Human alpha-defensin 5 (PDB: 4RBW) from Homo sapiens
Created by: Jenna Zschaebitz
Human alpha-defensin 5 (PDB ID: 4RBW) is a protein found in Homo sapiens, which is primarily involved in the human immune response (1). Defensins are peptide components that play a crucial role in the innate host defense of humans and plants (2,3). They have the ability to quickly recognize and neutralize specific foreign pathogens (3). These proteins are particularly useful in fighting bacterial, fungal, and viral infections, which can range from common colds to serious infections caused by bacterium such as Escherichia coli (4). They recognize foreign microbial invaders by binding to pathogen-associated patterns-recognition molecules, such as Toll-like receptors (3). After a pathogen is recognized, an acute antimicrobial response is generated by the protein, which recruits leukocytes and causes production of other antimicrobial molecules (3). Alpha-defensins show an amphiphilic arrangement, which means it contains both hydrophilic and hydrophobic residues. This topology is thought to be very important for the interaction of target membranes during an immune response (3). Recently, it has been proven that defensins can act directly on an entire virus particle, known as a virion, or they can affect a specific target cell, which can indirectly fight infections caused by certain pathogens (4). However, the exact mechanism by which the virion is inactivated is not clear and continues to be a point of investigation (4). Human alpha-defensins are becoming increasingly recognized for their importance in the human immune systems. As more pathogens are becoming resistant to traditional antibiotics, new remedies are being sought to combat infections around the world. Human alpha-defensin 5 is a protein that can be developed into an antiobiotic and it has the potential to fight infections caused by various pathogens, such as Escherichia coli and Human papillomavirus (1, 4).
Structurally, human alpha-defensin 5 is a short polypeptide containing 32 amino acid residues (1). It is a cationic, tridisulfide peptide that has been isolated from leukocytes (3). While human alpha-defensin 5 (HD5) is mainly produced in Paneth cells in the small intestine, it is in female genitalia tract, in salivary glands and others organs in the human body (4). The molecular weight of Human alpha-defensin 5 is 3,670.34 Da and it has an isoelectric point of 9.80, which is the point where the sum of the negative and positive electrical charges of a protein is equal to zero (5, 6). Human alpha-defensin 5’s secondary structure is a dimer and is predominantly characterized by β-sheets (1). At low salt concentrations, HD5 has substantial antibacterial activity, but at higher concentrations the function is drastically altered (4). The interactions between cells and defensins is very complex, but there are two proposed theories. One theory highlights the potential of defensins to interrupting cell-signaling pathways that are required in viral replication (4). Another theory proposes that the defensin can directly connect with cell surface glycoproteins (4).
Through several mutations and analyses it was confirmed that the critical residue for dimer formation in human alpha-defensin 5 is Glu-21. Glu-21 is important because it is located near the active region of the protein at the C-terminus (1). The active region is the site where substrates bind and where catalyzed reactions occur (6). Glu-21 was found to be the only electronegative residue in Human alpha-defensin 5 and substituting Glu-21 lead to loss of quaternary structure (1). Quaternary structure is the arrangement of two or more chains into a complete unit (5). Quaternary structure is very important, because it increases stability of a protein, genetic economy and efficiency (6). It also brings catalytic sites closer together, and leads to cooperativity at binding sites (6). The loss of quaternary structure illustrates the importance that the electronegative Glu residue has in the structure of human alpha-defensins.
Human alpha-defensin 5 has six Arginine residues and four of these residues are paired on opposite sides of the molecule (7). Arg-25 is the only Arginine residue that is not paired and Arg-6 forms a salt bridge with the Glu-14 resideue (7). Arg-9 and Arg-28 are two critical residues in human alpha-defensin 5 (7). Substitution of Arg-9 and Arg-28 by Lys is detrimental to the function of the protein (7). Specific mutations have demonstrated enhancement of the peptide’s antiviral activity (1). While the substitution of Glu-21 lead to structural confirmation, it did not affect the antibacterial activity of the protein. Substitution of Glu-21 with an Arg residue increased the antibacterial function of the protein significantly. Arginine is an important residue, because the atoms surrounding it are less sterically hindered by the side chain. In addition, arginine is found to have effective antibacterial efficiency (1).
There are two associated ligands in human alpha-defensin 5, which are important to the structure and function of the protein. One of the associated ligands is a chloride ion, which is used as a “bridging ligand” and can connect two or more atoms (1). While the chloride ion can contribute to ionic interactions, there is no specific interaction known. Another associated ligand that is found in HD5 is the sulfate ion, which plays an important role in disulfide pairings with cysteine residues. Defensins have six cysteine residues that can form disulfide bridges - sulfate ion acts as a chelate or bridge for the protein (3, 1). Previously, the tridisulfide structure of alpha-defensin was thought to be involved in the antimicrobial activity of the protein, but recent evidence indicates that the disulfide acts to protect the backbone of the protein (3). There is data that illustrates that the antimicrobial function of alpha-defensins is not affected by tridisulfide arrangement, the cytotoxity and the overall topology of the protein (8). Human alpha-defensin 5 does not have any alternate conformations, associated metal ions or drug complexes. However, a calcium-gated potassium channel, mIKCa1, regulates the Paneth cells. And the mIKCa1 channel controls the uptake of calcium ions from the extracellular matrix (3). Human alpha-defensins do not contain prosthetic groups and are not attached to any substrates or products.
The Position-Specific Iterated Basic Local Assignment Search Tool (PSI-BLAST) is a program that can be used to find proteins that have a similar sequence (primary structure) to the query protein, which can be an amino acid or nucleotide (9). PSI-BLAST generates gapped alignments and compares these to a vast database of protein residues (9). Through the database, several proteins are identified that have similar primary structures and are assigned E values (9). If the E value is lower than 0.05, the proteins similarities are more significant (9). In addition to the PSI BLAST, the Dali Server is also a relevant tool that can be used to compare proteins based on tertiary structure. The Dali Server is used to obtain Z-Scores, which are based on comparative predictions of protein folding (10). The Dali Server works by using a sum-of-pairs method, which produces a value of similarity between two structures based on their intramolecular distances (10). If the Z-Score is above 2, then the proteins are said to have a greater amount of similar folds (10). The Dali Server measures the similarity of two predicted protein structures. Both the PSI-BLAST and the Dali server assess comparison proteins that have similar sequence and tertiary structures.
Using the PSI-BLAST and Dali server, mucosal alpha-defensin (PDB ID: 2K1I) in Macaca mulatta, also known as Rhesus monkeys, was found to be structurally similar to HD5 (9). This comparison protein had an E value of 0.012 found using the PSI-BLAST and a Z score of 4.4 using the Dali server (9, 10). Human alpha-defensin 5 is similar in tertiary structure to the mucosal alpha-defensin found in Macaca mulatta. A protein’s tertiary structure provides information on how a protein folds to obtain a globular shape, which is characterized by the lowest surface-to-volume ratio (6). The mucosal alpha-defensin is also classified as an antimicrobial peptide with six cysteine residues that contribute to tridisulfide linkages (8). Mucosal alpha-defensin is characterized by disulfide pairing and alpha-defensin folding, which is visualized through NMR Spectroscopy (8). The secondary structure of mucosal alpha-defensin is dominated by anti-parallel β-sheets (8). The basic amino acid residues on mucosal alpha-defensins can interact with the negatively charged phosphate groups on a membrane, which allows them to anchor into a bilayer membrane of a cell (8). A comparison of both proteins through the PSI-BLAST indicates that mucosal alpha-defensin has a sequence that is 60% identical to human alpha-defensin 5 (8).
A protein’s secondary structure is predominantly determined by noncovalent forces such as hydrogen bonds, hydrophobic interactions, ionic interactions and van der Waals interactions (6). The protein can arrange itself into pleated fragments and are stabilized by hydrogen bonds between adjacent amino acid residues (6). In terms of secondary structure, Human alpha-defensin 5 is characterized by beta sheets and random coils. It consists of one subunit made up of four distinct chains. As demonstrated by understanding the amino acid content of human alpha-defensin 5, the protein structure is critical to understanding the folding and therefore function of an antimicrobial protein. Antimicrobial peptides, specifically human alpha-defensins, are one of the most ancient contributors to the human immune system. Human alpha-defensin 5 acts as an antimicrobial peptide that has the potential to be used in new drug developments to combat antibiotic resistance (8).