Computational Protein-Ligand Modelling of the Enzymes DNA gyrase and IcaB Ahmad Alaa-El-Din ...

139 Pages·2015·5.96 MB·English

Computational Protein-Ligand Modelling
of the Enzymes DNA gyrase and IcaB

Ahmad Alaa-El-Din Abd-El-Aziz

Master of Science by Research

University of York

Biology

March 2015
Abstract

Computational modelling of proteins and their interactions with small molecule ligands
is a growing field of research. Such studies provide an understanding of how protein
structure relates to mechanism and function as well as informing drug discovery and
design.

This thesis had two main aspects: computational modelling of ciprofloxacin derivatives
binding to DNA gyrase and homology modelling of the protein IcaB based on sequence
alignment with a related protein, PgaB.

The inhibitory activity of synthetic ciprofloxacin derivatives (with various linkage to
citrate groups) was experimentally assessed by gel electrophoresis to examine the effect
on DNA gyrase binding to a target DNA strand. Overall, the derivative which possessed
the greatest inhibition compared to the unmodified ciprofloxacin was the c-gly-
ciprofloxacin derivative, which had a 2 atom linker between the ciprofloxacin and citrate
groups. This correlated with the change in interactions seen between ciprofloxacin
derivatives as computationally modelled by molecular mechanics methods.

The second aspect of the thesis was to generate a model of the protein IcaB to test the
hypothesis that it is a deactylase of poly-N-acetyl-glucosamine (PNAG) during maturation
of the poly-glycan in the extracellular matrix responsible for biofilm generation for
bacteria. An initial review of deacetylase enzyme structures identified the conserved
features required for activity. A homologous protein, Pga,B was then used as a template
to generate a homology model of IcaB. The model maintained the orientation and
positioning of the metal-binding and catalytic residues critical for proper deacetylase
function. However, the PNAG binding groove, believed to be involved in the transport of
the PNAG to the active site of PgaB, was not properly replicated in the IcaB model.
Further modelling would require improved characterization of the binding groove of
IcaB.

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Table of Contents

Abstract ii
List of Figures vi
List of Tables x
List of Schemes xi
List of Equations xii
Acknowledgements xiii
Author’s Declaration xiv
Chapter 1: Introduction and Methods 1
1.1 Background 1
1.2 Protein Structure 2
1.3 Protein-Ligand Interactions 6
1.4 Computational Modelling of Protein Structure 9
1.5 Computational Modelling of Protein-Ligand Interactions 10
1.6 Molecular Mechanics Modelling 13
1.7 Homology Modelling 14
1.8 Outline of Thesis Contents 16
Chapter 2: Modelling of Citric Acid-Ciprofloxacin Conjugates 17
2.1 Cystic Fibrosis and Opportunistic Infections 17
2.2. DNA Gyrase and Ciprofloxacin Activity 19
2.3 Structure of Ciprofloxacin Derivatives 23
2.4 DNA Gyrase Inhibition Assays 24
2.4.1 Methodology 25
2.4.2 Results 25
2.4.3 Discussion 29
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2.5. Energy Minimization of Gyrase-Ciprofloxacin Complexes 29
2.5.1 Methodology 31
2.5.2 Results 32
2.5.3 Discussion 40
Chapter 3: Review of Deacetylase Enzymes 43
3.1 Introduction 43
3.2 Histone Deacetylases and Sirtuins 45
3.2.1 Histone Deacetylases 46
3.2.2 Sirtuins 47
3.3 Carbohydrate Esterases 48
3.3.1 CE-2 49
3.3.2 CE-3 50
3.3.3 Carbohydrate Esterase Family 4 51
3.3.3.1 Chitin Deacetylase 51
3.3.3.2 GlcNAc Peptidoglycan Deacetylase 52
3.3.3.3 MurNAc Peptidoglycan Deacetylase 54
3.3.3.4 Chitooligosaccharide Deacetylase 54
3.3.3.5 Poly-N-acetylglucosamine deacetylases (PgaB and IcaB) 56
3.3.4 CE-7 59
3.3.5 CE-8 61
3.3.6 CE-9 64
3.3.7 CE-10 66
3.3.8 CE-11 66
3.3.9 CE-12 and CE-13 (Pectin acetylesterases) 68
3.3.10 CE-14 69
3.3.11 CE-15 72
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3.3.12 CE-16 73
3.4 Citrulline Deacetylase 73
3.5 Common Features of Deacetylase Enzymes 74
3.6 Discussion of deacetylase review with focus on IcaB 76
Chapter 4: Homology Modelling Of IcaB 79
4.1 Biofilm Formation In staphylococcus Infections 79
4.2 Structure and Activity of Partially Deacetylated Poly-N-acetylglucosamine 79
4.3 The ica Operon of staphylococcus 80
4.4. Structure and Function of IcaB and PgaB 81
4.4.1 IcaB and PgaB domain structure 81
4.4.2 Structures of PgaB 82
4.5 IcaB Alignment with PgaB 85
4.5.1 Methods 86
4.5.2 Sequence Alignment 86
4.6. Homology Model of IcaB 88
4.6.1 Methods 91
4.6.2 Results 91
4.7 Discussion and Analysis of IcaB Model 92
Chapter 5: Conclusions 102
References 105

v
List of Figures

Figure 1.1: Peptide α-helix, β-sheet, and β-turn in stick diagrams 3
Figure 1.2: Structure of haemoglobin (colours represent individual
monomers and haem prosthetic groups are shown in stick format) pdbID:
1GZX 4
Figure 1.3: Structure of adenosine, a nucleotide consisting of a deoxyribose
sugar, phosphate, and nitrogenous base (adenine) 8
Figure 2.1: Top view of structure of Staphylococcus aureus DNA gyrase
complexed to ciprofloxacin 20
Figure 2.2: Side view of structure of Staphylococcus aureus DNA gyrase
complexed to ciprofloxacin 20
Figure 2.3: The ciprofloxacin-binding site with annotated adjacent amino acid
and nucleotide residues 20
Figure 2.4: Sequence alignment, derived using ClustalX 2.1, of S. aureus DNA
gyrase with E. coli DNA gyrase 22-23
Figure 2.5: DNA gyrase assay electrophoresis gel a) ciprofloxacin; b) c-
ciprofloxacin; c) c-gly-ciprofloxacin; d) c-ava-ciprofloxacin 25-26
Figure 2.6: Effect of ciprofloxacin concentration on gyrase activity 27
Figure 2.7: Effect of c-ciprofloxacin concentration on gyrase activity 27
Figure 2.8: Effect of c-gly-ciprofloxacin concentration on gyrase activity 28
Figure 2.9: Effect of c-ava-ciprofloxacin concentration on gyrase activity 28
Figure 2.10: Computationally derived structure of the ciprofloxacin-DNA
gyrase complex 33
Figure 2.11: Hydrogen bonding interactions of ciprofloxacin-DNA gyrase
complex 33
Figure 2.12: Computationally derived structure of the c-ciprofloxacin-DNA
gyrase complex 34
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Figure 2.13: Hydrogen bonding interactions of the c-ciprofloxacin-DNA gyrase
complex 34
Figure 2.14: Computationally derived structure of the c-gly-ciprofloxacin-DNA
gyrase complex 35
Figure 2.15: Hydrogen bonding interactions of the c-gly-ciprofloxacin-DNA
gyrase complex 35
Figure 2.16: Computationally derived structure of conformation 1 of the c-
ava-ciprofloxacin-DNA gyrase complex 36
Figure 2.17: Hydrogen bonding interactions of the conformation 1 of c-ava-
ciprofloxacin-DNA gyrase complex 37
Figure 2.18: Computationally derived structure of conformation 2 of the c-
ava-ciprofloxacin-DNA gyrase complex 38
Figure 2.19: Hydrogen bonding interactions of the conformation 2 of c-ava-
ciprofloxacin-DNA gyrase complex 39
Figure 3.1: Histone deacetylase 4 bound to a triflouromethylketone
inhibitor 46
Figure 3.2: Structure of CjCE2B at 2.0Å resolution (pdbID: 2W9X) 49
Figure 3.3: Structure of the CtCes3 N-terminal domain at 2.0Å resolution
(pdbID: 2VPT) 50
Figure 3.4: Structure of SpPgdA from S. pneumoniae at 1.75Å from (pdbID:
2C1G) 53
Figure 3.5: Structure of the chitooligosaccharide deacetylase homodimer
from Vibrio cholerae at 1.88Å (pdbID: 4NY2) 55
Figure 3.6: Structure of PgaB with Ni2+ as the active site metal from E. coli
from pdbID: 4f9d 57
Figure 3.7: Hydrophobic PgaB binding groove surface 59
Figure 3.8: Structure of the cephalosporin C deacetylase dimer from Bacillus
subtilis with acetate in the active site at 1.9Å resolution (pdbID: 1ODT) 60
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Figure 3.9: Structure of pectin methylesterase from Erwinia chrysanthemi
containing a hexamer of D-galacturonic acid in stick form (green) and binding
site residues (grey)(pdbID: 2NTB) 62
Figure 3.10: Binding cleft residues with labelled D-galacturonic subunit
positions set to +1 at the position adjacent to Gln177 63
Figure 3.11: Structure of NAG-6-P deacetylase from Bacillus subtilis at 2.05Å
resolution (pdbID: 1UN7) 65
Figure 3.12: Structure of LpxC homotrimer from Pseudomonas aeruginosa
containing inhibitor BB-78485 (sticks) and zinc ions (balls) in the active site
at 1.9Å resolution (pdbID: 2VES) 67
Figure 3.13: Structure of pectin acetylesterase at 2.1Å resolution from B.
subtilis (pdbID: 2O14) 69
Figure 3.14: Structure of MshB at 1.85Å (pdbID: 4EWL) 70
Figure 3.15: Active site of MshB containing glycerol and acetate to simulate
mycothiol binding 71
Figure 3.16: Structure of citrulline deacetylase from Xanthomonas campestris
at 1.75Å (pdbID: 2F7V) 73
Figure 4.1: Proposed functions of gene products of the ica Operon 80
Figure 4.2: a) IcaB domain structure from Staphylococcus epidermidis; b)
PgaB domain structure from Escherichia coli (strain K12). The domains are
represented by Yellow = signal sequence, light green = coiled coil, green =
deacetylase domain, GHL13 = Glycoside Hydrolase-Like C-terminal
domain 82
Figure 4.3: Alignment of all sequences of PgaB in Protein Databank (3vus,
4f9d, and 4f9j) 83
Figure 4.4: Comparison of PgaB structures available in the Protein Data Bank
(3vus in blue, 4f9d in green, and 4f9j in red) 84
Figure 4.5: Active site and N-terminal binding groove of PgaB from 4f9d 84
viii
Figure 4.6: a) Sequence alignment of IcaB with 4f9d N-terminal domain; b)
Sequence alignment of IcaB aligned with PgaB N-terminal domain NCBI
sequence and truncated to match 4f9d (residues 1-42 removed); c) Sequence
alignment from b) manually adjusted to preserve α7 helix 87
Figure 4.7: Active site and N-terminal groove residues in the final
alignment 88
Figure 4.8: Homology model of IcaB using manually adjusted 4f9d NCBI
alignment as a template 92
Figure 4.9: a) Charge surface of IcaB homology model (left) and 4f9d
template (right) in which red indicates regions of negative charge and blue
indicates regions of positive charge b) Charge surface from a) rotated by
180° 93
Figure 4.10: a) Hydrophobic moments in IcaB model (left) and PgaB (pdbID:
4f9d, right); b) Hydrophobic moments from a) rotated 180° 94
Figure 4.11: Regions of PgaB (red, 4f9d) that differed most from the IcaB
model (blue) 95
Figure 4.12: Overlap between active site residues of PgaB (red, 4f9d) and the
IcaB homology model (blue) 96
Figure 4.13: Comparison of manually adjusted IcaB alignment with PSIPRED
predicted secondary structure 97
Figure 4.14: Structure of IcaB from Ammonifex degensii at 1.70Å reproduced
from pdbID: 4WCJ 98
Figure 4.15: Alignment of IcaB sequences from S. epidermidis (top) and A.
degensii (bottom) (* = identical, : = conserved, .= semiconserved) 99
Figure 4.16: Comparison of S. epidermidis IcaB homology model (blue) and A.
degensii IcaB crystal structure from pdbID: 4WCJ (green) 100
Figure 4.17: IcaB active site residue overlap. Numbering is in the format S.
epidermidis structure (blue)/A. degensii structure (green) 100

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List of Tables

Table 2.1: IC calculated for each ciprofloxacin derivative based on
50
electrophoresis gel assay data 29
Table 2.2: Computationally determined energies of minimized DNA gyrase-
ciprofloxacin derivative complexes 40
Table 3.1: Sequences and structures of known families of deacetylase
enzymes as of June 8, 2015 45
Table 3.2: Deacetylase enzyme summary 77
Table 4.1: RMSD comparison between multiple models of IcaB with manually
adjusted 4f9d template 92

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