Assessment of differences in the conformational flexibility of hepatitis B virus core-antigen and e-antigen by hydrogen deuterium exchange-mass spectrometry.
Hepatitis B virus core-antigen (capsid protein) and e-antigen (an immune regulator) have almost complete sequence identity, yet the dimeric proteins (termed Cp149d and Cp(-10)149d , respectively) adopt quite distinct quaternary structures. Here we use hydrogen deuterium exchange-mass spectrometry (HDX-MS) to study their structural properties. We detect many regions that differ substantially in their HDX dynamics. Significantly, whilst all regions in Cp(-10)149d exchange by EX2-type kinetics, a number of regions in Cp149d were shown to exhibit a mixture of EX2- and EX1-type kinetics, hinting at conformational heterogeneity in these regions. Comparison of the HDX of the free Cp149d with that in assembled capsids (Cp149c ) indicated increased resistance to exchange at the C-terminus where the inter-dimer contacts occur. Furthermore, evidence of mixed exchange kinetics were not observed in Cp149c , implying a reduction in flexibility upon capsid formation. Cp(-10)149d undergoes a drastic structural change when the intermolecular disulphide bridge is reduced, adopting a Cp149d -like structure, as evidenced by the detected HDX dynamics being more consistent with Cp149d in many, albeit not all, regions. These results demonstrate the highly dynamic nature of these similar proteins. To probe the effect of these structural differences on the resulting antigenicity, we investigated binding of the antibody fragment (Fab E1) that is known to bind a conformational epitope on the four-helix bundle. Whilst Fab E1 binds to Cp149c and Cp149d , it does not bind non-reduced and reduced Cp(-10)149d , despite unhindered access to the epitope. These results imply a remarkable sensitivity of this epitope to its structural context.
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands; Netherlands Proteomics Centre, The Netherlands.
Jessica Z
JZ
Bereszczak
Norman R
NR
Watts
Paul T
PT
Wingfield
Alasdair C
AC
Steven
Albert J R
AJ
Heck
D041961:Deuterium Exchange Measurement / Q000379:methods*; D006512:Hepatitis B Core Antigens / Q000737:chemistry* / Q000378:metabolism; D006513:Hepatitis B e Antigens / Q000737:chemistry* / Q000378:metabolism; D006515:Hepatitis B virus / Q000737:chemistry*; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D011487:Protein Conformation; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary
D006512:Hepatitis B Core Antigens; D006513:Hepatitis B e Antigens; D007140:Immunoglobulin Fab Fragments
EX1 kinetics; e-antigen; hepatitis B virus; hydrogen deuterium exchange mass spectrometry