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

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.

Background:

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. Proteins generally unfold above the basal temperature of the organism in which they evolved. In some missense mutations, unfolding transitions induce protein denaturation in tertiary or secondary structure and/or toxic aggregation, leading to extensive, serious and prevalent human diseases. However, the origin of the heat unfolding pathway is still elusive because different biophysical measurements arrive at distinct conclusions. A good example is the most common cystic fibrosis-causing mutation F508del in the first nucleotide binding domain (hNBD1) from the human cystic fibrosis transmembrane conductance regulator (hCFTR) at the apical cell surface of epithelia. CFTR is a multi-domain membrane protein, consisting of transmembrane domain 1 (TMD1) and 2 (TMD2), nucleotide binding domains 1 (NBD1) and 2 (NBD2) and a unique regulatory (R) domain between NBD1 and TMD2. Despite belonging to the family of ATP-binding cassette (ABC) transporters, it functions as an ATP-gated anion channel. When the regulatory insert (RI) (405–436) and extension (RE) (647–678) are deleted to promote the formation of a head-to-tail homodimer and the biogenesis of F508del-CFTR in tissue culture cells, hNBD1-Δ(RI,RE) (387–646(Δ405–436)) exhibits various melting thresholds in the presence of 0.125 mM ATP and 10% glycerol. Although circular dichroism (CD) at 230 nm indicates a significant melting temperature threshold (Tm) of 51°C, CD at 297 nm and the Trp fluorescence signal reveal it as 45°C, which is consistent with the differential scanning calorimetry (DSC) unfolding curve. Similarly, when F508 is also deleted, (F508del)hNBD1-Δ(RI,RE) has a Tm of 46°C for far-UV CD but a Tm of 40°C for near-UV CD, DSC and intrinsic Trp fluorescence assays. These results demonstrate that heat unfolding of hNBD1-Δ(RI,RE) with or without F508 actually starts with a partial change in tertiary structure, leading to an ATP-free aggregation-prone “molten globule” intermediate in the presence of physiological concentrations of ATP.

Methods:

Recently, a graph theory approach has been developed to introduce a novel concept, model, direction, and feasibility for understanding the temperature sensitivity of biomacromolecules. By mapping networks of temperature-dependent noncovalent interactions such as H-bonds, salt bridges and π interactions from high-resolution 3D structures, well-organized fluidic-like grids of various sizes can be comprehensively constrained and defined as thermo-sensitive rings or thermorings. In this study, the thermoring structures of hNBD1 with Mg/ATP bound were analyzed in response to various site-mutation perturbations. The X-ray structure (PDB, 2BBO) reveals that the regulatory insertion (RI) (F405-L435) is disordered. With Mg/ATP bound to hNBD1, Mg2+ connected T465, Q493 and D572 together, creating the smallest thermoring with a zero-residue size. Meanwhile, ATP linked W401, K464, S466 and L475 together. Even without other hCFTR domains, F508 still formed a strong core grid mesh network through W496-F508, Y507-Y563-R516 and Y517-Y512 π bridges, as well as the R516-N505 and Y512-K503 H-bonds.

Results:

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. Identification of the biggest thermoring with a corresponding melting threshold in hNBD1 revealed that, with a total of 52 noncovalent interactions and 100 grid sizes, the calculated systematic thermal instability matched the initial experimental melting thresholds. 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. Although the deletion of F508 decreased the calculated melting temperature threshold (Tm) of hNBD1, the subsequent deletion of (RE, RI) or three second-site suppressor mutations (3S, F429S/F494N/Q637R) increased it.

Data Summary:

Quantitative results include the melting temperature thresholds: for hNBD1-Δ(RI,RE), far-UV CD Tm of 51°C, near-UV CD and Trp fluorescence Tm of 45°C; for (F508del)hNBD1-Δ(RI,RE), far-UV CD Tm of 46°C, near-UV CD, DSC and intrinsic Trp fluorescence Tm of 40°C. In the thermoring analysis, the biggest thermoring in hNBD1 comprised a total of 52 noncovalent interactions and 100 grid sizes. The smallest thermoring had a zero-residue size, formed by Mg2+ connecting T465, Q493 and D572.

Conclusions:

This groundbreaking discovery significantly advances our understanding of protein activity, thermal stability, and molecular pathology. 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, 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.

Practical Significance:

The C-terminal region of hNBD1, before the 3S mutations, is a crucial determinant of the thermal stability of hNBD1 and a potential drug target to rescue the interfacial thermal defect of the F508del mutant, offering a real-world application for alleviating the thermal defect caused by the most common cystic fibrosis-causing mutation.