Assessing the conformational changes of pb5, the receptor-binding protein of phage T5, upon binding to its Escherichia coli receptor FhuA

Abstract

Within tailed bacteriophages, interaction of the receptor-binding protein (RBP) with the target cell triggers viral DNA ejection into the host cytoplasm. In the case of phage T5, the RBP pb5 and the receptor FhuA, an outer membrane protein of Escherichia coli, have been identified. Here, we use small angle neutron scattering and electron microscopy to investigate the FhuA-pb5 complex. Specific deuteration of one of the partners allows the complete masking in small angle neutron scattering of the surfactant and unlabeled proteins when the complex is solubilized in the fluorinated surfactant F6-DigluM. Thus, individual structures within a membrane protein complex can be described. The solution structure of FhuA agrees with its crystal structure; that of pb5 shows an elongated shape. Neither displays significant conformational changes upon interaction. The mechanism of signal transduction within phage T5 thus appears different from that of phages binding cell wall saccharides, for which structural information is available.

Keywords: Bacteriophage; Biophysics; Electron Microscopy (EM); Fluorinated Surfactant; Membrane Proteins; Small Angle Neutron Scattering; Viral Protein.

SCHEME 1.

SANS contrast strategy. A, F₆-DigluM chemical structure; B, SANS contrast strategy to highlight specific parts of the protein-detergent complexes. Partially deuterated proteins are represented in dark gray. Hydrogenated proteins and the F₆-DigluM micelles are represented in light gray as is the 46% D₂O buffer in which they are contrast-matched.

FIGURE 1.

SANS of F₆-DigluM and validation of SANS strategy. A, Guinier plot of F₆-DigluM (10 mg/ml) in 0% (triangles) or 100% D₂O (squares). B and C, scattering curves, before buffer subtraction, of F₆-DigluM (17.6 mg/ml; B, triangles) and hFhuA (4.5 mg/ml; C, triangles) in F₆-DigluM (8 mg/ml free micelles from AUC), measured in 46% D₂O buffer, compared with the scattering curve of the 46% D₂O buffer (squares in B and C) and dFhuA in F₆-DigluM (circles in B and C). dFhuA was at 4.6 (B) and 0.8 (C) mg/ml, and AUC estimated 9 (B) and 2.3 (C) mg/ml free micelles. Insets, enlargements of the buffer curves.

FIGURE 2.

AUC and SEC quality control for dFhuA and dFhuA-dpb5 sample following SANS. A and B, superposition of experimental and fitted sedimentation velocity profiles obtained in 3-mm optical path length cells during 2 h at 42,000 rpm at 20 °C (top subpanels) and their differences (bottom subpanels), at 284 nm (A) and using interference optics (B), for dFhuA SANS sample in 46% D₂O (second protocol) after 4-day storage at 4 °C. C, Sedimentation coefficient distributions c(s). The distributions are normalized to the main protein peak value. D, SEC of the dFhuA-dpb5 SANS sample (second protocol), loaded after the SANS measurements onto an SD200 5/150 column in 20 mm Tris-HCl, pH 8, 150 mm NaCl, 0.7 mm F₆-DigluM, 46% D₂O, run at 0.45 ml/min. Void volume (V₀) = 1.12 ml; total volume (V_t) = 2.83 ml.

FIGURE 3.

SANS analysis of FhuA, pb5, and complex. A, calculated and measured scattering curve of dFhuA solubilized in F₆-DigluM (triangles) at 46% D₂O. The 2FCP curve was calculated with the program Cryson, considering a 46% D₂O buffer and a 72% deuterated protein (solid line). χ² = 1.173 on the whole Q range. B, experimental scattering curves of isolated dFhuA solubilized in F₆-DigluM (triangles) or in complex with hpb5 (diamonds). Inset, Guinier analysis of the two data sets. C, scattering curves of dpb5 alone (crosses) or in complex with hFhuA solubilized in F₆-DigluM (squares) measured in 46% D₂O. Inset, Kratky representation of the dpb5 sample. D, pair distance distribution function, p(r), calculated using Gnom, of hFhuA-dpb5 (squares), dFhuA-hpb5 (diamonds), and dFhuA-dpb5 (circles) samples measured in a 46% D₂O buffer.

FIGURE 4.

Ab initio envelopes from SANS. Ab initio envelopes (at 46% D₂O) of dFhuA-hpb5 (red mesh), superimposed with the crystal structure of FhuA (2FCP, green) (A); dpb5 (orange) and hFhuA-dpb5 (blue) (B); and dFhuA-dpb5 (C). 10 (dFhuA-hpb5) and 20 (dpb5, hFhuA-dpb5, and dFhuA-dpb5) independent, very similar structures, calculated using Dammif, were averaged using DAMAVER. Side and top views are shown.

FIGURE 5.

Negative stain electron microscopy. A, negative stain electron microscopy of purified FhuA (left), pb5 (middle), and FhuA-pb5 complex (right). The insets are their corresponding averages of the major classes. Scale bar, 50 nm; inset base line, 20 nm. B, FhuA and pb5 proteins are identified on the FhuA-pb5 complex.

FIGURE 6.

Comparison of the EM and SANS projections. Left, EM; right, SANS. The overall dimensions of the SANS and EM molecules coincide. Two SANS projections are presented, rotated by 180° because the relative position of FhuA and pb5 is not determined by the resolution. Projections are drawn to scale.