The flexible C-terminal arm of the Lassa arenavirus Z-protein mediates interactions with multiple binding partners

Abstract

The arenavirus genome encodes for a Z-protein, which contains a RING domain that coordinates two zinc ions, and has been identified as having several functional roles at various stages of the virus life cycle. Z-protein binds to multiple host proteins and has been directly implicated in the promotion of viral budding, repression of mRNA translation, and apoptosis of infected cells. Using homology models of the Z-protein from Lassa strain arenavirus, replica exchange molecular dynamics (MD) was used to refine the structures, which were then subsequently clustered. Population-weighted ensembles of low-energy cluster representatives were predicted based upon optimal agreement of the chemical shifts computed with the SPARTA program with the experimental NMR chemical shifts. A member of the refined ensemble was identified to be a potential binder of budding factor Tsg101 based on its correspondence to the structure of the HIV-1 Gag late domain when bound to Tsg101. Members of these ensembles were docked against the crystal structure of human eIF4E translation initiation factor. Two plausible binding modes emerged based upon their agreement with experimental observation, favorable interaction energies and stability during MD trajectories. Mutations to Z are proposed that would either inhibit both binding mechanisms or selectively inhibit only one mode. The C-terminal domain conformation of the most populated member of the representative ensemble shielded protein-binding recognition motifs for Tsg101 and eIF4E and represents the most populated state free in solution. We propose that C-terminal flexibility is key for mediating the different functional states of the Z-protein.

Fig. 1

Comparison of models of the core and C-terminus. (a) Core configurations of model 1 for each of the initial homology models. (b) Core Configurations of the five most populated members of H^Full. (c) Core Configuration of the four most populated members of the R^Core. (d) C-terminus orientations of the seven most populated conformations of R^31–99 (res:51–99 are shown).

Fig. 2

Hierarchal clustering of the core (res:31–67). Only the first four (of eight) levels of clustering are shown. The numbers indicate the population size of the clusters. Clusters whose lowest energy structure had a helix from Leu51 to Leu57 are colored gray.

Fig. 3

CS-rmsd versus energy for R^Core, data points are colored on the basis of helical content over residues Leu51-Leu57.

Fig. 4

Workflow diagram describing the methodology of weighted ensemble construction.

Fig. 5

Evaluating the effect of zinc ions on ensemble construction by excluding atoms around the zincs from the CS calculations. (a) The percentage of the ensemble made from members of R^Full versus the size of the exclusion shell. (b) The averaged signed CS error of atoms within a specified distance from the zincs for R^Full (No Shell) and the ensembles with varying exclusion shells.

Fig. 6

Conformation of the late domain (PTAPP) that is favorable for binding to Tsg101. (a) Superposition of PTAPPs from Z (magenta) and HIV-1 Gag (gray) bound to Tsg101 (1M4P). (b) The 2^nd most populated member of R^Full (cyan) presents a conformation of the late domain (magenta) that is favorable for binding Tsg101. The most populated member of R^Full is shown in gray. When these two conformations are superimposed over the core, a distance of 25 Å separates the PTAPP motifs. The gray conformation has extensive contacts between the C-terminus and the core, while the cyan gray conformation has very few.

Fig. 7

Protein-protein interactions between eIF4E and three binding partners, known native interactions are shown in (a), predicted interactions shown in (b) and (c). (a) NMR structure (1RF8) of eIF4E (gray) and a fragment of eIF4G (magenta), which is the native protein-protein interaction formed in the heterotrimeric eIF4F initiation complex. Shown in blue is the crystal structure of the fragment 4E-BP1 (1WKW) bound to eIF4E. (b–c) Predicted structures of Z bound to eIF4E from protein-protein docking. 4E-BP1 (blue) is superimposed with the helix of Z for comparision. The chain of Z is colored rainbow from blue to red to show the orientation of the C-terminus, but the N-terminal residues 1–30 are removed for clarity, as they do not interact significantly. The best static structure of Z is shown in (b) and the best dynamics structure in (c).

Fig. 8

Detailed interactions of the protein-protein interface of eIF4E (gray) and Z (rainbow) from the best static structure complex (a) and best dynamics structure complex (b) predicted from protein-protein docking. In both of these binding modes the first zinc center binds in close proximity to W73 (cyan) in the eIF4E structure. In both binding modes, the predicted complex of Z is shown compared to the bound structure of 4E-BP1 (blue). In the best static structure complex (a) residues from the α-helix of Z isosterically replaces several important residues in the α-helical recognition motif. R56 and L59 are shown for 4E-BP1, along with the residues N52 and L59 and from Z that replace these interactions. In the best dynamics structure complex (b), Z-protein residues I66 and K68 on a loop replace these interactions. The NZ atom from the K68 side chain of Z is in very close proximity to the guanidino group of R56 of 4E-BP1.

Fig. 9

Comparison of predicted binding structures of Z (to eIF4E) with the most populated conformation of Z from R^Full (predicted solution structure). Residues L56 on the α-helix and I92 on the flexible C-terminal arm both contribute contacts to the eIF4E binding surface. The best static structure (a) and the best dynamics structure (b) of Z in the predicted complex (red) are superimposed with the most populated solution conformation (cyan). In the solution conformation, residue I92 forms a strong hydrophobic contact with L56. In both binding conformations of Z, the flexible C-terminal arm swings out, allowing both L56 and I92 to form strong hydrophobic contacts with the surface of eIF4E (gray).

Fig. 10

Three conformational states of the C-terminus: (left) conformation predicted to bind eIF4E, (center) the most populated conformation in solution and (right) the conformation predicted to bind Tsg101.