, resulting in Lys1.2minF. They also examined its structure using X-ray diffraction. Lys1.2minF has a sequence length of 45 nucleotides. Its structure may serve as an adaptable protein-binding platform, capable of binding lysozyme with high affinity. It inhibits the cleavage of large substrates by lysozyme, and the bound aptamer has no effect on the catalysis of small substrates^[1].

SELEX

RNA aptamers were internally labeled with [α32P]-GTP and allowed to bind to lysozyme for 1h at room temperature. The bound complex was passed through a dual filter dot blot system attached to a vacuum manifold. The nitrocellulose membrane captures the aptamer–protein complexes while unbound aptamers are trapped by the nylon filter directly beneath. Both filters are visualized by phosphor storage imaging, and the fraction protein bound was calculated by determining the volume of radioactivity retained on the nitrocellulose divided by the total radioactivity retained by both filters. Sedimentation velocity experiments were performed on a Beckman XL-I using a standard procedure. Determining the S20, w and D20, w values of the RNA aptamers under each buffer condition specified by analytical ultracentrifugation. A standard lysozyme activity assay was used to determine the enzymatic activity^[1].

Detailed information are accessible on SELEX page.

Structure

2D representation

In 2013, Girvin et al. utilized the SELEX method to isolate an RNA aptamer, Lys1.2minE, which demonstrated high affinity for hen egg white lysozyme. This 59-nucleotide aptamer effectively inhibited lysozyme's catalytic activity on large cell wall substrates but not on small model substrates. Subsequently, they shortened the aptamer to Lys1.2minF, a 45-nucleotide variant, which retained its high binding affinity and selective inhibition of lysozyme's activity on large substrates. Here we use ribodraw to complete the figures, through the 3D structure information. The Lys1.2minF aptamer and Lys1.2minE aptamer were named by M. E. Girvin et al. in the article^[1].

Lys1.2minF: 5'-GGGCGGCUAAAGAGUGCAGAGUUACUUAGUUCACUGCAGACGCCC-3'
Lys1.2minE: 5'-GGGUUCAUCAGGGCUAAAGAGUGCAGAGUUACUUAGUUCACUGCAGACUUGACGAACCC-3'

3D visualisation

Girvin et al. resolved the 2.68 Å resolution X-ray crystal structure of the aptamer minF-lysozyme complex. The structures showed that both aptamers interact with the enzyme's active site, inhibiting its catalytic activity. Specifically, Lys1.2minE, with its 59-nucleotide sequence, binds strongly and selectively to large substrates, while Lys1.2minF, a shorter 45-nucleotide variant, retains similar binding properties but with a more compact structure. The PDB ID of this structure is 4M6D^[1].

Additional available structures that have been solved and detailed information are accessible on Structures page.

Girvin et al. resolved the 2.00 Å resolution X-ray crystal structure of the aptamer minE-lysozyme complex. The structures showed that both aptamers interact with the enzyme's active site, inhibiting its catalytic activity. Specifically, Lys1.2minE, with its 59-nucleotide sequence, binds strongly and selectively to large substrates, while Lys1.2minF, a shorter 45-nucleotide variant, retains similar binding properties but with a more compact structure. The PDB ID of this structure is 4M4O^[1].

Additional available structures that have been solved and detailed information are accessible on Structures page.

Binding pocket

Left: Surface representation of the binding pocket of the aptamer generated from PDB ID: 4M6D at 2.68 Å resolution. Lysozymes(shown in vacuumm electrostatics), blue is positive charge, red is negative charge. Right: The hydrogen bonds of binding sites of the aptamer bound with Lysozymes.

Left: Surface representation of the binding pocket of the aptamer generated from PDB ID: 4M4O at 2 Å resolution. Lysozymes(shown in vacuumm electrostatics), blue is positive charge, red is negative charge. Right: The hydrogen bonds of binding sites of the aptamer bound with Lysozymes.

Ligand information

SELEX ligand

M. E. Girvin et al. distinguished structures Lys1.2 and Lys1. 3 by trimming nucleotides predicted by each to be largely unstructured. Removing different nucleotides to test whether it can binding with lysozyme, and testing its affinity. RNA aptamers were internally labeled with [α-32P]-GTP and allowed to bind to lysozyme for 1 h at room temperature. The bound complex was passed through a dual filter dot blot system attached to a vacuum manifold. The nitrocellulose membrane captures the aptamer–protein complexes while unbound aptamers are trapped by the nylon filter directly beneath. Both filters are visualized by phosphor storage imaging, and the fraction protein bound was calculated by determining the volume of radioactivity retained on the nitrocellulose divided by the total radioactivity retained by both filters^[1].

Structure ligand

Lysozymes have primarily a bacteriolytic function; those in tissues and body fluids are associated with the monocyte-macrophage system and enhance the activity of immunoagents. Has bacteriolytic activity against M. luteus. Lysozyme C is capable of both hydrolysis and transglycosylation; it shows also a slight esterase activity. It acts rapidly on both peptide-substituted and unsubstituted peptidoglycan, and slowly on chitin oligosaccharides.-----From Uniprot

UniProt ID: uniquely identifies protein sequences in the UniProt database, a resource for protein information.

Pfam: a widely recognised database of protein families and domains.

GenBank: maintained by NCBI(National Center for Biotechnology Information), is a database of nucleotide sequences from various organisms, vital for genetic and molecular biology research.

Mass: an intrinsic property of a body.

Uniprot ID	Pfam	Mass	Protein sequence	PDB ID	GenBank
P00698	PTHR11407	14.31 kDa	GGPYLQ ...... KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDVQAWIRGCRL	4WM6	396218

Similar compound

We used the Dail server website to compare the structural similarities of ligand proteins, and chose the top 10 in terms of similarity for presentation.

Dail server website: a network service for comparing protein structures in 3D. Dali compares them against those in the Protein Data Bank (PDB).

Z-score: a standard score that is converted from an original score. The list of neighbours is sorted by Z-score. Similarities with a Z-score lower than 2 are spurious.

RMSD: (Root Mean Square Deviation) is used to measure the degree to which atoms deviate from the alignment position.

PDB: PDB ID+ chain name.

PDB	Z-score	RMSD	Description
4WM6-A	0	1	Hen Egg White Lysozyme
1LSG-A	23	1	Hen Egg White Lysozyme
1NF5-C	20.6	1.4	Alpha-Lactalbumin
4OWD-A	8.1	2.7	Membrane-Bound Lytic Murein Transglycosylase f
4C5F-A	8	2.5	Membrane-Bound Lytic Murein Transglycosylase c
6TA9-A	7.9	2.5	Slt Domain-Containing Protein
1QTE-A	7.9	2.6	Soluble Lytic Transglycosylase Slt70
3BKH-A	7.8	2.8	Lytic Transglycosylase
5E27-A	7.8	2.5	Resuscitation-Promoting Factor Rpfb
4YIM-B	7.7	2.8	Putative Soluble Lytic Murein Transglycosylase
7LAM-A	7.5	3.1	Lytic Transglycosylase Domain-Containing Protein

References

[1] An RNA aptamer possessing a novel monovalent cation-mediated fold inhibits lysozyme catalysis by inhibiting the binding of long natural substrates.
Padlan, C. S., Malashkevich, V. N., Almo, S. C., Levy, M., Brenowitz, M., & Girvin, M. E.
RNA, 20(4), 447–461. (2013)