Dissecting the Energetics of Intrinsically Disordered Proteins via a Hybrid Experimental and Computational Approach

Abstract

Intrinsically disordered proteins (IDPs) play important roles in biology, but little is known about the energetics of their inter-residue interactions. Methods that have been successfully applied to analyze the energetics of globular proteins are not applicable to the fluctuating partially ordered ensembles populated by IDPs. A combined computational experimental strategy is introduced for analyzing the energetic role of individual residues in the free state of IDPs. The approach combines experimental measurements of the binding of wild-type and mutant IDPs to their partners with alchemical free energy calculations of the structured complexes. These data allow quantitative information to be deduced about the free state via a thermodynamic cycle. The approach is validated by the analysis of the effects of mutations upon the binding free energy of the ovomucoid inhibitor third binding domain to its partners and is applied to the C-terminal domain of the measles virus nucleoprotein, a 125-residue IDP involved in the RNA transcription and replication of measles virus. The analysis reveals significant inter-residue interactions in the unbound IDP and suggests a biological role for them. The work demonstrates that advances in force fields and computational hardware have now led to the point where it is possible to develop methods, which integrate experimental and computational techniques to reveal insights that cannot be studied using either technique alone.

Figure 1.

Illustration of the approach used to deduce the energetics of the free IDP. The thermodynamic cycle describes the binding of a wild-type IDP and a mutant IDP to its partner. The capped tripeptide has the same residues adjacent to the mutation site as found in the full protein. ΔG_bind and ΔG’_bind are the binding free energies measured by experiment for wildtype and mutant respectively (red text). ΔG_com and ΔG_frag are calculated using alchemical free energy calculations (blue text). The value for ΔG_free is obtained from ΔG_free = ΔG_bind - ΔG’_bind + ΔG_com. The effect of secondary structure and long-range interactions on the mutations is obtained from ΔΔG_inter = ΔG_free - ΔG_frg. ΔΔG_inter and ΔG_free (green text) cannot be measured by either experiments or calculations alone but can be obtained by combining the experimental and computational measurements.

Figure 2.

Free energy cycle of L-to-A mutations in the binding of SGPB and OMTKY3. Ribbon structures represent SGPB (pink), OMTKY3 (blue) and the SGPB/OMTKY3 complex. Leu18 (top) and Ala18 (bottom) of OMTKY3 are shown in stick format. ΔG values in red are binding free energies measured by experiment. ΔG values in blue are free energies calculated using TI.

Figure 3.

Scatter plot of experimental (ΔΔG_exp) and calculated (ΔΔG_calc) ΔΔG values for the binding of SGPB with different OMTKY3 variants. ΔΔG_exp = ΔG_bind -ΔG’_bind. ΔΔG_calc = ΔG_free - ΔG_com. The point labeled CARL is for the binding of subtilisin Carlsberg with OMTKY3 variants. Blue and orange dots indicate calculated ΔΔG values calculated using different starting structures. For example, the value for the blue dot for A to G was calculated using the structure of SGPB/OMTKY3-Ala18 (PDB code 1SGP) and the value for the orange dot for A to G was calculated using the structure of SGPB/OMTKY3-Gly18 (PDB code 1SGQ). There is no structure for subtilisin Carlsberg/OMTKY3-Ala18 so only one ΔΔG values using the structure of subtilisin Carlsberg/OMTKY3-Leu18 (PDB code 1R0R) was calculated. Similarly, only one ΔΔG value for S-to-C mutation in SGPB/OMTKY3 was calculated. Three outliers, V-to-A, S-to-C and Y-to-F are labeled as crosses. The solid line represents ΔΔG_exp = ΔΔG_calc. The dashed red line indicates the results of the linear regression of the data (y=0.86x+0.31, R²=0.82 p<10⁻⁹).

Figure 4.

Ribbon representation of the NTAIL (486–504)/XD (458–506) complex from the X-ray structure (PDB code 1T6O). The X-ray structure includes the region shown in ribbons, and an artificial linker which was deleted in simulations and is not shown in this figure. Residue A494, L495 and L498 are shown in stick format. The disordered N and C-terminal regions of NTAIL were not included in the X-ray structure and MD simulations and are depicted schematically as thin lines.