Determination of the solution-bound conformation of an amino acid binding protein by NMR paramagnetic relaxation enhancement: use of a single flexible paramagnetic probe with improved estimation of its sampling space

Abstract

We demonstrate the feasibility of elucidating the bound ("closed") conformation of a periplasmic binding protein, the glutamine-binding protein (GlnBP), in solution, using paramagnetic relaxation enhancements (PREs) arising from a single paramagnetic group. GlnBP consists of two globular domains connected by a hinge. Using the ligand-free ("open") conformation as a starting point, conjoined rigid-body/torsion-angle simulated annealing calculations were performed using backbone (1)H(N)-PREs as a major source of distance information. Paramagnetic probe flexibility was accounted for via a multiple-conformer representation. A conventional approach where the entire PRE data set is enforced at once during simulated annealing yielded poor results due to inappropriate conformational sampling of the probe. On the other hand, significant improvements in coordinate accuracy were obtained by estimating the probe sampling space prior to structure calculation. Such sampling is achieved by refining the ensemble of probe conformers with intradomain PREs only, keeping the protein backbone fixed in the open form. Subsequently, while constraining the probe to the previously found conformations, the domains are allowed to move relative to each other under the influence of the non-intradomain PREs, giving the hinge region torsional degrees of freedom. Thus, by partitioning the protocol into "probe sampling" and "backbone sampling" stages, structures significantly closer to the X-ray structure of ligand-bound GlnBP were obtained.

Figure 1

Ligand-free, open conformation of GlnBP (PDB ID: 1GGG). The large domain is displayed at the bottom and the small domain on top. The color scheme indicates H^N-PRE values observed on the bound GlnBP S51C sample: red, PRE > 40 s⁻¹ or completely broadened peak; orange, 40 ≥ PRE > 30 s^{– 1}; yellow, 30 ≥ PRE > 20 s⁻¹; green, 20 ≥ PRE > 10 s⁻¹; grey, PRE ≤ 10 s⁻¹ or missing due to overlapped/unassigned peak. An arrowhead indicates the position of the S51C point mutation. All molecular graphics were generated with MOLMOL.

Figure 2

Difference between C^α and C^β secondary chemical shifts (ΔC^α–ΔC^β) for bound GlnBP versus residue number. The secondary structure of ligand-free GlnBP is indicated by arrows (β-strand) and curls (α-helix).

Figure 3

PRE-based structures of bound GlnBP (red) superimposed to the X-ray model (blue; PDB ID: 1WDN) via the large domain (bottom). (A) Structures calculated by simultaneous optimization of probe conformers and protein backbone. (B) Structures calculated with previously optimized paramagnetic probe conformers using intra-domain PRE data on the fixed, open backbone coordinates. The 10 lowest-PRE energy structures (out of 200) are shown.

Figure 4

Correlation between experimental and calculated ¹H^N-PRE values for structures computed via optimization of the protein backbone with simultaneous (A and B) or previous (C) optimization of the paramagnetic probe conformer(s). Plot A stems from a single-conformer probe representation, while plots B and C result from a three-conformer ensemble. The average overall Q-factor is indicated. See Table 1 for details.

Figure 5

Paramagnetic probe sampling as sensed by the PRE data during different simulated annealing protocols. Only the oxygen atom of the nitroxide group is shown (sphere), against the backbone conformation of bound, closed GlnBP (PDB ID: 1WDN), displayed with the large domain at the bottom. In green are atomic positions optimized assuming the fixed closed conformation. In yellow and red are positions optimized with a flexible hinge, using a 1- or 3-conformer representation of the probe, respectively. All PREs were used (green, yellow, and red). In blue are positions optimized with intra-domain PREs only, assuming a fixed, open conformation. In all cases, results of the 10 lowest PRE energy optimizations are shown (out of 200). Views A and B differ by a 90° rotation.