Temperature dependence of molecular interactions involved in defining stability of glutamine binding protein and its complex with L-glutamine

Abstract

The temperature dependence of dynamic parameters derived from nuclear magnetic resonance (NMR) relaxation data is related to conformational entropy of the system under study. This provides information such as macromolecules stability and thermodynamics of ligand binding. We studied the temperature dependence of NMR order parameter of glutamine binding protein (GlnBP), a periplasmic binding protein (PBP) highly specific to L-glutamine associated with its ABC transporter, with the goal of elucidating the dynamical differences between the respective ligand bound and free forms. We found that the protein-ligand interaction, which is stabilized at higher temperature, has a striking effect on the stability of the hydrophobic core of the large domain of GlnBP. Moreover, in contrast to what was found for less specific PBPs, the decreasing backbone motion of the hinge region at increasing temperature supports the idea that the likelihood that GlnBP can adopt a ligand free closed conformation in solution diminishes at higher temperatures. Our results support the induced-fit model as mode of action for GlnBP. In addition, we found that the backbones of residues involved in a salt bridge do not necessarily become more rigid as the temperature rises as it was previously suggested [Vinther, J. M., et al. (2011) J. Am. Chem. Soc., 133, 271-278]. Our results show that for this to happen these residues have to also directly interact with a region of the protein that is becoming more rigid as the temperature increases.

Figure 1

Comparison between fitted S² (open symbols) and S²_R (filled symbols) for residues V114 (circles) and F164 (squares) taken as reference examples from the GlnBP bound form. The error bars signify the experimental errors.

Figure 2

Temperature dependence of averaged order parameter S²_R for bound (circles) and free (squares) forms of Glutamine Binding Protein. The error bars correspond to standard deviations of the S²_R.

Figure 3

Free GlnBP residues with decreased order parameter with respect to the bound form at 30°C (colored in blue) plotted on the bound GlnBP structure. A) Surface location of more flexible residues, B) active site residues with decreased flexibility in the bound form and C) hinge region (Y85-S88 and E181-Y185) with amide backbones of residues with decreased flexibility identified in blue.

Figure 4

Percentage difference of S²_R as a function of temperature for the three different classes of residues for both GlnBP bound and free form. Horizontal dashed lines represent the maximum percent error in S²_R associated with the respective form of GlnBP. Order parameter temperature dependence is shown as positive, constant and negative and colored in red, green and blue, respectively. A) Bound form example residues: F27 in red, R197 in green and Y43 in blue. B) Free form example residues: D106 in red, V176 in green and T11 in blue.

Figure 5

Space filling representation of temperature dependence of active site residues. “Positive” residues having their S²_R increasing at increasing temperature are highlighted in red; “negative” residues displaying a negative temperature dependence of their S²_R are shown in blue; “constant” residues associated with constant S²_R are colored in green. Glutamine ligand is shown in gold.

Figure 6

Location comparison of “positive” residues (displaying a positive temperature dependence of S²_R) in bound GlnBP. Glutamine ligand is shown in gold, hydrophobic residues in grey and polar or charged residues in cyan. A) Space filling representation of “positive” residues in the large domain of bound GlnBP. B) 90°rotation of bound GlnBP large domain. C) Space filling representation of “positive” residues in the small domain of bound GlnBP. D) Enlargement of aromatic hydrophobic pocket (residues in cyan) located in the large domain showing the details of parallel and perpendicular stacking interactions. E) Enlargement of hydrophobic interaction between an aromatic ring and aliphatic carbons of hydrophobic – non aromatic – residues (cyan).

Figure 7

Location of residues involved in a salt bridge on GlnBP bound form structure. Glutamine ligand is shown in gold, “negative” residues are colored in blue and “positive” residues in red. Also, location of the aromatic hydrophobic core is provided as grey space filling atoms.

Figure 8

Different location of “positive” (panels A and B) and “negative” (panels C and D) residues on the structure of bound (left) and free (right) GlnBP. “Positive” residues are shown in space filling and colored in red while glutamine ligand (when present) is colored in gold. “Negative” residues are shown in blue. Fitting of the Cα of the large domain was performed in order to have the same orientation of the proteins.