Bioinformatics and Molecular Modeling studies of Membrane
Proteins
Shiva Amiri
Problem: Difficult to obtain high resolution crystallographic images of membrane proteins
Structure Determination
Unwin et.al, Nature, 26 June 2003
Getting there?
Some success using cryo-electron microscopy coupled with Fourier Transforms, i.e. Unwin’s 4 Å image of the TM region.
But still no full structure
Unwin et al., Nature, 26 June 2003
My Project
To design structure determination
software for Ligand Gated Ion Channels
(LGICs) i.e. nAChR, GABAA and GABAC receptors, 5HT3 receptor, Glycine receptor
Main focus so far:
The α-7 Nicotinic Acetylcholine Receptor (nAChR)
cationic channel homopentamer four transmembrane regions (M1-M4)
Transmembrane Domain Alignment
Homology modeling – Transmembrane domain
Homology modeling – Transmembrane domain
The homology model of the TM region with the Torpedo Marmorata structure
(PDB: 1OED - 4 Å) and the Chick α-7 sequence using MODELLER
Ligand Binding Domain Alignment
Homology Modeling – Ligand Binding domain
Homology Modeling – Ligand Binding domain
The homology model of the LBD with
Acetylcholine Binding Protein (AChBP) as the structure (PDB: 1I9B – 2.7 Å) and the Chick α-7 sequence using MODELLER
The software …
Combining the Transmembrane domain with the Ligand Binding domain
Failed first attempt: Minimizing distances between target residues in the LBD and the TM domains - 6 degrees of freedom (rotations and translations on all three axes)
- Models were not straight
Second attempt …
New algorithm:
z-axis
b. Align each domain onto the z-axis
a. straighten each domain with respect to the z-axis
c. Rotate and translate about z-axis- angle of rotation and steps of translations are
user- defined
z-axis
Theta (angle of rotation)
Second Attempt continued…
At each rotation and for each translation the Unwin distance, the Termini distance the number of bad contacts is calculated
Scoring Functions
1. Unwin Distance – the distance between residues from the TM domain and the LB domain that are meant to come into close proximity
2. Termini Distance – the distance between the C-terminus of the LB domain and the N-terminus of the TM domain
Scoring Functions continued …
3. Bad Contacts – Number of residues that are closer than a certain cut-off distance (user-defined), currently set to 5 Å
Scoring Functions continued…
Plots of Scoring FunctionsUnwin Distance Termini Distance
Bad contacts
Linear Combinations of Scoring Functions
Unwin + Termini Unwin + Termini + Bad contacts
Choosing the Best Model
Model chosen based on scoring function data
Once a good model was decided on, energy minimization using GROMACS was carried out to ensure the electrochemical legitimacy of the model
Model of the α-7 nAChR
Gaussian Network Model (GNM) Analysis
A course-grained model to approximate molecular motions of proteins
Current code cannot allocate memory for the 1660 residues of the α-7 nAChR
Analysis has been done using the TM domain and the LB domain separately
GNM was also run on one subunit of the model B-values generally in agreement with crystallographic
data but modeled structures are difficult to analyze using present code
AChBP – one subunit
0
20
40
60
80
100
120
140
160
1 16 31 46 61 76 91 106 121 136 151 166 181 196
Number of Residues
B-V
alu
e Series1
Series2
α-7 nAChR Transmembrane
0
50
100
150
200
250
300
350
1 39 77 115 153 191 229 267 305 343 381 419 457 495 533 571 609
Number of Residues
B-v
alu
es
Series1
Series2
α-7 nAChR model – 2 subunits
0
20
40
60
80
100
120
1 43 85 127 169 211 253 295 337 379 421 463 505 547 589 631
Number of Residues
B-v
alu
e
Series1
Series2
CONCOORD Analysis
Generates protein conformations around a given structure based on distance restrictions
Principal Component Analysis (PCA) is applied on the 500 resulting structures from CONCOORD
First eigenvector shows opening and closing of the pore as the subunits rotate
First eigenvector
Eigenvector Plot Covariance lines
Future work… Looking at the hydrophobic girdle (M2) of LGICs to study
patterns of conservation and the behaviour of these residues during gating
Further verification and analysis of models Other models of LGICs
Summary
Software designed to determine structure of LGICs
Structure of α-7 nAChR
Used various methods (GNM, CONCOORD) to look at possible motions using the hypothesized structure
Thanks to:
Prof. Mark S.P. Sansom Sundeep DeolDr. Phil Biggin Yalini PathyDr. Kaihsu Tai Jonathan CuthbertsonDr. Paul Barrett Pete Bond
Jeff CampbellDr. Alessandro Grotessi Katherine CoxDr. Daniele Bemporad Jennifer JohnstonDr. Jorge Pikunic Robert D’RozarioDr. Shozeb Haider Loredana VaccaroDr. Andy Hung John HolyoakeDr. Bing Wu Tony IvetacOliver Beckstein Sylvanna HoSyma Khalid Samantha KayeZara Sands George Patargias
Hydrophobic girdle
M2 alignment
b. Align each domain onto the z-axis
c. Rotate and translate about z-axis- angle of rotation and steps of translations are
user- defined
α-7 nAChR LBD – 3 subunits
0
50
100
150
200
250
300
350
1 37 73 109 145 181 217 253 289 325 361 397 433 469 505 541 577 613
Number of residues
B-v
alu
e Series1
Series2
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