Part I: Structure and Function in the NMDA Ligand Binding Domain Part II: Comparison of Paclitaxel Analogs through Molecular Dynamics Simulation; Solution Conformations of Cyclic Peptides Open Access

Geballe, Matthew Thomas (2009)

Permanent URL: https://etd.library.emory.edu/concern/etds/r207tp578?locale=en
Published

Abstract

Part I:
The NMDA receptor is an ionotropic glutamate receptor found throughout the CNS.
The receptor functions as a tetramer comprised of two NR1 subunits and two NR2
subunits. There are four subtypes of NR2, NR2A through NR2D, and functional
properties of the assembled receptor vary with the identity of the NR2 subunits. The
ligand binding domain of this receptor binds either the agonist glutamate or co-agonist
glycine, and triggers the gating process which allows ions to flow through the channel.
Crystal structures of the ligand binding domain have recently been solved, and these
structures provided the basis for molecular dynamics simulations of ligand binding
domains of different NR2 subtypes. Structures of different NR2 ligand binding domains
were prepared by homology modeling, and simulations were performed of just the NR2
ligand binding domain, as well as simulation of the ligand binding domain dimer of
NR1/NR2. Comparison of simulations of different subtypes as well as the same subtype
with different ligands bound reveal structural differences and changes that may shed
light on how the receptor functions.

Part II:
The dynamic equilibrium between free α,β-tubulin and assembled microtubules plays
a important role in cellular structure. Alteration of this equilibrium by paclitaxel
(PTX) and other similar compounds is a critical method of anti-cancer treatment, and
recently analogs were discovered that induce polymerization at levels much greater
than paclitaxel. These analogs were placed into a crystal structure of ,-tubulin
with PTX and subjected to molecular dynamics simulation. Over the simulation the
analogs induced changes in the critical M-loop and strengthened theories of how the
M-loop facilitates tubulin polymerization.


A cyclic pentapeptide was analyzed using NAMFIS (NMR Analysis of Molecular Flex-
ibility In Solution), a method which combines NMR data and modeling-based con-
formational searching. This peptide was well-studied in the literature, providing an
opportunity to evaluate NAMFIS alongside other methods for predicting conforma-
tions of small molecules in solution. NAMFIS was able to identify the conformations

Table of Contents

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Part I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2 The NMDA Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.1 Subunit Topology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.2 Variation by Subtype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.2.1 Regional and Temporal Distribution . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.2.2 Functional Variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.3 Ligand Binding Domains: Structures and Simulations . . . . . . . . . . . . . . 14
2.3.1 Crystal Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.3.2 Modeling and Molecular Dynamics Simulations . . . . . . . . . . . . . . . 18
2.3.3 Obstacles to Molecular Dynamics Approach . . . . . . . . . . . . . . . . . . 19
3 Ligand Binding Domain Simulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.1 Homology-based Partial Agonist Simulations . . . . . . . . . . . . . . . . . . . . . . 24
3.1.1 Structure Preparation and Simulation Parameters . . . . . . . . . . . . . 25
3.1.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.2 Crystal-Based Simulations of NR2A and NR2D . . . . . . . . . . . . . . . . . . . . 34
3.2.1 Structure Preparation and Simulation Parameters . . . . . . . . . . . . . 34
3.2.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.3 Synopsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

4 NR1/NR2 Ligand Binding Domain Dimer Simulations . . . . . . . . . 48
4.1 Structure Preparation and Simulation Parameters . . . . . . . . . . . . . . . . . . 49
4.1.1 Model Construction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
4.1.2 Simulation Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
4.2 Comparing NR2A and NR2D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
4.2.1 Average Structure Comparison . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
4.2.2 Ligand Binding Site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4.2.3 Interdomain Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
4.3 SYM2081 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
4.4 NR2C and D-cycloserine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
4.5 Synopsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
5.1 Agonist Binding Site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
5.2 Inter-domain Contacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
5.3 Displacement of Helix F and Efficacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
5.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Part II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
6 Comparison of Paclitaxel Analogs through Molecular
Dynamics Simulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
6.1.1 Bridged Paclitaxel Analogs and Improved MT Assembly . . . . . . 101
6.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
6.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
6.3.1 Ligand Positioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
6.3.2 M-loop Displacement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
6.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
6.5 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
7 Solution Conformations of a Cyclic Peptide . . . . . . . . . . . . . . . . . . . . 116
7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
7.1.1 NAMFIS Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
7.1.2 D-Pro-Ala4, a Cyclic Pentapeptide . . . . . . . . . . . . . . . . . . . . . . . . . 119
7.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
7.2.1 D-Pro-Ala4 NMR Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
7.2.2 Conformational Searching and Clustering . . . . . . . . . . . . . . . . . . . 121
7.2.3 NAMFIS Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
7.2.4 DFT Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
7.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
7.3.1 MD Conformers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
7.3.2 NAMFIS Conformers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
7.3.3 Post-NAMFIS Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
7.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
7.4.1 Use of NAMFIS on small cyclic peptides . . . . . . . . . . . . . . . . . . . . 131
7.4.2 Conformation, Energy, and Solvation . . . . . . . . . . . . . . . . . . . . . . . 133
7.5 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

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