Transcript Slide 1

Disrupting Multimerization of Hermes Transposase by Single
Amino Acid Mutation
http://www.engr.ucr.edu/~dmorikis/
Chelsea Vandegrift, Aliana López De Victoria, Ronald D. Gorham, Dimitrios Morikis,
Hermes transposase is a hexamer of three heterodimers native to Musca domestica1.
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.
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.
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Electrostatic free energy values were calculated using the Analysis of Electrostatics of Proteins (AESOP)
protocol developed by our group6, 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 ΔΔGsolvation.
Heterotetramer
B
2
Figure 3A
A
2
Retrieval & cleaning of coordinates for parent protein complex
C
F
ΔG
Generation of coordinates for mutants
ΔGAF
1
ΔGBC
ΔGComplex
3
Generation of coordinate files with partial charges & vdW radii
2
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.
4
Calculation of electrostatic potentials
εprotein= 2
εsolvent= 80
κ≠0
AF
BC
Complex
5
Electrostatic potential
visualization
PDB
WHATIF
PDB2PQR
APBS
6
Free energy
calculation
Figure 2
126K
Interface 3
90
105
120
135
150
165
180
195
210
225
240
255
270
285
300
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.
Acknowledgements
Dr. Dimitrios Morikis, Aliana López De Victoria, Ronald Gorham, Chris Kieslich
Chimera
ΔGsolvation = ΔGsolution – ΔGvacuum
Δ ΔGsolvation = ΔGSolvation Complex – ΔGsolvation AF – ΔGsolvation BC
91K
Future work will include experimental validation of the free energy predictions by
Prof. Peter Atkinson's group, Dept. of Entomology, UCR.
ΔG
+
139E
Future Work
E
Solution
122K
105D
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.
+
εsolvent= 2
κ=0
D
2
93E
84K
We are grateful to Prof. Atkinson for suggesting the project and for exciting and
insightful discussions.
BRITE and the National Science Foundation
APBS
Thermodynamic cycle used to calculate electrostatic free energies
of association and solvation.
References
1Craig,
C-alpha-trace of the
heterotetramer (red)
with the theoretical
full model (blue).
N.L., Dyda, F., Hickman, A.B., (2005) Molecular architecture of a eukaryotic DNA transposase,
Nature Structural & Molecular Biology 12:715-721.
Results and Discussion
C-alpha-trace of
the theoretical full
model
(red)
exactly covering
four chains of the
spiral
hexamer
(blue).
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).
Free Energy 0mM Chain B
Figure 4
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).
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Parent 1250.00
Free Energy (KJ/mol)
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.
1200.00
1150.00
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97
K 122
K
92
K
84 91
104
K K
R
107
R
1000.00
96
E
89 93
D E
950.00
900.00
850.00
82
E
800.00
75
150
K
126
K
133
E
138E
/139
E
369R
497D
119
D
530E
549K
537D
175
275
375
Mutation
475
575
F
Mutation
Near Vacuum
εprotein= 2
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75
Figure 3B
1
C
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The procedures and protocols used to calculate these free energies are shown in Fig. 3B.
1
A
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138E
F
Interfaces
B
107R
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These calculations were repeated for the following combination of monomers and dimers (letters indicate the
chains):
AF-BC
A-B
B-C
AB-CF
A-C
B-F
AC-BF
A-F
C-F
A
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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.
Figure 1: The letters indicate the chain and the numbers label the
interfaces.
1
Free Energy ΔΔG 0mM chain B, part 1
Electrostatic Free Energy Calculations:
•The available crystallographic structure is a heterotetramer, (used in this
research).
Hexamer
Figure 5
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•It is functionally active as a hexamer.
3
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).
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.
•Hermes Transposase catalyze DNA breakage and rejoining reactions.2
C
Chris A. Kieslich
Methods
Background
B
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.
Free Energy (kJ/mol)
Abstract
2Craig,
N.L., Dyda, F., Hickman, A.B., (2005) Purification, crystallization and preliminary
crystallographic analysis of Hermes transposase, Acta Crystallographic F61:587–590.
3Guex
N and Peitsch MC: SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative
protein modeling. Electrophoresis 18: 2714-2723, 1997.
4Hickman
A, Perez Z, Zhou L, et al. (2005). Molecular architecture of a eukaryotic DNA tranposase.
Nature Structural &Molecular Biology. 12:715-721.
5Humphrey
W, Dalke A, Schulten K (1996). VMD: visual molecular dynamics. Journal of Molecular
Graphics. 14: 33-37.
6Kieslich,
CA, Yang, J., and Morikis, D (2009) AESOP: Analysis of Electrostatoc Properties of Proteins,
To be Published.
7MOLMOL:
8UCSF
a program for display and analysis of macromolecular structures
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.