Solution structure of the IIAChitobiose-IIBChitobiose complex of the N,N'-diacetylchitobiose branch of the Escherichia coli phosphotransferase system

Abstract

The solution structure of the IIA-IIB complex of the N,N'-diacetylchitobiose (Chb) transporter of the Escherichia coli phosphotransferase system has been solved by NMR. The active site His-89 of IIA(Chb) was mutated to Glu to mimic the phosphorylated state and the active site Cys-10 of IIB(Chb) was substituted by serine to prevent intermolecular disulfide bond formation. Binding is weak with a K(D) of approximately 1.3 mm. The two complementary interaction surfaces are largely hydrophobic, with the protruding active site loop (residues 9-16) of IIB(Chb) buried deep within the active site cleft formed at the interface of two adjacent subunits of the IIA(Chb) trimer. The central hydrophobic portion of the interface is surrounded by a ring of polar and charged residues that provide a relatively small number of electrostatic intermolecular interactions that serve to correctly align the two proteins. The conformation of the active site loop in unphosphorylated IIB(Chb) is inconsistent with the formation of a phosphoryl transition state intermediate because of steric hindrance, especially from the methyl group of Ala-12 of IIB(Chb). Phosphorylation of IIB(Chb) is accompanied by a conformational change within the active site loop such that its path from residues 11-13 follows a mirror-like image relative to that in the unphosphorylated state. This involves a transition of the phi/psi angles of Gly-13 from the right to left alpha-helical region, as well as smaller changes in the backbone torsion angles of Ala-12 and Met-14. The resulting active site conformation is fully compatible with the formation of the His-89-P-Cys-10 phosphoryl transition state without necessitating any change in relative translation or orientation of the two proteins within the complex.

FIGURE 1.

Binding of IIA^Chb*(H89E) and IIB^Chb(C10S). Backbone amide chemical shift perturbations upon titrating unlabeled IIA^Chb*(H89E) into a solution of ¹⁵N-labeled IIB^Chb(C10S) at 20 °C. The chemical shifts were monitored using ¹H-¹⁵N HSQC spectroscopy at a spectrometer ¹H frequency of 600 MHz. Δδ_H/N = [(Δδ¹⁵N)²/25 + (Δδ¹H)²)/2]^1/2 (78). The IIA^Chb*(H89E):IIB^Chb(C10S) molar ratios, expressed in terms of subunit concentration of IIA^Chb*(H89E), are 0, 0.2, 0.4, 1.0, 1.5, and 2.0, with corresponding subunit concentrations of IIA^Chb*(H89E) of 0, 0.10, 0.20, 0.47, 0.68, and 0.88 mm, respectively, and concentrations of IIB^Chb(C10S) of 0.5, 0.49, 0.49, 0.47, 0.45, 0.44 mm, respectively. The solid lines represent the results of a global non-linear least squares best-fit to all the titration data simultaneously, using a simple equilibrium binding model. The optimized K_D value is 1.3 ± 0.3 mm.

FIGURE 2.

Intermolecular NOEs in the IIA^Chb*(H89E)-IIB^Chb(C10S) complex. NOEs in a three-dimensional ¹²C-filtered/¹³C-separated NOE experiment recorded in D₂O are specifically observed from protons attached to ¹²C (in the F₁ dimension) to protons attached to ¹³C (in the F₃ dimension). Strips are shown for NOEs involving the ¹³Cδ methyl groups of Ile-72 and Ile-26 of IIA^Chb*(H89E). The amino acid specific labeling scheme used for [¹H-AA]/[²H, ¹²C, ¹⁴N]-IIB^Chb(C10S) is shown above each strip.

FIGURE 3.

Solution structure of the IIA^Chb*(H89E)- IIB^Chb(C10S) complex. A, stereoview of a superposition of the backbone (N, Cα, C) atoms of the final 90 simulated annealing structures with the A, B, and C subunits of the IIA^Chb*(H89E) symmetric trimer in blue, gold, and green, respectively, and IIB^Chb(C10S) in red. The active site residues, H89E and C10S are shown in purple and cyan, respectively, and the pink meshes represent the reweighted atomic density probability map (69) for these two residues (drawn at a value of 20% maximum). Only a single IIB^Chb molecule binding at the interface of the A and C subunits of IIA^Chb*(H89E) is shown; because IIA^Chb*(H89E) is a symmetric trimer there are three identical binding sites formed at the interfaces between the A and C subunit, the C and B subunits and the B and A subunits. B, ribbon diagram of the complex showing two IIB^Chb(C10S) molecules bound to the IIA^Chb*(H89E) trimer. C, ribbon diagram of the complex with a view orthogonal to that shown in B depicting three molecules of IIB^Chb(C10S) bound to the IIA^Chb*(H89E) trimer. The color coding in B and C is the same as that in A. D, stereoview showing a reweighted atomic probability density map (drawn at a value of 20% maximum and calculated from the final 90 simulated annealing structures) for some of the interfacial side chains displayed as purple and green meshes for IIB^Chb(C10S) and the C chain of IIA^Chb*(H89E), respectively. The backbones are shown as tubes color coded as in A; the side chains of the restrained regularized mean structure are color coded according to atom type (carbon, gray; oxygen, red; and nitrogen, blue). Residues of IIB^Chb(C10S) are labeled in italics.

FIGURE 4.

The IIA^Chb*(H89E)-IIB^Chb(C10S) interface. A, stereoview of the interface between the A subunit of IIA^Chb*(H89E) and IIB^Chb(C10S) with the respective backbones shown as transparent blue and red ribbons, respectively. B, stereoview of the interface between the C subunit of IIA^Chb*(H89E) and IIB^Chb(C10S) with the respective backbones shown as transparent green and red ribbons, respectively. The dashed lines indicate intermolecular hydrogen bonds or salt bridges. The side chain atoms are colored according to atom type; carbon, gray; nitrogen, blue; oxygen, red. Residues of IIB^Chb(C10S) are labeled in italics. C, diagrammatic representation of the intermolecular contacts between the A and C subunits of IIA^Chb*(H89E) and IIB^Chb(C10S). Residues involved in side chain-side chain electrostatic interactions are colored in blue (donor) or red (acceptor). Ile-72 of the C subunit of IIA^Chb*(H89E) is colored in orange because its backbone carbonyl accepts a hydrogen bond from Ser-17 of IIB^Chb(C10S). The active site residues, H89E of IIA^Chb*(H89E), and C10S of IIB^Chb(C10S) are colored purple.

FIGURE 5.

Interaction surfaces for the IIA^Chb*(H89E)-IIB^Chb(C10S) complex. The left panel display the interaction surface (formed by the A and C subunits) on IIA^Chb*(H89E) for IIB^Chb(C10S); the right panel shows the interaction surface on IIB^Chb(C10S) for IIA^Chb*(H89E). The surfaces are color coded as follows: hydrophobic residues, green; uncharged residues bearing a polar functional group, cyan; negatively charged residues, red; positively charged residues, blue. Relevant portions of the backbone and active site residue of the interacting partner are displayed as tubes and bonds, respectively. Residues of IIB^Chb(C10S) are labeled in italics. Residues from the C subunit of IIB^Chb*(H89E) are indicated by an apostrophe after the residue number; in addition, the surfaces of the A and C subunits of IIA^Chb* that do not constitute the interaction surface are colored in dark gray and light gray, respectively.

FIGURE 6.

The phosphoryl transition state of the IIA^Chb*-IIB^Chb complex. A, stereoview displaying the conformational change within the active site loop of IIB^Chb accompanying formation of a phosphoryl transition state. Superposition of the active site loop of the IIA^Chb*-P-IIB^Chb transition state (solid colors) and the IIA^Chb*(H89E)-IIB^Chb(C10S) complex (transparent colors) with the backbones of IIA^Chb* and IIB^Chb in red and blue, respectively (see text for further details). B, stereoview of the environment surrounding the His-89-P-Cys-10 transition state. The backbone is displayed as transparent tubes with IIB^Chb in red, and the A and C subunits of IIA^Chb* in blue and green, respectively. C, schematic of the interactions stabilizing the transition state. The thick dashed lines indicate hydrogen bonds, the thin dashed lines indicate electrostatic interactions that are too long to be classified as hydrogen bonds. It should be noted that only side chain torsion angle changes confined within a single rotamer would be required to permit hydrogen-bonding interactions from His-93^A (Chain A) and Gln-91^C (Chain C) of IIA^Chb* to the phosphoryl group in phospho-IIA^Chb*. Color coding: red, IIB^Chb; blue, A subunit of IIA^Chb*; green, C subunit of IIA^Chb*. Residues of IIB^Chb are labeled in italics.

FIGURE 7.

Comparison of the IIA^Chb*-IIB^Chb and IIA^Mtl-IIB^Mtl complexes. A, overall stereoview and, B, stereo close up of the His-P-Cys phosphoryl transition state, with IIB^Chb and IIB^Mtl superimposed. The backbone is displayed as a ribbon diagram and the His-P-Cys transition state as a stick diagram. IIA^Mtl and IIB^Mtl are shown in green and gray, respectively; IIA^Chb* and IIB^Chb are shown in transparent blue and red, respectively; and the His-P-Cys phosphoryl transition state is shown in gold for the IIA^Chb*-IIB^Chb complex and in purple for the IIA^Mtl-IIB^Mtl complex. The coordinates of the IIA^Mtl-IIB^Mtl complex are taken from Ref. (PDB code 2FEW). The superposition of IIB^Chb and IIB^Mtl was carried out using the program O (79).