1. 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

2. 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 

3. 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 

4. 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

5. - + Salt Bridges – Interactions between charged amino acids
ASP
LYS - +
Strength of Interaction Bond Distance
Strong – 3.5 Å
Medium – 5.0 Å
Weak – 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

6. - + 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

7. 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

8. Calculation of Electrostatic Potential8
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

9. 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

10. 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

11. 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 Å Å
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.

12. 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

13. Free Energies 9 different calculations each at 0mM and 150mM ABCF ABAF BC BF CF A B C F
0 ° ° ° °
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

14. 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

15. 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

16. 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
~ necessary for binding
~The region between amino acids 551 and 569
1Hickman A. et al. Nature Structural & Molecular Biology  vol. 12  (2005) 
2Atkinson, P.W. et al. Insect Biochem. MOL. Biol. 33, (2003).

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

18. 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.

19. 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: , 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:
Humphrey W., Dalke A., Schulten K. (1996). VMD: visual molecular dynamics. Journal of Molecular Graphics. 14:
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, (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 Oct;25(13):

20. Questions?