Ligand-free open-closed transitions of periplasmic binding proteins: the case of glutamine-binding protein

Abstract

The ability to undergo large-scale domain rearrangements is essential for the substrate-binding function of periplasmic binding proteins (PBPs), which are indispensable for nutrient uptake in Gram-negative bacteria. Crystal structures indicate that PBPs typically adopt either an "open" unliganded configuration or a "closed" liganded one. However, it is not clear whether, as a general rule, PBPs remain open until ligand-induced interdomain closure or are in equilibrium with a minor population of unliganded, closed species. Evidence for the latter has been recently reported on maltose-binding protein (MBP) in aqueous solution [Tang, C., et al. (2007) Nature 449, 1078-1082] via paramagnetic relaxation enhancement (PRE), a technique able to probe lowly populated regions of conformational space. Here, we use PRE to study the unliganded open-closed transition of another PBP: glutamine-binding protein (GlnBP). Through a combination of domain structure knowledge and intermolecular and concentration dependence PRE experiments, a set of surface residues was found to be involved in intermolecular interactions. Barring such residues, PRE data on ligand-free GlnBP, paramagnetically labeled at two sites (one at a time), could be appropriately explained by the unliganded, open crystal structure in that it both yielded a good PRE fit and was not significantly affected by PRE-based refinement. Thus, contrary to MBP, our data did not particularly suggest the coexistence of a minor closed conformer. Several possibilities were explored to explain the observed differences in such closely structurally related systems; among them, a particularly interesting one arises from close inspection of the interdomain "hinge" region of various PBPs: strong hydrogen bond interactions discourage large-scale interdomain dynamics.

Figure 1

Conformational change of GlnBP upon ligand binding. The crystal structures of unliganded (PDB ID 1GGG) and liganded (PDB ID 1WDN) GlnBP are superimposed via the large domain, colored grey (only that of liganded GlnBP is shown). The small domain and hinge region of unliganded and liganded GlnBP are colored red and blue, respectively. The L-Gln ligand is shown in “balls-and-sticks” representation. The mutation sites used for spin labeling are indicated. All molecular representations were generated with MOLMOL (19).

Figure 2

PRE profiles for unliganded and liganded GlnBP. In both, the unliganded (A) and liganded (B) cases, experimental transverse ¹H^N-PREs (circles) are shown against the calculated values (lines) obtained via ensemble paramagnetic probe optimization using the indicated PRE data set (S51C or D122C; single-headed arrows denote the mutation/paramagnetic labeling sites) and the corresponding crystal structure. Each calculated PRE value is the average over the 10 lowest-PRE energy results out of 200 independent optimizations. Double-headed arrows (top) indicate the large and small domains, respectively. Open circles involve residues 143–149, 168, 169, and 173, of which 144 is highlighted in the unliganded data sets (see text).

Figure 3

Correlations between calculated and experimental PRE values. Both the unliganded (A) and liganded (B) cases are shown. Each calculated PRE value is the average over the 10 lowest-PRE energy results out of 200 independent paramagnetic probe optimizations. Intradomain PREs are shown in red, interdomain in blue. Open circles involve residues 143–149, 168, 169, and 173, of which 144 is highlighted in the unliganded data sets (see text).

Figure 4

Intermolecular interactions on unliganded GlnBP. Residues 143–149, 168, 169, and 173 (magenta) form a continuous, solvent exposed surface on the small domain of unliganded, open GlnBP (A). For each residue, the PRE concentration dependence in unliganded, spin-labeled (i.e., MTSL-derivatized) GlnBP S51C is expressed in terms of the square of the PRE difference, (ΔΓ₂)², between two protein concentrations (0.5 and 0.25 mM), divided by the square of the average PRE error, σ² (B). Intermolecular PREs arising from the interaction of wild-type (WT) ¹⁵N-enriched GlnBP and natural isotopic abundance, spin-labeled GlnBP D122C (both proteins unliganded) are shown (C). Residue 144 is indicated throughout.

Figure 5

Effect of a minor population of closed GlnBP conformer on the fit of PREs arising from unliganded GlnBP S51C (full lines) and D122C (dashed lines). Overall, intra-, and interdomain Q-factors are indicated in black, red, and blue, respectively. The backbone coordinates for the closed conformer are those of the liganded GlnBP crystal structure (PDB ID 1WDN).

Figure 6

Interdomain Q-factor surfaces for the fit of the unliganded GlnBP S51C (left) and D122C (right) PRE data sets as a function of the relative population and extent of domain closure of the minor species. Domain closure is expressed in terms of the iteration number of the morphing protocol that gradually converts the unliganded open structure (PDB ID 1GGG, iteration 0) into the liganded closed one (PDB ID 1WDN, iteration 99).

Figure 7

Hinge segments from the crystal structures of open–unliganded GlnBP (PDB ID 1GGG) (34), MBP (PDB ID 1OMP) (43), GGBP (PDB ID 2FW0) (41), and ChoX (PDB ID 3HCQ) (42). The hinges of GlnBP and ChoX have two segments, while those of MBP and GGBP have three; the residues flanking each segment are indicated. Backbone heavy atoms are shown, in addition to those of side chains involved in hydrogen bonding. Covalent bonds are colored according to the liked atoms (carbon, grey; oxygen, red; nitrogen, blue). Hydrogen bonds are colored green, and were detected as detailed in Materials and Methods. Arrowheads indicate regions reported to support the major conformational change upon ligand binding; such a specific region in ChoX has been neither reported nor observed here after backbone torsion-angle analysis (see text).