Biophysical studies of dynamic CD4 changes implicated in HIV-1 infection

Channell, Jennifer Anne

Abstract

Cluster of differentiation 4 (CD4) is a receptor protein found on the surface of cells of the immune system and is the primary receptor of the human immuno-deficiency virus-1 (HIV-1) envelope glycoprotein 120 (gp120). The extracellular portion of CD4 is comprised of four immunoglobulin-like domains of which domains 1, 2 and 4 contain disulphide bonds. The disulphide bond in domain 2 is an allosteric disulphide bond which can alter protein function through redox shuffling. Reduction of this bond is essential for gp120 binding and subsequent HIV-1 host cell entry. However, reduced monomeric CD4 does not have any known physiological function in CD4’s normal immune response. CD4 reduced in domain 2 is a functionally distinct redox isoform of CD4 implicated in HIV-1 infection and is therefore, a target for an anti-HIV-1 vaccine.
The aim of this work was to obtain biophysical and small angle X-ray and neutron scattering (SAXS and SANS) data which would explain how reduction of the second domain disulphide bond affects CD4 structure and therefore its ability to bind to gp120. A novel cell-free protein expression (CFPE) protocol was developed to produce recombinant deuterium labelled and un-labelled wild-type two domain CD4 (2dCD4-WT). 2dCD4-WT produced using CFPE is demonstrated to be functional, correctly folded and suitable for SAXS and SANS by a series of biochemical and biophysical techniques. Ablation of the second domain disulphide bond is shown to cause relaxation of the domain so that 2dCD4-WT reduced in domain 2 has a smaller hydrodynamic volume than its fully oxidised and fully reduced counterparts. For the first time, deuterium labelled 2dCD4-WT in the gp120-bound and -unbound state is described by SANS contrast variation analysis. Finally, size-exclusion chromatography coupled to SAXS data on gp120-bound 2dCD4-WT is presented and validates the use of CFPE for the production of recombinant protein for small-angle scattering studies.

Citation

Channell, J. A. Biophysical studies of dynamic CD4 changes implicated in HIV-1 infection. (Thesis). Keele University. https://keele-repository.worktribe.com/output/413133

• Thesis Type: Thesis
• Publicly Available Date: May 26, 2023
• Public URL: https://keele-repository.worktribe.com/output/413133
• Award Date: 2019-03