The structure of myristoylated Mason-Pfizer monkey virus matrix protein and the role of phosphatidylinositol-(4,5)-bisphosphate in its membrane binding

Abstract

We determined the solution structure of myristoylated Mason-Pfizer monkey virus matrix protein by NMR spectroscopy. The myristoyl group is buried inside the protein and causes a slight reorientation of the helices. This reorientation leads to the creation of a binding site for phosphatidylinositols. The interaction between the matrix protein and phosphatidylinositols carrying C(8) fatty acid chains was monitored by observation of concentration-dependent chemical shift changes of the affected amino acid residues, a saturation transfer difference experiment and changes in (31)P chemical shifts. No differences in the binding mode or affinity were observed with differently phosphorylated phosphatidylinositols. The structure of the matrix protein-phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] complex was then calculated with HADDOCK software based on the intermolecular nuclear Overhauser enhancement contacts between the ligand and the matrix protein obtained from a (13)C-filtered/(13)C-edited nuclear Overhauser enhancement spectroscopy experiment. PI(4,5)P(2) binding was not strong enough for triggering of the myristoyl-switch. The structural changes of the myristoylated matrix protein were also found to result in a drop in the oligomerization capacity of the protein.

Fig. 1

Superposition of the 19 best structures of myristoylated M-PMV MA. The helices are marked by roman numbers, and the myristate is shown in red.

Fig. 2

Comparison of the myristoylated MA (green) and non-myristoylated MA (red). The myristoyl group is represented as sticks. The structures were superimposed with the PyMOL program.

Fig. 3

¹H and ¹⁵N CCSD histograms of myristoylated and non-myristoylated MA residues calculated from the ¹H/¹⁵N HSQC spectra of the MAs at a concentration range of c₀ to c₀/10.

Fig. 4

Dependence of the MA L31 residue ¹H and ¹⁵N CCSD on the PIP:MA molar ratio. Myristoylated MA (blue diamonds) and non-myristoylated MA (red squares).

Fig. 5

Structure of the myristoylated MA with the residues significantly affected by interaction with C₈-PI(4,5)P₂. 1H/15N CCSDs greater than 1SD above the average CCSD are highlighted. The myristoyl group is shown in blue, and the affected residues are shown in red and pink (the CCSDs of the red residues were greater than 1SD above the average CCSD in all steps of the titration, whereas the CCSDs of the pink residues were greater in at least four points).

Fig. 6

¹H STD spectrum of a C₈-PI(4,5)P₂:MA 50:1 (molar) mixture (black) together with the assigned 1H spectrum of pure C₈-PI(4,5)P₂ (red).

Fig. 7

Structure of the myristoylated MA (green)– C₈-PI(4,5)P₂ (blue) complex calculated by HADDOCK. Phosphates are shown in orange and red.

Fig. 8

The myrMA and C₈-PI(4,5)P₂ complex showing the interaction between myrMA lysines (green) and C₈-PI(4,5)P₂ phosphates (red).

Fig. 9

Structure of the MA (a) and myrMA (b) as van der Waals surface representations (gray) with lysine residues highlighted in blue to show the binding cavity created by the first, second and third helices of myrMA bound to a molecule of C₈-PI(4,5)P₂ (cyan).