FC0065
preS1 Hepatitis B virus  -  EAR domain of γ2-adaptin

Biological function
Infectious HBV virions comprise an icosahedral nucleocapsid formed by the capsid-forming core protein HBc, within which the viral polymerase, genome and host proteins are encapsulated. Nucleocapsids are enveloped by three viral surface proteins: the small (S), middle (M) and large (L) surface proteins. L adopts two distinct orientations, i-preS L and e-preS L. In i-preS L, preS domains are cytoplasmically oriented and have essential roles in nucleocapsid envelopment. In e-preS L, preS domains are located within the endoplasmic reticulum lumen, eventually displaying preS1 on the exterior of the virion envelope, where it mediates infection via a cell-surface receptor (or receptors). PreS1 also regulates genome amplification after infection, has transactivation activity in host cells, regulates viral particle secretion and interacts with host chaperones to establish the dual topology of L. Yeast two-hybrid and co-immunoprecipitation studies show i-preS L interacts with γ2-adaptin but not with γ1- adaptin.

Domain organization/sequence features
PreS1 contains multiple motifs that mimic the γ-adaptin recognition motifs of host cargo proteins that assist transport between membrane compartments. These motifs allowed preS1 to bind γ2-EAR and therefore HBV to gain access to the host protein- export apparatus. HBV has evolved conserved motifs (Sites 1, 3 and 5) that mimic host two-pin plug motifs and allow preS1 to bind γ2-EAR or γ2-adaptin and ’piggyback’ on the host membrane-trafficking apparatus to facilitate nucleocapsid envelopment.

Structural evidence
preS1 did not have substantial preformed secondary structure, and γ2-EAR binding did not induce further secondary structure formation. NOE enhancements, which probed fast, picosecond backbone dynamics, showed that residues 11–44 (encompassing Site 1, Site 2 and Site 3), though not as rigid as a folded protein, although were less dynamic than expected for an IDP. γ2-EAR–binding motifs and other regions of preS1 fall into discrete clusters flanked by proline residues with coherent dynamic properties.

Biochemical evidence
Affinity with which preS1and preS1 (29–36) bound γ2-EAR was lower by a factor of ten than that of full-length preS1 (Kd~ 0.1 mM). ITC showed that binding of preS1(9–16), a Site 3 peptide, to 2-EAR was weaker by a factor of ~20 than that of full-length preS1. Deletions spanning preS1 Site 1, Site 3 or Site 5 impair HBV virion secretion and clearly ablate virus infectivity.

Structure/Mechanism
It is possible that flanking preS1 sequences make additional, cryptic contributions to binding. The ’dynamic clusters’ arise from conformational restrictions, wherein flanking prolines act as motif ’start’ and ’stop’ signals, whereas the motif sequences determine specificity and affinity.

Mechanism category
Tethering

Significance
Albeit the motif-based interaction is weak it can be enhanced by the flanking fuzzy regions, which also enable the combinatorial usage of motifs.