Biology subjects

Loughlin, B. J.

Publications and source records attributed to Loughlin, B. J..

3 recordsLinked to original sources

bioRxiv2025

Nanobody-Driven Stabilization Synergistically Rescues F508del-CFTR and Reveals an Alternative Active State of the Channel

Defects in protein trafficking underlie many genetic diseases, including cystic fibrosis (CF), where the predominant F508del mutation destabilizes the cystic fibrosis transmembrane conductance regulator (CFTR) channel, leading to its degradation. To provide a protein-specific chaperone, we used lipid nanoparticles to deliver mRNA encoding T2a, a nanobody that thermally stabilizes CFTR via high-affinity binding to nucleotide-binding domain 1 (NBD1). When combined with clinically-approved correctors, T2a considerably improved F508del-CFTR maturation, plasma membrane expression, and channel activity. Single-channel recordings revealed that nanobody binding sustained channel activity by promoting both full open and sub-conductance gating states and protecting F508del-CFTR against thermal deactivation. Because T2a binding to NBD1 prevents the ATP-dependent NBD1-NBD2 association that drives canonical channel opening, the observation of channel activity in the presence of the nanobody suggested adoption of a novel open-channel CFTR structure. This inference was confirmed by cryo-EM analyses of CFTR in the presence of T2a, which revealed a novel open-channel conformation in which NBD1 binds to an alternative site on CFTR incompatible with NBD dimerization. Our findings establish a new paradigm to correct protein trafficking by stabilizing misfolded domains with targeted nanobodies and demonstrate a broadly applicable framework to treat CF and related protein misfolding diseases.

biochemistry↗

bioRxiv2023

Systematic enhancement of protein crystallization efficiency by bulk lysine-to-arginine (KR) substitution

Structural genomics consortia established that protein crystallization is the primary obstacle to structure determination using x-ray crystallography. We previously demonstrated that crystallization propensity is systematically related to primary sequence, and we subsequently performed computational analyses showing that arginine is the most overrepresented amino acid in crystal-packing interfaces in the Protein Data Bank. Given the similar physicochemical characteristics of arginine and lysine, we hypothesized that multiple lysine-to-arginine (KR) substitutions should improve crystallization. To test this hypothesis, we developed software that ranks lysine sites in a target protein based on the redundancy-corrected KR substitution frequency in homologs. We demonstrate that three unrelated single-domain proteins can tolerate 5-11 KR substitutions with at most minor destabilization and that these substitutions consistently enhance crystallization propensity. This approach rapidly produced a 1.9 [A] crystal structure of a human protein domain refractory to crystallization with its native sequence. Structures from bulk-KR-substituted domains show the engineered arginine residues frequently make high-quality hydrogen-bonds across crystal-packing interfaces. We thus demonstrate that bulk KR substitution represents a rational and efficient method for probabilistic engineering of protein surface properties to improve protein crystallization.

biophysics↗

bioRxiv2022

Mechanism of dual pharmacological correction and potentiation of human CFTR

Cystic fibrosis (CF) is caused by mutations in a chloride channel called the human Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR). We used cryo-EM global conformational ensemble reconstruction to characterize the mechanism by which the breakthrough drug VX445 (Elexacaftor) simultaneously corrects both protein-folding and channel-gating defects caused by CF mutations. VX445 drives hCFTR molecules harboring the gating-defective G551D mutation towards the open-channel conformation by binding to a site in the first transmembrane domain. This binding interaction reverses the usual pathway of allosteric structural communication by which ATP binding activates channel conductance, which is blocked by the G551D mutation. Our ensemble reconstructions include a 3.4 [A] non-native structure demonstrating that detachment of the first nucleotide-binding domain of hCFTR is directly coupled to local unfolding of the VX445 binding site. Reversal of this unfolding transition likely contributes to its corrector activity by cooperatively stabilizing NBD1 and the transmembrane domains of hCFTR during biogenesis. SummaryCryo-EM global conformational ensemble reconstruction has been used to characterize the mechanism-of-action of a breakthrough pharmaceutical that corrects fatal protein-folding and channel-gating defects in the human cystic fibrosis transmembrane conductance regulator (CFTR).

biophysics↗