Disrupting Multimerization of Hermes Transposase Chelsea Vandegrift, Ronald D. Gorham, Aliana López De Victoria, Chris A. Kieslich, Dimitrios Morikis Department of Bioengineering, University of California, 900 University Avenue, Riverside, CA 92521 Aug. 20, 2009

Background Hermes Transposase Hermes Transposase is native to Musca domestica (the housefly) and carries out transposition of DNA segments. It works using a cut-and-paste mechanism and the DNA flanking the excised element transiently forms hairpins before the gap is repaired. Hickman et al. Nature Structural & Molecular Biology (2005)  vol. 12  715-721

Heterotetramer Homohexamer Hermes Transposase is a hexamer, but the largest crystallographic structure available is the heterotetramer. Homohexamer Heterotetramer A F C B D E DO I NEED TO EXPLAIN WHAT CRYSTALLOGRAPHIC DATA IS? Hickman et al. Nature Structural & Molecular Biology (2005)  vol. 12  715-721

Objective: To delineate the physicochemical properties that hold the different chains together. 1. Hydrophobicity (SASA – Solvent Accessible Surface Area) 2. Charge Coulombic Interactions Salt Bridges Hydrogen Bonds

- + Salt Bridges – Interactions between charged amino acids ASP LYS - + Strength of Interaction Bond Distance Strong 0.0 – 3.5 Å Medium 3.5 – 5.0 Å Weak 5.0 – 8.0 Å Acidic Amino Acids (-) Basic Amino Acids (+) Aspartic Acid (ASP or D) Lysine (LYS or K) Glutamic Acid (GLU or E) Arginine (ARG or R) Histidine (HIS or H) Atom measured from: ASP CG ARG CZ GLU CD LYS NZ

- + pKa Values HA  H+ + A- Favorable Unfavorable The pKa represents the pH at which Hydrogen ions are 50% dissociated. If a basic amino acid is found to have a higher pKa value than typical, it indicates that it is in a favorable location and the hydrogen is less likely to be dissociated. The opposite is true for acidic amino acids. A pKa value lower than the model indicates a favorable environment where the hydrogen is more likely to be dissociated. + - Unfavorable Favorable Basic Acidic

Electrostatic Free Energies Procedure Calculate electrostatic free energies of association and solvation ΔGsolvation BC = Gsolution BC – Gvacuum BC Δ ΔGsolvation = ΔGSolvation Complex – ΔGsolvation AF – ΔGsolvation BC = ΔGsolution - ΔGvacuum Vacuum εprotein= 2 εsolvent= 2 κ=0 Solution εsolvent= 80 κ≠0 + ΔG BC AF Complex ΔGBC ΔGAF ΔGComplex

Calculation of Electrostatic Potential 8 Calculation of Electrostatic Potential Linearized Poisson-Boltzmann Equation (LPBE): Accounts for different dielectric coefficients within the protein and solvent, ionic strengths, and protein charges Calculated at discrete grid points within and surrounding protein and extrapolated to individual atoms ε = dielectric coefficient κ = ion accessibility I = ionic strength e = charge z = ionic valence φ = electrostatic potential

Two-step binding model McCammon et al (1986) J. Phys. Chem proposed two-step model for association of proteins Recognition Initial collision of the two proteins free in solution through diffusive motion Driven and or accelerated by long range electrostatic interactions Weak encounter complex Binding Short to medium range electrostatic interactions, van der Waals interactions, as well as entropic affects Specific final complex. Essential in understanding why mutations away from the binding interface can affect binding

AESOP Calculation of Free Energy PDB WHATIF PDB2PQR APBS 1 2 3 4 5 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 Free energy calculation

Intermolecular Salt Bridges ≤5 Å A F C B Model pKa Values Asp (D) 3.9 Glu (E) 4.3 Arg (R) 12 Lys (K) 10.5 Number of salt bridges at each interface   3.5Å 3.6-5.0Å 5.1-8.0Å AF/BC 1 4 AC/BF 9 2 AB ≤5 Å Intermolecular Salt Bridges – AC Chain Residue pKa Å A 84 K 10.43 C 89 D 3.85 4.6 Intermolecular Salt Bridges – BF Chain Residue pKa Å B 84 K 10.43 F 89 D 3.85 4.6 Intermolecular Salt Bridges – AB Chain Residue pKa Å B 549 K 10.29 A 537 D 1.96 3.4 369 R 11.31 497 D 3.58 5.0 Red = unusual pka value (20% off) Bold = important salt bridge as indicated by pka value and/or distance Searched all salt bridges on other document. All ones that had a residue with an abnormal pka value were under 5A (except residue 550, which is 5.8A and not included in the tables in this powerpoint. Some residues are invloved in multiple salt bridges, and not all are shown here.

Intermolecular Salt Bridges – BC and AF Model pKa Values Asp (D) 3.9 Glu (E) 4.3 Arg (R) 12 Lys (K) 10.5 Intermolecular Salt Bridges continued… Intermolecular Salt Bridges – BC and AF Chain pKa Å B/A 91 K 10.15 C/F 139E 4.42 3.9 104 R 11.87 119 D 3.14 5.0 13.37 -1.93 4.7 122 K 11.45 138 E 2.88 4.0 3.49 4.1 126 K 13.06 93 E 1.85 4.3 10.01 4.27 4.2 150 K 13.13 96 E 4.84 4.9 10.22 4.05

Free Energies 9 different calculations each at 0mM and 150mM ABCF AB AF BC BF CF A B C F 0 ° 90° 180 ° 270 ° 0mM Charge +10 +8 +5 +2 +4 +1 Part 1 Part 2 Complex AF BC ABCF AB CF ABCF AC BF ABCF + = Tetramers Part 1 Part 2 Complex A B AB A C AC A F AF B C BC B F BF C F CF + = Dimers

A F C B BC AF Shaded = ±50 KJ/mol from the parent, Bold = less than 5.0Å, Blue = not in a salt bridge BC AF A F C B 97K 104R 81R 84K 92K 107R 91K 122K 126K 149R 150K 156K 154D 82E 119D 133E 93E 105D 130E 139E 138E Parent 96E 157E 89D 97K 104R 81R 84K 92K 107R 126 150K 122K 149R 156K 91K 158K 82E 119D 133E 157E 154D 93E 130E 105D 138E 139E Parent 89D 96E

BC AF A F C B 92K 81R 97K 122K 150K 84K 91K 104R 126K 205R 107R 96E 138E/139E 165E 217E 93E 89D 133E 154D 203E 206D 119D 82E 609K 573R Bold indicates in a salt bridge mentioned in this presentation. 82E is in a 6.1A salt bridge with 92K Make images 539K 605K 372K 369R 530E 550K 595D 342E 536E 542E 584E 497D 549K 537D Shaded = ±50 KJ/mol from the parent, Red = less than 3.5Å, Bold = less than 5.0Å, Blue = not in a salt bridge

Previous Studies continued… Mutations known to disrupt multimerization: ~Triple mutant: Mutation of R369A, F501A and F504A together disrupts domain swapping interface.1 Regions known to be important to multimerization2: ~The first 252 amino acids from the N-terminus ~252-380 necessary for binding ~The region between amino acids 551 and 569 1Hickman A. et al. Nature Structural & Molecular Biology  vol. 12  715-721 (2005)  2Atkinson, P.W. et al. Insect Biochem. MOL. Biol. 33, 959-970 (2003).

A F C B 107R 82E 149R A F 93E 84K 139E 122K 105D 126K 91K 138E 586R 308R 312K 585K 588R 584E

Conclusions Mutations within the first 250 amino acids and at the C-terminus have the most effect on the electrostatic free energy calculations. Mutation of an acidic amino acid to alanine decreases stability while mutation of a basic amino acid increases stability. The free energy calculations agree with the salt bridge analysis. Future Work The data will continue to be analyzed and these theoretical predictions can be tested in a wet lab to confirm their validity.

Acknowledgements Dr. Dimitrios Morikis, Ron Gorham, Aliana López De Victoria, Chris Kieslich Dr. Atkinson for suggesting the project Jun Wang and the BRITE program National Science Foundation References Craig, N.L., Dyda, F., Hickman, A.B., Musingarimi, P., Perez Z. (2005) Purification, crystallization and preliminary crystalographic analysis of Hermes transposase, Acta Crystalographica F61:587–590. Guex N and Peitsch MC: SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18: 2714-2723, 1997. Hickman A., Perez Z., Zhou L., Musingarimi P., Ghirlando R., Hinshar J., Craig N., Dyda F. (2005). Molecular architecture of a eukaryotic DNA transposase. Nature Structural &Molecular Biology. 12:715-721. Humphrey W., Dalke A., Schulten K. (1996). VMD: visual molecular dynamics. Journal of Molecular Graphics. 14: 33-37. Kieslich, CA, Yang, J., and Morikis, D (2009) AESOP: Analysis of Electrostatic Properties of Proteins, To be Published. Michel, K., O’Brochta, D.A. and Atkinson, P.W. The C-terminus of the Hermes transposase contains a protein multimerization domain. Insect Biochem. MOL. Biol. 33, 959-970 (2003). MOLMOL: a program for display and analysis of macromolecular structures 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.

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