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A solution state NMR study of the structure and ligand binding properties of the human C-type lectin DC-SIGNR
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Probert, Fay (2012) A solution state NMR study of the structure and ligand binding properties of the human C-type lectin DC-SIGNR. PhD thesis, University of Warwick.
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WRAP_THESIS_Probert_2012.pdf - Submitted Version Download (217Mb) | Preview |
Official URL: http://webcat.warwick.ac.uk/record=b2680866~S1
Abstract
The protein DC-SIGNR (Dendritic-cell specific ICAM3 grabbing non-integrin
related) is a C-type (calcium-dependent) lectin, which binds highly-branched
mannose oligosaccharides. DC-SIGNR interacts with a range of deadly diseases via
surface glycans on pathogenic glycoproteins, and the ability of DC-SIGNR to
increase the rate of infection of viruses including human immunodeficiency virus
(HIV) and hepatitis C virus (HCV) makes the study of DC-SIGNR/oligosaccharide
interactions very attractive. The research described in this thesis sought to gain
insight into the calcium and ligand binding properties of the DC-SIGNR
carbohydrate recognition domain (CRD) in solution by utilising solution state
nuclear magnetic resonance spectroscopy (NMR).
A protocol for the production of uniformly 15N /13C labelled DC-SIGNR CRD
was developed, allowing the acquisition of heteronuclear NMR experiments and
the first assignment of the calcium-bound (holo) DC-SIGNR CRD to be reported.
The assignment has allowed investigation of calcium and glycan binding, as well
as the pH dependence of the DC-SIGNR CRD.
The data presented in this thesis reveal that the DC-SIGNR CRD is highly
dynamic in the calcium-free state, with the addition of calcium resulting in global
conformational and dynamic changes throughout the CRD. While calcium binding
hinders the protein dynamics (particularly in the calcium binding regions), a large
degree of mobility remains. The evidence that ligands are released at low pH
suggests that DC-SIGNR may act as an endocytic receptor.
In addition to calcium binding, interactions of the DC-SIGNR CRD with a
range of ligands were investigated. In particular, interactions with the
oligosaccharide Man9GlcNAc (present on the HIV viral envelope) are described,
representing the first direct study of the CRD interacting with a diseaseassociated
ligand. The glycans employed in this study all bind to the primary
calcium binding site, supporting previous crystal data. However, each glycan
displays distinct patterns of chemical shift perturbations implying that they each
have different, extended binding modes. Particularly striking is the difference
between the disease-associated Man9GlcNAc ligand and the ligand present in a
previously published crystal structure, (GlcNAc)2Man3.
An investigation of the dynamics of the CRD in the holo form and bound
to the ligand Man5 shows that the CRD is highly dynamic and that glycan binding
further hinders, but does not abolish, the molecular motions. The dynamics data
also suggests that a ligand-induced conformational change may occur and
indicates potential new binding sites which are not present in any published
crystal structures. The dynamic nature of the DC-SIGNR CRD may explain the wide
range of ligand specificities and affinities of the C-type lectin scaffold and
suggests that the study of the ligand binding properties and dynamics of proteins
such as DC-SIGNR in solution is essential to further understanding of this class of
proteins.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QP Physiology | ||||
Library of Congress Subject Headings (LCSH): | Lectins, Cell receptors, Carbohydrates, Calcium-binding proteins, Ligands (Biochemistry), Nuclear magnetic resonance | ||||
Official Date: | December 2012 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | Molecular Organisation and Assembly in Cells | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Dixon, Ann M.; Mitchell, Daniel Anthony | ||||
Sponsors: | Engineering and Physical Sciences Research Council (EPSRC) | ||||
Extent: | xvii, 202 leaves : illustrations. | ||||
Language: | eng |
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