1. Hermes transposase is a hexamer of three heterodimers native to Musca domestica 1. The function of hermes transposase is to catalyze DNA breakage and rejoining reactions. 2 Domain Swapping, where a secondary or tertiary structure of one chain is replaced with the same element of another chain, is present at three interfaces where two alpha helixes are swapped. The goal of this research is to delineate which residues are essential to multimerization of the hexamer and to predict what specific mutations will prevent formation of the hexamer. Structural analysis of a tetrameric crystallographic structure indicated many intermolecular hydrogen bonds and salt bridges at each of the six interfaces that are present. Hydrophobicity also plays a role, especially in the domain swapping interfaces. The individual importance of every charged amino acid, with emphasis on those participating in salt bridges, was further investigated by performing a computational alanine scan on the parent protein in which each charged amino acid was replaced, one by one, with an alanine. The effect of each mutation was observed by calculating the electrostatic free energy of association for each mutant and comparing these values with the free energy of association of the parent. Many of the mutants whose electrostatic free energy deviated greatly from the parent were the same residues that had previously been identified as participating in intermolecular salt bridges. Depending on the calculated increase or decrease of the electrostatic free energy of association, we are able to predict which mutations result to better or worse stability of individual dimers. Our method provides an efficient computational screening of contributions to multimerization for all charged amino acids and can be used as a guide to select a small subset of amino acids to be tested with more elaborate experimental studies. Background Hermes Transposase catalyze DNA breakage and rejoining reactions. 2 It is functionally active as a hexamer. The available crystallographic structure is a heterotetramer, (used in this research). A theoretical model, containing four equal length chains (A, B, C and D) is also available, but it deviates substantially from the planar crystallographic heterotetramer. The theoretical model aligns very closely with the spiral version of the hexamer and for this reason was not utilized in this investigation. C-alpha-trace of the heterotetramer (red) with the theoretical full model (blue). C-alpha-trace of the theoretical full model (red) exactly covering four chains of the spiral hexamer (blue). The longer chains of the heterotetramer are not complete, and in addition to a 17-residue gap between 480A and 497D in chains B and A of the heterotetramer, the first 78 residues were removed to allow for crystallization. Methods Salt Bridge Analysis: Salt bridges were identified using MOLMOL and were defined as being between oppositely charged amino acids (R, K and D, E). A bond distance of 0-3.5Å was defined as strong, 3.5-4.5Å as mid-range in strength, and 4.5-5.0Å as weak. Pairs of amino acids whose distance was between 5.0Å and 8.0A were included as well to account for electrostatic interactions that may arise due to protein dynamics. Once the residues involved in salt bridges were identified, the pKa, a measurement of the likelihood of a proton being dissociated from a molecule, for each of these residues was calculated using PROPKA 2.0. The location of the individual residues (surface or buried) was also determined by PROPKA and was compared to the SASA (Solvent Accessible Surface Area), calculated by MOLMOL. Electrostatic Free Energy Calculations: Electrostatic free energy values were calculated using the Analysis of Electrostatics of Proteins (AESOP) protocol developed by our group 6, shown in Fig. 3B. Using a high-throughput computational approach, an alanine scan, in which each charged amino acid is replaced one by one with an alanine, was used to generate mutants based on the PDB file containing atomic coordinates of the heterotetramer. PDB2PQR was used to add charges and van der Waals radii to PDB files, and APBS was used to calculate the electrostatic potential within and surrounding the protein. The electrostatic free energy of each mutant was calculated based on the thermodynamic cycle shown in Fig. 3A. Change in energy of both the horizontal process of association and the vertical process of solvation are taken into account, giving ΔΔG solvation. The Linearized Poisson-Boltzmann Equation (LPBE) is used to calculate the electrostatic potentials (see step 4 in Fig. 3b) and this has several advantages. First, it is able to account for the different dielectric constants within the protein and solvent. It also takes into account the ionic strength and protein charges. The protein is placed in a three-dimensional grid that is 129 x 129 x 129 the LPBE is calculated at discrete grid points within and surrounding the protein and extrapolated to individual atoms. These calculations were repeated for the following combination of monomers and dimers (letters indicate the chains): AF-BCA-BB-C AB-CFA-CB-F AC-BFA-FC-F The procedures and protocols used to calculate these free energies are shown in Fig. 3B. Results and Discussion There was substantial agreement between the salt bridge data and the electrostatic free energy calculations. All the residues identified as participating in one of the intermolecular salt bridges with a bond distance of 5.0Å or less were shown to have a significant effect on the energy of association when mutated to an alanine (Fig. 4). References 1 Craig, N.L., Dyda, F., Hickman, A.B., (2005) Molecular architecture of a eukaryotic DNA transposase, Nature Structural & Molecular Biology 12:715-721. 2 Craig, N.L., Dyda, F., Hickman, A.B., (2005) Purification, crystallization and preliminary crystallographic analysis of Hermes transposase, Acta Crystallographic F61:587–590. 3 Guex N and Peitsch MC: SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18: 2714-2723, 1997. 4 Hickman A, Perez Z, Zhou L, et al. (2005). Molecular architecture of a eukaryotic DNA tranposase. Nature Structural &Molecular Biology. 12:715-721. 5 Humphrey W, Dalke A, Schulten K (1996). VMD: visual molecular dynamics. Journal of Molecular Graphics. 14: 33-37. 6 Kieslich, CA, Yang, J., and Morikis, D (2009) AESOP: Analysis of Electrostatoc Properties of Proteins, To be Published. 7 MOLMOL: a program for display and analysis of macromolecular structures 8 UCSF Chimera--a visualization system for exploratory research and analysis. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE. J Comput Chem. 2004 Oct;25(13):1605-12. Acknowledgements Dr. Dimitrios Morikis, Aliana López De Victoria, Ronald Gorham, Chris Kieslich We are grateful to Prof. Atkinson for suggesting the project and for exciting and insightful discussions. BRITE and the National Science Foundation http://www.engr.ucr.edu/~dmorikis/ Disrupting Multimerization of Hermes Transposase by Single Amino Acid Mutation Chelsea Vandegrift, Aliana López De Victoria, Ronald D. Gorham, Dimitrios Morikis, Chris A. Kieslich Figure 2 Another trend visible in the free energy data is that mutation of a basic (positive) amino acid results in an increase in stability (higher free energy values than parent) while mutation of an acidic (negative) amino acid results in a decrease in stability (lower free energy values than parent). All chains of Hermes Transposase are positively charged, thus removing a positively charged amino acid helps decrease the repulsive forces whereas mutation of a negative amino acid increases positive character and inhibits multimerization. It is interesting to note that mutation of even a basic amino acid can result in greater instability if that residue in involved in a particularly strong salt bridge. This is the case with 549K (shown in red in Fig. 4). The free energy calculations also help to indicate what interfaces are most influenced by which amino acids. Fig. 5 shows free energy calculations for association of dimer BA with dimer CF. Association of these dimers is dependent mainly on interface 3 and, as can be seen in the graph, mutation of the basic amino acids involved in salt bridges at that interface result in a decrease in stability along with acidic amino acids. These basic amino acids are shown to increase the stability of the heterotetramer upon mutation in other free energy calculations not involving interface 3. Thus the free energy calculations are able to pinpoint how a mutation will affect a particular area as well as show how the total charge of the chains effect multimerization. AB C D F E Figure 1: The letters indicate the chain and the numbers label the interfaces. Hexamer Heterotetramer A FC B 1 2 1 1 2 2 Interfaces 3 84K 91K 105D 93E122K 126K 138E 139E 107R A F C B Interface 3 Figure 5 Future Work Future work will include experimental validation of the free energy predictions by Prof. Peter Atkinson's group, Dept. of Entomology, UCR. The BioMoDeL lab will perform computational alanine scans of charged amino acids and clustering of spatial distributions of electrostatic potentials. This study will use a structural bioinformatics method developed in BioMoDeL to locally perturb the physicochemical makeup of hermes transposase and cluster the resulted mutants according to their similarity/dissimilarity to parent protein. The electrostatic clusters will be ranked according to the calculated free energies presented here. The graph shows the energy of association of dimer AF with dimer BC. The first dot represents the energy of the parent while the rest of the graph shows the energy of association after various mutations. All labeled residues are amino acids involved in a salt bridge, those in bold have a distance of 5.0Å or less while those in red are in a salt bridge of less that 3.5Å. Mutation of any amino acid involved in a salt bridge causes a change in energy greater that ±50 KJ/mol (shown by the gray bar). 119 D 82 E 122 K 150 K 84 K 89 D 91 K 93 E 96 E 138E /139 E 126 K 104 R 133 E 97 K 92 K 107 R 497D 537D 549K 369R 530E Parent Figure 4 Thermodynamic cycle used to calculate electrostatic free energies of association and solvation. ΔG solvation = ΔG solution – ΔG vacuum Δ ΔG solvation = ΔG Solvation Complex – ΔG solvation AF – ΔG solvation BC Near Vacuum ε protein = 2 ε solvent = 2 κ=0 Solution ε protein = 2 ε solvent = 80 κ≠0 + + ΔGΔG AF BC Complex ΔG AF ΔG BC ΔG Complex ΔGΔG Figure 3AFigure 3B PDB WHATIF PDB2PQR APBS 1 2 3 4 6 Retrieval & cleaning of coordinates for parent protein complex Generation of coordinates for mutants Generation of coordinate files with partial charges & vdW radii Calculation of electrostatic potentials Chimera APBS 5 Electrostatic potential visualization Free energy calculation Abstract