Solution structure of the phosphoryl transfer complex between the signal transducing proteins HPr and IIA(glucose) of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system

Abstract

The solution structure of the second protein-protein complex of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system, that between histidine-containing phosphocarrier protein (HPr) and glucose-specific enzyme IIA(Glucose) (IIA(Glc)), has been determined by NMR spectroscopy, including the use of dipolar couplings to provide long-range orientational information and newly developed rigid body minimization and constrained/restrained simulated annealing methods. A protruding convex surface on HPr interacts with a complementary concave depression on IIA(Glc). Both binding surfaces comprise a central hydrophobic core region surrounded by a ring of polar and charged residues, positive for HPr and negative for IIA(Glc). Formation of the unphosphorylated complex, as well as the phosphorylated transition state, involves little or no change in the protein backbones, but there are conformational rearrangements of the interfacial side chains. Both HPr and IIA(Glc) recognize a variety of structurally diverse proteins. Comparisons with the structures of the enzyme I-HPr and IIA(Glc)-glycerol kinase complexes reveal how similar binding surfaces can be formed with underlying backbone scaffolds that are structurally dissimilar and highlight the role of redundancy and side chain conformational plasticity.

Fig. 1. Intermolecular NOEs. 2D ¹⁵N-filtered/¹³C-separated NOE spectra (120 ms mixing time) recorded on (A) a 1:1 HPr(¹⁵N)–IIA^Glc(¹³C) complex and (B) a 1:1 HPr(¹³C)–IIA^Glc(¹⁵N) complex, specifically illustrating intermolecular NOE contacts from protons attached to ¹³C (F₁ axis) to amide protons attached to ¹⁵N (F₂ axis). Residues from HPr are denoted in italic. Intermolecular NOEs were assigned with the aid of complementary 2D ¹⁵N-separated/¹³C-filtered and ¹⁵N-filtered/¹³C-filtered NOE spectra. Note that the aromatic ¹³C resonances are folded.

Fig. 2. The structure of the E.coli HPr–IIA^Glc complex. Three sets of superpositions are shown. (A) The structures are best fitted to all the backbone atoms; the backbone and side chains of HPr are shown in green and magenta, respectively, and the backbone and side chains of IIA^Glc are shown in blue and red, respectively. (B) The structures are best fitted to the backbone atoms of IIA^Glc only. (C) The structures are best fitted to the backbone atoms of HPr only. The latter two superpositions illustrate the precision with which the orientation of one molecule is determined relative to the other. Residues from HPr are denoted in italic. The axis of the alignment tensor is shown at the bottom of the figure.

Fig. 3. Ribbon diagram showing two views of the E.coli HPr–IIA^Glc complex. HPr is in green, IIA^Glc in blue and the location of the active site histidines, His15 of HPr and His90 of IIA^Glc, is indicated. Residues from HPr are denoted in italic. The secondary structure elements in the vicinity of the interface are indicated.

Fig. 4. HPr–IIA^Glc interactions in the unphosphorylated complex and in the transition state. (A) Stereoview of the unphosphorylated HPr–IIA^Glc interface. The backbones of HPr and IIA^Glc, depicted as ribbon diagrams, are shown in blue and light green, respectively, the side chains of HPr and IIA^Glc are shown in dark green and red, respectively, and the active site histidines (His15 of HPr and His90 of IIA^Glc) are depicted in purple. (B) Detailed view around the active site histidines, illustrating the backbone and side chain positions in the unphosphorylated complex, the dissociative transition state and the associative transition state. The color coding is the same as in (A) except that the active site histidines and pentacoordinate phosphoryl group (in the case of the transition states) are shown in purple for the unphosphorylated complex, in light blue for the dissociative transition state (Nδ1–Nε2 distance between His15 and His90 of ∼6 Å) and in orange for the associative transition state (Nδ1–Nε2 distance between His15 and His90 of ∼4 Å). The backbones for the unphosphorylated and dissociative transition state complexes are identical and only the positions of the active site histidines are different; in the associative transition state complex there are small alterations in the backbone of residues 13–17 of HPr and 89–91 of IIA^Glc (see text for details). (C) Detailed view of the active site during the putative associative transition state illustrating the interactions that stabilize the phosphoryl group. The color coding is the same as in (A) and the phosphoryl group is shown in yellow. Residues from HPr are denoted in italic.

Fig. 5. Summary of interactions between (A) HPr and its partner proteins EIN and IIA^Glc and (B) between IIA^Glc and its partner proteins HPr and glycerol kinase (GK). Side chains that participate in analogous hydrophobic and electrostatic interactions in both partners are depicted in blue and red, respectively.

Fig. 6. Surface representations illustrating the binding surfaces involved in the (A and B) EIN–HPr, (C and D) HPr–IIA^Glc and (E and F) IIA^Glc–GK complexes. The HPr binding surface on EIN and IIA^Glc are shown in (A) and (C), respectively; the EIN and IIA^Glc binding surfaces on HPr are shown in (B) and (D), respectively; the GK binding surface on IIA^Glc is shown in (E); the IIA^Glc binding surface on GK is shown in (F). The binding surfaces are color coded with hydrophobic residues in green, polar residues in light blue, the active site histidines in purple, positively charged residues in dark blue and negatively charged residues in red. The relevant portion of the backbone of the partner protein is shown as a gold ribbon with positively charged side chains in dark blue and negatively charged ones in red. Only charged residues and the active site histidines are labeled, with residues from HPr and GK denoted in italic. Note that although the active site histidines of EIN (His189) and IIA^Glc (His90) are in close contact with His15 of HPr, their direction of approach is different: His189 (EIN) approaches His15 from above (B), while His90 (IIA^Glc) approaches His15 from below (D). The coordinates for the EIN–HPr and IIA^Glc–GK complexes are taken from Garrett et al. (1999) (RCSB accession code 3EZA) and Hurley et al. (1993) (RCSB accession code 1GLA).

Fig. 7. Conformational side chain plasticity. Stereoviews of superpositions of selected regions of the interfaces of the HPr–IIA^Glc and EIN–HPr complexes illustrating alternative conformations of (A) Phe48 and (B) Arg17 of HPr in the two complexes. HPr in the HPr–IIA^Glc and EIN–HPr complexes is shown in red and blue, respectively; EIN is shown in purple and IIA^Glc in green.