Created by: Mehron Kouhestani 

          Hermes transposase (PDB ID: 4D1Q) is a member of the histone acetyltransferase (hAT) superfamily that has active representatives from the organism Musca domestica (1). The hAT enzymes work by acetylating conserved lysine amino acids on histone proteins by transferring an acetyl group from acetyl-CoA to form N-acetyl-lysine. The name "Hermes" is derived from the gene that codes for one of the largest families of RNA-binding proteins that stands for Heart RNA Recognition Motif Expressed Sequence (2). Gene activation is triggered when DNA wraps around histones, transferring an acetyl group onto the histone subsequently increasing gene expression (1,2). A transposase protein regulates the movement of genes from one position on the chromosome to another. Barbara McClintock et. al. suggested this idea that an organism's genome is not stationary, but rather is subject to chromosomal breakage and recombination (3). Transposable elements and genes encoding transposase proteins serve vital biological purpose and evolutionary significance as they populate genomes of nearly all organisms. Proteins like recombination activation protein-1 (RAG1), an essential component of the adaptive immune system, are derived from transposases. Understanding how transposon movement and amplification have changed genomes is correlated to understanding how genomic organization and remodeling have driven evolution (4,5). This is significant since the genes encoding transposase proteins can be mutagenic and be beneficial to their hosts. Although there are only a few hAT transposons and their transposases have been identified, they seem to share common mechanisms and structures. In fact, the only available structure of a hAT transposase is the portion of Hermes transposase from Musca domestica. By analyzing the primary, secondary, and tertiary structures of Hermes transposase, insight into the function and relevance of other transposase proteins could be revealed. 

          According to ExPASy, Hermes transposase has a molecular weight of 70.11 kDa and an isoelectric point (pI) of 8.64. Because proteins are the least soluble at its isoelectric point due to the absence of charges, this pI value ensures the solubility of Hermes transposase at physiological pH (6). This shows possible indications to its function as its solubility allows it to travel freely in the hemolymph of Musca domestica rather than being membrane bound or protein bound. The crystal structure Hermes transposase was obtained by x-ray crystallography after a single isomorphous replacement with anomalous scattering (SIRAS) at 3.4 angstom resolution using a Ta₆Br₁₂ derivative. This structure of Hermes transposase-DNA complex revealed that Hermes forms an octameric ring organized as a tetramer of dimers of its terminal inverted repeats (TIR) (4). In each dimer, the secondary structure consists of 53% alpha-helices, 7% beta pleated sheets, and 40% random coils (2). The alpha-helix is a common secondary structure of proteins and is a right hand-spiral conformation in which every backbone amine group donates a hydrogen bond to the backbone carbonyl group of the amino acid located three or four residues earlier along the protein sequence. When an alpha helix runs along the surface of the protein, one side of it will show polar side chains while the other side will show non-polar side chains (1). The abundance of these alpha-helices as compared to beta pleated sheets show that there is little rigidity in the protein supporting the free-floating protein theory. In addition to that alpha-helices often serve as signaling transmitting pathways between receptor and effector domains on proteins which is vital for Hermes as it directs histones towards genes for remodeling (7). 

          The primary structure of Hermes transposase is divided into four subunits: A, B, G, and H. From A, G, and B are all part of the insertion domain for transposase between residues 266-565. This insertion domain makes up the vast majority of the alpha-helices as it is part of the signaling mechanism (1). There are also two overlapping domains that do not cover the whole protein but are still quintessential to the activity and function of the protein. They are the domains of the hAT family C-terminal dimerization region and Hermes transposase DNA-binding domain. Because little is known about the primary structure and its direct function, the active site residues must be analyzed to determine any possible catalytic traits (1). 

          A few of the functionally important residues include Asp-180, Asp-248, and Glu-572 which form the acidic catalytic site that can protonate the sulfur on acetyl-CoA allowing the transfer of the acetyl group by dissociation. Amino acids Glu-138, Glu-139, and Leu-141 are bound to a sodium ligand for stabilization during crystallization. The sodium ligand bonded to N-terminal sequence induces crystal growth by altering solubility. The sodium ion also acts as a counter-ion to a phosphate group of the DNA backbone, coordinated by the main chain carbonyls found on Glu-138, Glu-139, and Leu-141. The Trp-139 residue stacks against base Gly-1 of the transferred strand and caps the 3' end of DNA strands that are being affected by the protein; and Lys-372, Arg-573, Asp-587, Arg-318, and Ser-310 participate in terminal inverted repeat binding (4). Stabilizing hydrogen bonds are created between Asp-6, Lys-91, and Glu-139 with another set between Ser-143 and Asp-6 (1, 3, 4, 5). 

           To find comparable proteins to Hermes transposase, the protein of interest (POI), based on primary and tertiary structure, PSI-BLAST and Dali Server were run respectively. The PSI-BLAST database is a tool used to determine significant similarities between primary structure based on E values. E-values less than the value 0.05 indicates significant similarity between two proteins' primary structures. An E-value of 5e-50 was given for the three-dimensional structure of the Hermes DNA transposase from Musca domestica (PDB ID: 2BW3) (8). In contrast, the Dali Server compares the tertiary structure by calculating the differences in intramolecular distances of proteins using a Z-score quantifiably derived using the sum-of-pairs method. A Z-score for a protein is considered significant if it is greater than 2. The Z-score for three-dimensional structure of the Hermes DNA transposase is 8.7, thus indicating a significant degree of similarity in tertiary structure and folding (9). Based on the values from the servers and from observation of the structure, there is minimal difference between these two proteins. This is expected because they are both derived from the same protein from the same organism. However, out of the results given from the database searches, this was the only viable comparison. 

          In terms of primary structure, the full-length Hermes DNA transposase (residues 1-612) is soluble much like that of the POI. However, it forms globular aggregates in aqueous solutions when expressed as an Escherichia coli fusion protein containing a histidine tag (His-tag) unlike the POI (4). Only upon the removal of the His-tag was Hermes DNA transpose able to be successfully crystalized with the use of selenomethionyl ligand for stability. The Trp-139 residue is highly conserved between the POI and Hermes DNA transposase indicating a vital role in the active site binding to histones. Because Trp-139 provides stabilization and a binding site for the 3' hairpin end of DNA, changing this residue between the two proteins would completely alter the protein function. The secondary structure of Hermes DNA transposase consists of 58% alpha-helices and 8% beta pleated which is nearly identical to the ratio found in the POI. This indicates the same importance in flexibility and movement in the hemolymph for signaling. 

          The tertiary structure of Hermes DNA transposase gives clues as to the function of the POI. Although the isolated dimers of Hermes transposase are active in vitro for all the chemical steps of transposition, only octamers are active in vivo. The structure of Hermes DNA transposase is comprised only of trimers rather than octamers. The aggregation of this protein when expressed in vivo in E. coli shows a resulting loss of function proving the need for an octamer transposase when functioning physiologically. Additionally, substitution of histidine in hAT with alanine in Hermes resulted in severe limitations for in vitro transposition. This indicated its requirement for function. The reason for the importance of the octamer is that the multiple subunits can provide for multiple specific and nonspecific DNA-binding domains to recognize repeated subterminal sequences within the transposon ends. The recognition of DNA at multiple sites aids in the binding specificity and surface activity for the target capture. Interactions between multiple sites could allow a transposase to locate its transposon ends amidst DNA (3,7). Physically, the Hermes transposase octamer works by looping the insertion on the DNA, creating a hairpin, to put the flanking DNA strands on the end in close proximity to then be excised generating double-strand breaks (4). 

          Although little is known about Hermes transposase, the information derived from the primary, secondary, and tertiary structures can provide some insight into its physiological function. Additionally, the comparison between this POI and Hermes DNA transposase highlights the importance of multiple subunits for function in vivo. By continuing to conduct research on Hermes transposase, deep insight into DNA rearrangement and its evolutionary implications can be found. Not only will this information expand the knowledge of the human genome, it will revolutionize the categorization of organisms on an evolutionary basis.