Conformational Plasticity of Hepatitis B Core Protein Spikes Promotes Peptide Binding Independent of the Secretion Phenotype

Abstract

Hepatitis B virus is a major human pathogen, which forms enveloped virus particles. During viral maturation, membrane-bound hepatitis B surface proteins package hepatitis B core protein capsids. This process is intercepted by certain peptides with an “LLGRMKG” motif that binds to the capsids at the tips of dimeric spikes. With microcalorimetry, electron cryo microscopy and peptide microarray-based screens, we have characterized the structural and thermodynamic properties of peptide binding to hepatitis B core protein capsids with different secretion phenotypes. The peptide “GSLLGRMKGA” binds weakly to hepatitis B core protein capsids and mutant capsids with a premature (F97L) or low-secretion phenotype (L60V and P5T). With electron cryo microscopy, we provide novel structures for L60V and P5T and demonstrate that binding occurs at the tips of the spikes at the dimer interface, splaying the helices apart independent of the secretion phenotype. Peptide array screening identifies “SLLGRM” as the core binding motif. This shortened motif binds only to one of the two spikes in the asymmetric unit of the capsid and induces a much smaller conformational change. Altogether, these comprehensive studies suggest that the tips of the spikes act as an autonomous binding platform that is unaffected by mutations that affect secretion phenotypes.

Binding of a Pocket Factor to Hepatitis B Virus Capsids Changes the Rotamer Conformation of Phenylalanine 97

“…The purification of HBc-CLPs was previously described [10]: In brief, wt HBc and HBc-F97L, HBc-P5T and HBc-L60V were overexpressed in BL21 (DE3) Star cells. After harvesting by centrifugation, the cells were disrupted by microfluidization (M-110P Microfluidizer, Westwood, MA, USA) at 1500 bar.…”

“…It is plausible that such determinants for a maturation or blocking signal are encoded in the structure of the capsids. Even though many structures of capsids have been determined [10][11][12][13][14][15][16][17][18][19], the nature of the blocking signal remained enigmatic.…”

“…This SS-bridge forms rapidly in capsid like particles (CLPs) [26] and separates the protruding part of the spike from the part, which is embedded in the continuous capsid shell. The protruding part is structurally highly dynamic [27,28] and adapts its conformation to binding of inhibitory peptides [10,29] or fragments of HBs [30] by splaying the spike helices apart. In contrast, the embedded part of the spike is structurally rigid and shows little response to conformational challenges at the tips of the spikes.…”

“…Naturally occurring point mutations like HBc-F/I97L with an immature secretion phenotype [32,33] or point mutations with a low secretion phenotype, such as HBc-L60V and HBc-P5T [34], are located in the area surrounding the pocket. Regardless of the differences in phenotype, the structural changes between these mutants are restricted to the respective residue without long-range impact on the protein backbone [10,13] offering no plausible hint of the possible mode of action of maturation signals or blocking signals.…”

“…We asked what the underlying conformational changes were and whether these changes differed between CLPs of wt HBc, mutants with immature secretion phenotype (HBc-F97L) or mutants with low secretion phenotypes (HBc-P5Tand HBc-L60V). To address this question, we used electron cryo-microscopy and image processing and compared HBc-CLPs from detergentfree purifications [10] with those purified in the same way with added TX100 after the purification process.…”