Helix packing and orientation in the transmembrane dimer of gp55-P of the spleen focus forming virus

Abstract

gp55-P is a dimeric membrane protein with a single transmembrane helix that is coded by the env gene of the polycythemic strain of the spleen focus forming virus. gp55-P activates the erythropoietin (Epo) receptor through specific transmembrane helix interactions, leading to Epo-independent growth of erythroid progenitors and eventually promoting erythroleukemia. We describe the use of magic angle spinning deuterium NMR to establish the structure of the transmembrane dimer of gp55-P in model membranes. Comparison of the deuterium lineshapes of leucines in the center (Leu(396-399)) and at the ends (Leu(385), Leu(407)) of the transmembrane sequence shows that gp55-P has a right-handed crossing angle with Leu(399) packed in the dimer interface. We discuss the implications of the structure of the gp55-P transmembrane dimer for activation of the Epo receptor.

FIGURE 1

(A) Sequence of the TM domain of gp55-P. (B) Polarized IR spectra of gp55-P TM peptides reconstituted into DMPC bilayers exhibit an amide I vibration at 1657 cm⁻¹ and a dichroic ratio of 3.0. These data indicate that the gp55-P TM domain has α-helical secondary structure with the helix axis oriented roughly perpendicular to the membrane surface.

FIGURE 2

Deuterium MAS NMR spectra and simulations of gp55-P deuterium labeled at Leu³⁹⁶-Leu³⁹⁹ (A–D). The MAS spectra were obtained at 3 kHz and a temperature of 25°C. Each spectrum represents the average of 600,000–800,000 transients. Exponential line broadening of 200 Hz was applied. The simulations to the right were carried out using the program SIMPSON version 1.1.0 (35). The MAS rate was set to 3 kHz, and the asymmetry parameter was set to 1.0. The best fits to the observed side band intensities were obtained using quadrupole coupling constants of 16 kHz (A), 14 kHz (B), 16 kHz (C), and 25 kHz (D).

FIGURE 3

Helical wheel plots for helix dimers having LH and RH crossing angles. These diagrams have either 3.5 (LH) or 3.9 (RH) amino acids per turn and provide a truer representation than a canonical helical wheel diagram, of which amino acids line the dimer interface when helices cross with LH or RH crossing angles, respectively. For example, in an LH coiled coil, if Leu³⁹⁹ and Leu³⁸⁵ are in the dimer interface, then Gly³⁹¹ and Leu⁴⁰⁷ would lie outside of the interface.

FIGURE 4

Deuterium MAS NMR spectra and simulations of gp55-P deuterium labeled at Leu³⁸⁵ and Leu⁴⁰⁷. The MAS spectra were obtained at 3 kHz and a temperature of 298 K. The spectra of Leu³⁸⁵ (A) and Leu⁴⁰⁷ (B) represent the average of 800,000 and 1,200,000 transients, respectively. An exponential line broadening of 200 Hz was applied. The simulations to the right were carried out using the program SIMPSON version 1.1.0 (35). The MAS rate was set to 3 kHz, the asymmetry parameter was set to 1.0, and the quadrupole coupling constant was changed to 16 kHz and 27 kHz to obtain the best fit to the side band intensities of Leu³⁸⁵ and Leu⁴⁰⁷, respectively.

FIGURE 5

Computational searches for low-energy structures of the gp55-P TM dimer. (A) Low-energy structures of the gp55-P TM dimer with RH helix crossing angles. The figure plots the final rotational angles of the two TM helices (φ₁ and φ₂) for those structures that fall into a “cluster”. Only a single cluster (circled) was observed along the diagonal and corresponds to a symmetric dimer structure. No symmetric dimer clusters were observed for comparable searches for gp55-P with LH crossing angles. The interhelical separation for this search was maintained at 10 Å. (B) Helix interaction energies for average TM dimer structure from the symmetric cluster.

FIGURE 6

Structural model of the gp55-P TM dimer. (A) Cross section of the gp55-P dimer showing the noninterfacial position of Leu³⁸⁵ (orange). (B) Cross section of the gp55-P dimer highlighting one helix turn with four consecutive leucines. Leu³⁹⁹ (red) is in the dimer interface.( C) Cross section of the gp55-P dimer showing the interfacial position of Leu⁴⁰⁷ (orange). (D) gp55-P TM dimer highlighting the leucine residues labeled in this study. Met³⁹⁰, Trp⁴⁰⁵, and His⁴⁰⁸ are oriented away from the dimer interface. Met³⁹⁰ is thought to interact with Ser²⁹⁸ of the Epo receptor.