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. 2023 Jan 17;122(2):279-289.
doi: 10.1016/j.bpj.2022.12.019. Epub 2022 Dec 16.

Thermodynamic analysis of an entropically driven, high-affinity nanobody-HIV p24 interaction

Jennifer C Brookes  1 , Eleanor R Gray  1 , Colleen N Loynachan  2 , Michelle J Gut  1 , Benjamin S Miller  1 , Alex P S Brogan  3 , Rachel A McKendry  4
Affiliations

Thermodynamic analysis of an entropically driven, high-affinity nanobody-HIV p24 interaction

Jennifer C Brookes et al. Biophys J. .

Abstract

Protein-protein interactions are fundamental to life processes. Complementary computational, structural, and biophysical studies of these interactions enable the forces behind their specificity and strength to be understood. Antibody fragments such as single-chain antibodies have the specificity and affinity of full antibodies but a fraction of their size, expediting whole molecule studies and distal effects without exceeding the computational capacity of modeling systems. We previously reported the crystal structure of a high-affinity nanobody 59H10 bound to HIV-1 capsid protein p24 and deduced key interactions using all-atom molecular dynamics simulations. We studied the properties of closely related medium (37E7) and low (48G11) affinity nanobodies, to understand how changes of three (37E7) or one (48G11) amino acids impacted these interactions; however, the contributions of enthalpy and entropy were not quantified. Here, we report the use of qualitative and quantitative experimental and in silico approaches to separate the contributions of enthalpy and entropy. We used complementary circular dichroism spectroscopy and molecular dynamics simulations to qualitatively delineate changes between nanobodies in isolation and complexed with p24. Using quantitative techniques such as isothermal titration calorimetry alongside WaterMap and Free Energy Perturbation protocols, we found the difference between high (59H10) and medium (37E7) affinity nanobodies on binding to HIV-1 p24 is entropically driven, accounted for by the release of unstable waters from the hydrophobic surface of 59H10. Our results provide an exemplar of the utility of parallel in vitro and in silico studies and highlight that differences in entropic interactions between amino acids and water molecules are sufficient to drive orders of magnitude differences in affinity.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Figure 1
Figure 1
Structural and genetic data of p24 and the nanobodies considered in this study. (a) Two views of the complex formed by 59H10 (yellow) and HIV-1 p24 (purple). Amino acids that differ between 59H10, 48G11, and 37E7 are highlighted. i) View from above 59H10 CDR loops. ii) Side view. (b) Sequences of 59H10, 48G11, and 37E7, where variant, and comparison of the structures of the three nanobodies showing secondary structure (β-sheets in yellow, α-helices in purple, and unstructured loops in blue). To highlight the differences, the variant amino acids are rendered in licorice. For full sequences, refer to Gray et al. (9). To see this figure in color, go online.
Figure 2
Figure 2
Effect of variations in CDR2 and CDR3 explored through circular dichroism and molecular dynamics simulations of each nanobody. (a) CD spectroscopy of the three nanobody variants. (b) Map of the flexibility of residues, with RMSF of the Cα carbon on each residue versus residue number for nanobody structure in isolation with CDR1 (purple band), CDR2 (red band), and CDR3 (blue band) regions denoted. (c) CD spectroscopy of p24 and 59H10, individually and as a bound pair. (d) Map of the flexibility of residues, with RMSF of the Cα carbon on each residue versus residue number for the nanobodies in complex with p24 with CDR1 (purple band), CDR2 (red band), and CDR3 (blue band) regions denoted. (e) The online tool BeStSel was used to analyze the relative proportions of the different types of secondary structure. Although 59H10 and 37E7 appear relatively similar in the proportion of antiparallel β-sheets, turns, and unclassified structures (others such as π-helices, β-bridges, and disordered loops), 48G11 is qualitatively distinct. To see this figure in color, go online.
Figure 3
Figure 3
Analysis of 59H10, 48G11, and 37E7 interaction with p24. Shown is the change in enthalpy as the molar ratio of reagents alters for (a) 59H10 and p24, (b) 48G11 and p24, and (c) 37E7 and p24. Graphs show combined mean results from at least three experiments performed per reagent set; error bars show standard deviations. Raw thermograms are shown in Fig. S3. Control titrations are shown in Fig. S4. To see this figure in color, go online.
Figure 4
Figure 4
WaterMaps of apo nanobody (no antigen bound) 59H10 versus 48G11. Comparing free energy density, a continuous water map, with a surface isovalue of 0.033 kcal mol−1Ȧ−3 of the free energy of the water molecules at the nanobody interface (a) 59H10 F93 (blue, high-affinity, solid isosurface) and 48G11 D93 (gray, binding abolished, mesh isosurface) zoomed into the residue site and phenylalanine shown in ball and stick. The energy distribution surrounding the phenylalanine in 59H10 demonstrates these waters would be favorably displaced by a hydrophobic interface (the p24 antigen), as doing so releases ∼6.6 kcal/mol –TΔS, indicating that this nanobody-antigen PPI is entropically driven for 59H10. (b) Shows the hydration sites (spheres) for waters surrounding the phenylalanine (in 59H10, blue) for those ΔG > |2| kcal/mol (colored in relative; red is unfavorable, green is favorable). To see this figure in color, go online.
Figure 5
Figure 5
WaterMaps of apo nanobody (no antigen bound) full view all. Comparing the WaterMaps of (a) 59H10 (in blue) (b) 48G11 (gray), and (c) 37E7 (purple), for those hydration sites ΔG > |2| kcal/mol, with directional depiction of the waters. In yellow are shown the hydrogen bonds between those waters at the hydration sites over the trajectory analysis of the simulation. Inset are zooms to the F93D site. Note the “ice-like” ring around F93 of 59H10. To see this figure in color, go online.
Figure 6
Figure 6
WaterMaps of apo and holo nanobodies (59H10 and 37E7). (a) Apo 59H10 and 37E7 (no antigen bound). i) Comparison of hydration sites of magnitude ΔG > |2| kcal/mol and 6 Å from the nanobody chain B for 59H10 (F55-D56-P57) in spheres and 37E7 (G55-Y56-A57) in pyramids (colored in relative, red: unfavorable, green: favorable), highlighting site 1 (the bigger sphere with no corresponding pyramid). The two nanobodies structures are overlaid and show the binding interface at complexation (59H10, light blue; 37E7, purple; p24, dark blue). For 59H10, the circled hydration sites are unstable and would be displaced by the binding of the p24 (sites 1 and 2) that arise in 59H10 but not 37E7 (there are no equivalent hydration sites as there are at other points in between the nanobody-antigen interface), and this approximates the difference in binding affinity. Releasing hydration site 1 (–TΔS = 1.27 kcal/mol) and 2 (–TΔS = 1.4 kcal/mol) upon complexation would expend approximately 2.67 kcal/mol –TdS, (Table S1), indicating the difference in this entropically driven interaction. ii) An alternative view, which shows site 1 in 59H10 would be gated by the hydrophobic F55 nearby with the unstable water displaced upon antigen binding, and this hydrophobic area protected by F55 sealing the interface. This F55 is replaced by G55 for 37E7. (b) Holo 59H10 and 37E7 (antigen bound). i) Comparison of water distributions around the binding site when the nanobodies 59H10 (light blue) and 37E7 (purple) are bound to p24 (dark blue). Shown are hydration sites of magnitude ΔG > |2| kcal/mol and 6 Å from the nanobody chain B for 59H10 (F55-D56-P57) in spheres and 37E7 (G55-Y56-A57) in pyramids (colored in relative, red: unfavorable, green: favorable), highlighting in particular site 1’. Site 1’ (circled) in the 37E7-p24 complex resides where the F55 in 59H10 is situated. ii) Depiction of the nanobody-p24 complex; note that the PPI interface is largely free of water with the main differences in hydration sites seen around the variable areas at the edge of the interface. iii) Depiction of F55 from a different viewpoint to permit comparison of the free energy density map for 59H10 F55-D56-P57 (solid) versus 37E7 G55-Y56-A57 (mesh). To see this figure in color, go online.
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