ATP-dependent thermoring basis for the heat unfolding of the first nucleotide-binding domain isolated from human CFTR

Abstract

Traditionally, the thermostability of a protein is defined by a melting temperature, at which half of the protein is unfolded. However, this definition cannot indicate the structural origin of a heat-induced unfolding pathway. Here, the thermoring structures were studied on the ATP-dependent heat-induced unfolding of the first nucleotide-binding domain from the human cystic fibrosis transmembrane conductance regulator. The results showed that initial theoretical and experimental melting thresholds aligned well after three structural perturbations including the F508del mutation, the most common cause of cystic fibrosis. This alignment further demonstrated that the heat-induced unfolding process began with the disruption of the least-stable noncovalent interaction within the biggest thermoring along the single peptide chain. The C-terminal region, which was related to the least-stable noncovalent interaction and the ATP-dependent dimerization of two nucleotide-binding domains, emerged as a crucial determinant of the thermal stability of the isolated protein and a potential interfacial drug target to alleviate the thermal defect caused by the F508del mutation. This groundbreaking discovery significantly advances our understanding of protein activity, thermal stability, and molecular pathology.

Keywords: cooperative misfolding; grid thermodynamic signature; least-stable interaction; melting threshold; noncovalent structure; partial unfolding.

Figure 1

The thermoring structures of hNBD1 with F508 in the Mg/ATP bound state at 7 °C. (a) The grid-like noncovalently interacting mesh network based on the X-ray structure of an isolated hNBD1 with F508 in a Mg/ATP bound state at 7 °C (PDB ID, 2BBO, 2.55 Å). Salt bridges, H-bonds and p interactions are colored purple, orange, and green, respectively. The constrained grid sizes required to control the least-stable noncovalent interactions in the grids are labeled with black numbers. The least-stable S605-R658 H-bond in the biggest Grid₁₆ is highlighted. The total grid sizes and the total grid size-controlled noncovalent interactions along the single peptide chain from T389 to P676 are shown in cyan and black circles, respectively. (b) The structure of the biggest Grid₁₆ with a 16-residue size at the RE/NBD1 interface to control the least-stable S605-R658 H-bond. The grid size and the equivalent basic H-bonds for the least-stable noncovalent interaction are shown in and near a red circle. (c) The sequence of the biggest Grid₁₆ to control the least-stable S605-R658 H-bond in the blue box.

Figure 2

The thermoring structures of hNBD1 without F508 in the Mg/ATP bound state at 4 °C. (a) The grid-like noncovalently interacting mesh network based on the X-ray structure of an isolated NBD1 without F508 in a Mg/ATP bound state at 4 °C (PDB ID, 1XMJ, 2.3 Å). Salt bridges, H-bonds and p interactions are colored purple, orange, and green, respectively. The constrained grid sizes required to control the least-stable noncovalent interactions in the grids are labeled with black numbers. The least-stable S605-R658 H-bond in the biggest Grid₂₀ is highlighted. The total grid sizes and the total grid size-controlled noncovalent interactions along the single peptide chain from E391 to A675 are shown in cyan and black circles, respectively. (b) The structure of the biggest Grid₂₀ with a 20-residue size at the RE/NBD1 interface to control the least-stable S605-R658 H-bond. The grid size and the equivalent basic H-bonds for the least-stable noncovalent interaction are shown in and near a red circle. (c) The sequence of the biggest Grid₂₀ to control the least-stable S605-R658 H-bond in the blue box.

Figure 3

The thermoring structures of hNBD1-D(RE,RI) with F508 in the Mg/ATP bound state at 8 °C. (a) The grid-like noncovalently interacting mesh network based on the X-ray structure of an isolated NBD1-D(RE,RI) with F508 in a Mg/ATP bound state at 8 °C (PDB ID, 2PZE, 1.7 Å). Salt bridges, H-bonds and p interactions are colored purple, orange, and green, respectively. The constrained grid sizes required to control the least-stable noncovalent interactions in the grids are labeled with black numbers. The least-stable S459-H620 H-bond and Y627-F446 p interaction in the biggest Grid₁₃ are highlighted. The total grid sizes and the total grid size-controlled noncovalent interactions along the single peptide chain from S386 to M645 are shown in cyan and black circles, respectively. (b) The structure of the biggest Grid₁₃ with a 13-residue size at the interface between N- and C- termini to control the least-stable S459-H620 H-bond and Y627-F446 p interaction. The grid size and the equivalent basic H-bonds for the least-stable noncovalent interactions are shown in and near a red circle. (c) The sequence of the biggest Grid₁₃ to control the least-stable S459-H620 H-bond and Y627-F446 p interaction in the blue box.

Figure 4

The thermoring structures of hNBD1-D(RE,RI) without F508 in the Mg/ATP bound state at 8 °C. (a) The grid-like noncovalently interacting mesh network based on the X-ray structure of an isolated NBD1-D(RE,RI) without F508 in a Mg/ATP bound state at 8 °C (PDB ID, 2PZF, 2.0 Å). Salt bridges, H-bonds and p interactions are colored purple, orange, and green, respectively. The constrained grid sizes required to control the least-stable noncovalent interactions in the grids are labeled with black numbers. The least-stable Y627-F446 p interaction in the biggest Grid₁₇ is highlighted. The total grid sizes and the total grid size-controlled noncovalent interactions along the single peptide chain from S386 to G646 are shown in cyan and black circles, respectively. (b) The structure of the biggest Grid₁₇ with a 17-residue size at the interface between N and C termini to control the least-stable Y627-F446 p interaction. The grid size and the equivalent basic H-bonds for the least-stable noncovalent interaction are shown in and near a red circle. (c) The sequence of the biggest Grid₁₇ to control the least-stable Y627-F446 p interaction in the blue box.

Figure 5

The thermoring structures of hNBD1–3S without F508 in the Mg/ATP bound state at 7 °C. (a) The grid-like noncovalently interacting mesh network based on the X-ray structure of an isolated hNBD1-F429S/F494N/Q637R without F508 in a Mg/ATP bound state at 7 °C (PDB ID, 2BBS, 2.05 Å). Salt bridges, H-bonds and p interactions are colored in purple, orange, and green, respectively. The constrained grid sizes required to control the least-stable noncovalent interactions in the grids are labeled with black numbers. The least-stable S388-D567 H-bind in the biggest Grid₁₆, is highlighted. The total grid sizes and the total grid size-controlled noncovalent interactions along the single peptide chain from S388 to L671 are shown in cyan and black circles, respectively. (b) The structure of the biggest Grid₁₆, with a 16-residue size to control the least-stable S388-D567 H-bind. The grid size and the equivalent basic H-bonds for the least-stable noncovalent interaction are shown in and near a red circle. (c) The sequence of the biggest Grid₁₆, to control the the least-stable S388-D567 H-bind in the blue box.

Figure 6

Effects of DRE and 3S mutations on the biggest thermorings in hNBD1 with or without F508. The x-ray structures of hNBD1 with (2BBO) and without F508 (1XMJ), hNBD1- D(RE, RI) with (2ZPE) and without F508 (2PZF), and hNBD1–3S without F508 (2BBS) are used for the models. RE is circled in blue and the biggest thermorings are shown in red. The residues responsible for the least-stable noncovalent interactions in the biggest thermorings are shown in space fills.

Figure 7

Smaller highly conserved thermorings in hNBD1 upon structural perturbations. The X-ray structure of hNBD1-F508del with the 3S mutations (2BBS) was chosen for the models. The thermoring sizes are circled in red.