Part II: diffraction from two-dimensional cholera toxin crystals bound to their receptors in a lipid monolayer

Abstract

The structure of cholera toxin (CTAB(5)) bound to its putative ganglioside receptor, galactosyl-N-acetylgalactosaminyl (N-acetyl-neuraminyl) galactosylglucosylceramide (GM(1)), in a lipid monolayer at the air-water interface has been studied utilizing grazing incidence x-ray diffraction. Cholera toxin is one of very few proteins to be crystallized in two dimensions and characterized in a fully hydrated state. The observed grazing incidence x-ray diffraction Bragg peaks indicated cholera toxin was ordered in a hexagonal lattice and the order extended 600-800 A. The pentameric binding portion of cholera toxin (CTB(5)) improved in-plane ordering over the full toxin (CTAB(5)) especially at low pH. Disulfide bond reduction (activation of the full toxin) also increased the protein layer ordering. These findings are consistent with A-subunit flexibility and motion, which cause packing inefficiencies and greater disorder of the protein layer. Corroborative out-of-plane diffraction (Bragg rod) analysis indicated that the scattering units in the cholera layer with CTAB(5) shortened after disulfide bond reduction of the A subunit. These studies, together with Part I results, revealed key changes in the structure of the cholera toxin-lipid system under different pH conditions.

FIGURE 1

GIXD Bragg peaks obtained for layers of CTB₅ and CTAB₅ at pH = 8. The protein crystals nucleated under the DPPE:GM1 monolayer forming an ordered 2D protein monolayer with hexagonal packing. (a) Bragg peaks from CTB₅ and CTB₅ + DTT. Miller indices {h, k} of the observed peaks are indicated. Bragg peaks were integrated over the q_z region (–0.05 to 0.2 Å⁻¹). Peaks were fitted (solid lines) using Lorenzian curves (see Table 1 for details). (b) Bragg peaks from CTAB₅ and CTAB₅ + DTT. Intensities and positions of the Bragg peaks (especially higher order) in the case of CTAB₅ had larger uncertainties due to weaker in-plane ordering and a higher incoherent background contribution due to the A subunit.

FIGURE 2

(a) Bragg rod profiles corresponding to scattering from the cholera protein layer at pH = 8. Rods were produced by integrating through the 0.08 Å⁻¹ ≤ q_xy ≤ 0.12 Å⁻¹ region of the {1,0} Bragg peaks in Fig. 1. The profiles corresponding to CTAB₅ have been offset by 6 for clarity. The sharp peak at q_xy∼0.01 Å⁻¹ is the Vineyard-Yoneda peak (18). Error bars are smaller than the symbol size. Lines are fits to the profiles based on cylinders shown in b. (b) Schematic representation of the cylindrical objects used to approximate the scattering units. Structural parameters such as the cylinder's height, radius, and tilt angle of the cholera molecules are reported in Table 2. In the case of CTAB₅, two populations of cylinders were used to fit the data. After activation of CTAB₅ there was a shift from ∼75% to ∼40% for population 2.

FIGURE 3

Grazing incidence x-ray diffraction (GIXD) Bragg peaks obtained for layers of CTB₅ and CTAB₅ with a subphase at pH = 5. The crystals nucleated on the DPPE:GM₁ monolayer forming an ordered 2D protein monolayer. The observed GIXD Bragg peaks indicate packing of the toxins in a hexagonal 2D unit cell. (a) Bragg peaks from CTB₅ and CTB₅ + DTT. Miller indices {h, k} of the observed peaks are indicated. Bragg peaks were integrated over the qz region from −0.05 to 0.2 Å⁻¹. Peaks were fitted (solid lines) using Lorenzian curves (see Table 1 for details). (b) Bragg peaks from CTAB₅ and CTAB₅ + DTT. Intensities and positions of the Bragg peaks (especially higher order) in the case of CTAB₅ had larger uncertainties due to weaker in-plane ordering and higher incoherent background contribution.

FIGURE 4

Bragg rod profiles corresponding to scattering from the cholera protein layer at pH = 5. Rods were produced by integrating through the 0.08 Å⁻¹ ≤ q_xy ≤ 0.12 Å⁻¹ region of the {1,0} Bragg peaks in Fig. 3. Cylindrical objects were used to approximate the scattering units. Structural parameters such as the cylinder's height, radius, and tilt angle of the cholera molecules are reported in Table 2. In the case of CTAB₅, two populations of cylinders were used to fit the data. The curves corresponding to CTAB₅ have been offset for clarity. The sharp peak at q_xy = 0.01Å⁻¹ is the Vineyard-Yoneda peak (18).