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

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

Background

Hermes Transposase is a hexamer, but the largest crystallographic structure available is the heterotetramer.

Homohexamer

HeterotetramerA

FC

B

AB

C

D

F

E

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)

ASP CG

ARG CZ

GLU CD

LYS NZ

Atom measured from:

pKa Values

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

Unfavorable

Favorable

+--

++ -+

-

Basic

Acidic

HA   H+ + A-

Electrostatic Free EnergiesProcedure

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

εprotein= 2

εsolvent= 80

κ≠0

+

+

ΔG

BC AF Complex

ΔGBC ΔGAF ΔGComplex

ΔG

+

• 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 coefficientκ = ion accessibilityI = ionic strengthe = chargez = ionic valenceφ = electrostatic potential

Calculation of Electrostatic Potential

8

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

APBSFree energycalculation

Intermolecular Salt Bridges – AC

Chain Residue pKa Chain Residue pKa Å

A 84 K 10.43 C 89 D 3.85 4.6

Intermolecular Salt Bridges – BF

Chain Residue pKa Chain Residue pKa Å

B 84 K 10.43 F 89 D 3.85 4.6

Intermolecular

Salt Bridges

Intermolecular Salt Bridges – AB

Chain Residue pKa Chain Residue pKa Å

B 549 K 10.29 A 537 D 1.96 3.4

A 549 K 10.29 B 537 D 1.96 3.4

B 369 R 11.31 A 497 D 3.58 5.0

A 369 R 11.31 B 497 D 3.58 5.0

≤5 Å

Number of salt bridges at each interface

  3.5Å 3.6-5.0Å 5.1-8.0Å

AF/BC 0 1 4

AC/BF 0 9 2

AB 2 1 4

A

FC

B

Model pKa Values

Asp (D) 3.9

Glu (E) 4.3

Arg (R) 12

Lys (K) 10.5

Intermolecular Salt Bridges continued… A

FC

B

Model pKa Values

Asp (D) 3.9

Glu (E) 4.3

Arg (R) 12

Lys (K) 10.5

Intermolecular Salt Bridges – BC and AF

Chain pKaChain

pKa Å

B/A 91 K 10.15 C/F 139E 4.42 3.9

B/A 104 R 11.87 C/F 119 D 3.14 5.0

C/F 104 R 13.37 B/A 119 D -1.93 4.7

B/A 122 K 11.45 C/F 138 E 2.88 4.0

C/F 122 K 10.15 B/A 138 E 3.49 4.1

B/A 126 K 13.06 C/F 93 E 1.85 4.3

C/F 126 K 10.01 B/A 93 E 4.27 4.2

B/A 150 K 13.13 C/F 96 E 4.84 4.9

C/F 150 K 10.22 B/A 96 E 4.05 4.9

Free Energies9 different calculations each at 0mM and 150mM A

FC

B

Part 1 Part 2 Complex

A B AB

A C AC

A F AF

B C BC

B F BF

C F CF

+

+

+

+

+

+ =

=

=

=

=

=

Dimers

Part 1 Part 2 Complex

AF BC ABCF

AB CF ABCF

AC BF ABCF

+

+

+

=

=

=

Tetramers

ABCF

AB

AC

AF

BC

BF

CF

A

B

C

F

0 ° 90° 180 ° 270 °0mM Charge

+10

+8

+5

+5

+5

+5

+2

+4

+4

+1

+1

Free Energies ΔΔG 0mM Chain C

800.00

850.00

900.00

950.00

1000.00

1050.00

1100.00

1150.00

1200.00

74 84 94 104 114 124 134 144 154Mutation

Fre

e E

nerg

y (

kJ/m

ol)

Free Energy ΔΔG 0mM Chain F

800.00

850.00

900.00

950.00

1000.00

1050.00

1100.00

1150.00

1200.00

75 85 95 105 115 125 135 145 155

Mutation

Fre

e E

nerg

y (

kJ/m

ol)

82E

81R 84K

89D

91K

92K

93E

96E

97K 104R

105D

107R

119D

122K 126K

130E

133E

138E 139E

149R 150K

154D

156K

157E

82E

81R 84K

89D

91K

92K

93E

96E

97K 104R

105D

107R

119D

122K126

130E

133E

138E 139E

149R150K

154D

156K

157E

158K

A

FC

B

BC AF

Parent

Parent

Shaded = ±50 KJ/mol from the parent,

Bold = less than 5.0Å,

Blue = not in a salt bridge

Free Energy ΔΔG 0mM Chain B, part 1

800.00

850.00

900.00

950.00

1000.00

1050.00

1100.00

1150.00

1200.00

70 120 170 220 270

Mutation

Fre

e E

nerg

y (

kJ/m

ol)

119D82E

81R 122K150K84K

89D

91K

93E

96E138E/139E

126K104R

Free Energies ΔΔG 0mM Chain B, part 2

800.00

850.00

900.00

950.00

1000.00

1050.00

1100.00

1150.00

1200.00

1250.00

1300.00

295 345 395 445 495 545 595

Mutation

Fre

e E

nerg

y (

kJ/m

ol)

497D

537D

539K

549K

369R

573R

609K

Shaded = ±50 KJ/mol from the parent, Red = less than 3.5Å, Bold = less than 5.0Å, Blue = not in a salt bridge

133E

97K92K

372K

530E 550K

154D

165E203E 206D

217E

205R

342E 536E 542E 584E595D

605K

107R

A

FC

B

BC AF

Previous Studies continued… Mutations known to disrupt multimerization:

~Triple mutant: Mutation of R369A, F501A and F504A together disrupts domain swapping interface.1

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).

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

800.00

850.00

900.00

950.00

1000.00

1050.00

1100.00

1150.00

1200.00

1250.00

1300.00

295 345 395 445 495 545 595

Free E

nerg

y (

kJ/m

ol)

Free Energy ΔΔG 0mM Chain A, part 1

1400.00

1450.00

1500.00

1550.00

1600.00

1650.00

1700.00

1750.00

1800.00

75 90 105 120 135 150 165 180 195 210 225 240 255 270 285 300

Mutation

Fre

e E

ner

gy

(KJ/

mo

l)

Free Energy ΔΔG 0mM Chain A, part 2

1400.00

1450.00

1500.00

1550.00

1600.00

1650.00

1700.00

1750.00

1800.00

298 313 328 343 358 373 388 403 418 433 448 463 478 493 508 523 538 553 568 583 598

Mutation

Fre

e E

ner

gy

(KJ/

mo

l)

A

FC

B

82E

91K

93E

107R

105D122K

138E

126K

149R

139E84K

584E

308R312K 585K

586R

588R

A

F

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.

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.

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

Questions?