Post on 16-Jan-2017
Examining the role of quaternary structure for the catalysis and
regulation of DAH7PS from Neisseria meningitidis (Nme)
Vicky Zhang Supervisor: Prof. Emily Parker
1
IntroductionThe shikimate pathway
Neisseria meningitidis
(Trp) (Phe) (Tyr)
2DAH7PS: 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase
IntroductionNmeDAH7PS Tight dimer interface
Dimer-dimer interface
3PDB Entry: 4HSN; Cross, et al. Protein science, 2013, 22, 1087-1099
Project Aims
1. To disrupt the tetrameric structure of NmeDAH7PS by removing key interactions.
2. Characterise the interface variant and compare to the wild type NmeDAH7PS.
4
Dimer-dimer interface
5Glu27Arg126
Arginine (Arg)
Glutamate (Glu)
Serine (Ser)
Arg126Ser Mutation
Arg126Ser NmeDAH7PS
(MW~38 kDa)
kDa
260
40
30
6
1 2 3 4 5 6 7 8
The Arg126Ser variant protein
1. Analytical Size Exclusion Chromatography
1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.10
0.5
1
1.5
2
2.5
3
Ve / Vo
Log
(mol
ecul
ar w
eigh
t)Dimer vs Tetramer
7
0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.20
0.5
1
1.5
2
2.5
3
Ve / Vo
Log(
mol
ecul
ar w
eigh
t)
Wild-type protein MW~142kDa
Dimer vs Tetramer
8
8 9 10 11 12 13 14 15 16 17 18 0
100
200
300
400
500
WT
Protein elution volume (mL)
Abs
orba
nce
280
nm (
mA
u)
1. Analytical Size Exclusion Chromatography
0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.20
0.5
1
1.5
2
2.5
3
Ve / Vo
Log
(mol
ecul
ar w
eigh
t)
Wild-type protein MW~142kDa
Arg126Ser variant MW~ 66kDa
Dimer vs Tetramer
9
8 9 10 11 12 13 14 15 16 17 18 0
100
200
300
400
500
WT
Arg126Ser
Protein elution volume (mL)
Abs
orba
nce
280
nm (
mA
u)
1. Analytical Size Exclusion Chromatography
WT Arg126Ser
Dimer vs Tetramer
10
2. Small Angle X-ray Scattering (SAXS)Wild-type protein (Tetrameric)
Dimer vs Tetramer
11
Arg126Ser variant (Dimeric)
2. Small Angle X-ray Scattering (SAXS)
12
Characterisation1. Metal Ion Dependency
Mn Cd Co Zn Fe Cu Mg0%
20%
40%
60%
80%
100%
120%
Arg126SerWT protein
Divalent metal ions
Spec
ific
activ
ity
(%)
1. Cross, et al. Protein science, 2013, 22, 1087-1099
1
DAH7PS KmPEP (μM) Km
E4P (μM) kcat (S-1)
NmeDAH7PS-WT1
11 ± 1 43 ± 4 25.5 ± 0.5
NmeDAH7PS-Arg126Ser
100 ± 7 22 ± 3 26.3 ± 0.4
2. Michaelis-Menten Kinetics
Characterisation
131. Cross, et al. Protein science, 2013, 22, 1087-1099
0 100 200 300 400 500 600 700 800 900 10000%
20%
40%
60%
80%
100% PheTyrTrp
Inhibitor concentration (µM)
Spec
ific
act
ivit
y re
mai
nin
g (%
)
14
Characterisation3. Regulatory Properties
15
Characterisation3. Regulatory Properties
DAH7PS KmPEP(μM) Km
E4P(μM) kcat (S-1)
NmeDAH7PS-WT1 (With 300 μM Phe)
21 ± 1 92 ± 10 6.9 ± 0.4
NmeDAH7PS-Arg126Ser(With 300 μM Phe)
25 ± 2 121 ± 12 15.2 ± 1
1. Cross, et al. Protein science, 2013, 22, 1087-1099
16
PEP
Glu27
Arg126
Ser126
Mutation site
Active site
(rmsd=0.6 Å)
Crystal Structure
Yellow=WT structureGreen=Variant structure
17
Arg126SerMutation
Conclusions
Conclusions
18
The dimeric form of
NmeDAH7PS is a
FUNCTIONAL UNIT
Similar metal ion
dependency
Similar catalytic
efficiency
Similar regulatory properties
Conclusions
19
The dimeric form of
NmeDAH7PS is a
FUNCTIONAL UNIT
Similar metal ion
dependency
Similar catalytic
efficiency
Similar regulatory properties
Crystal structure with Phe binding
Protein flexibility
Acknowledgements
20
Prof. Emily Parker
Dr. Penelope Cross
Dr. Ali Nazmi
Dr. Marie Squire
Gerd Mittelstaedt
Dmitri Joseph
Logan Heyes
Nicky Blackmore
Sarah Wilson-Coutts
Tammie Cookson
21
Site-directed mutagenesis----Arg126Ser
5’ 3’
5’ 3’
3’ 5’
3’ 5’
5’ 3’3’ 5’
5’ 3’3’ 5’
Mutated plasmidParental plasmid
Transformation
Site-directed mutagenesis
Protein purification
(http://chromacademy.com/Introduction_to_Ion_Chromatography_Essential_Guide.asp)
• Anion-exchange chromatography
• Size-exclusion chromatographyProtein purification
Arg126Ser NmeDAH7PS(~38 kDa)
kDa 260
40
30
(http://en.wikipedia.org/wiki/Size-exclusion_chromatography)
Arg126Ser NmeDAH7PS
25
Trp Tyr Phe45
46
47
48
49
50
51
52
53
48.3 48.1
52.3
Mel
ting
Tem
pera
ture
(⁰C
)
0
X-ray Crystallography
Crystal growth: Hanging-drop diffusion at 20 °C for 6-8 days