Search for allosteric disulfide bonds in NMR structures

Abstract

Background: Allosteric disulfide bonds regulate protein function when they break and/or form. They typically have a -RHStaple configuration, which is defined by the sign of the five chi angles that make up the disulfide bond.

Results: All disulfides in NMR and X-ray protein structures as well as in refined structure datasets were compared and contrasted for configuration and strain energy.

Conclusion: The mean dihedral strain energy of 55,005 NMR structure disulfides was twice that of 42,690 X-ray structure disulfides. Moreover, the energies of all twenty types of disulfide bond was higher in NMR structures than X-ray structures, where there was an exponential decrease in the mean strain energy as the incidence of the disulfide type increased. Evaluation of protein structures for which there are X-ray and NMR models shows that the same disulfide bond can exist in different configurations in different models. A disulfide bond configuration that is rare in X-ray structures is the -LHStaple. In NMR structures, this disulfide is characterised by a particularly high potential energy and very short alpha-carbon distance. The HIV envelope glycoprotein gp120, for example, is regulated by thiol/disulfide exchange and contains allosteric -RHStaple bonds that can exist in the -LHStaple configuration. It is an open question which form of the disulfide is the functional configuration.

Figure 1

Distribution of disulfide strain energies in NMR and X-ray structures. A. Number of disulfide bonds for each dihedral strain energy (in 2.5 kJ.mol^-1 increments) for structures determined by NMR (total of 55,005 disulfides, Table 2) and X-ray (total of 42,690 disulfides, Table 1). B. Plot of the mean strain energy and 95% confidence intervals of each disulfide configuration versus the incidence of that configuration. The dotted lines are the linear least-squares fit to the NMR data (top line; Table 2) or single exponential least squares fit to the X-ray data (bottom line; Table 1). C. Plot of the mean strain energy and 95% confidence intervals of each disulfide configuration versus the incidence of that configuration for all X-ray disulfides (42,690 disulfides; see part B), a unique set of 6,874 X-ray disulfides described by Schmidt et al. [8] (data set 1) and the 16,225 disulfides of a culled set of X-ray structures described by Guoli Wang and Roland Dunbrack, Jr. [25] (data set 2).

Figure 2

Mean distance between the α-carbons of each of the 20 disulfide configurations in NMR and X-ray structures. The mean distance between the α carbons of all disulfides is 5.6 Å for both NMR (part A) and X-ray (part B) structures. The outliers with a short α carbon distance are the allosteric -RHStaple bonds in both NMR and X-ray structures and the -LHStaple bonds in NMR structures. The dotted lines are the linear least-squares fit to the data.

Figure 3

Distribution of strain energies and α-carbon distances for the -LHStaple disulfides in NMR and X-ray structures. A major fraction of the 1,805 -LHStaple bonds in NMR structures (part A) have a high strain energy (~50 kJ.mol^-1) and short α-carbon distance (~4 Å). The majority of the 599 -LHStaple bonds in X-ray structures (part B) have a low strain energy (~10 kJ.mol^-1) and long α-carbon distance (~6.5 Å). Example of a short, high energy -LHStaple (the Cys45–Cys56 bond in fibronectin, PDB ID 1o9a) and a long, low energy -LHStaple (the Cys133–Cys193 bond in urokinase plasminogen activator, PDB ID 2fd6) is shown in part C. The fibronectin disulfide is a NMR structure (Table 4), while the urokinase plasminogen activator disulfide is a X-ray structure with a resolution of 1.9 Å, a DSE of 2.9 kJ.mol^-1 and an α-carbon distance of 6.5 Å. The structures look at the side of the S-S bond, which is shown in the horizontal position. They were generated using PyMol [35].