MSACL 2024 Abstract

Self-Classified Topic Area(s): Proteomics > Proteomics

Podium Presentation in Steinbeck 3 on Thursday at 16:00 (Chair: Timothy Collier / David Colquhoun)

Discovery stage. How a Single Mutation in CFTR Causes the Systemic Disease Cystic Fibrosis: Interactions, PTMs, and Structure
John R. Yates III1, Pankow, Casimir Bamberger, Salvador Martínez-Bartolomé1, Sung-Kyu Park,
Department of Molecular Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA

John Yates, PhD (Presenter)
Scripps Research Institute

Presenter Bio: John R. Yates is the Ernest W. Hahn Professor in the Departments of Molecular Medicine and Neurobiology at The Scripps Research Institute. His research interests include development of integrated methods for tandem mass spectrometry analysis of protein mixtures, bioinformatics using mass spectrometry data, and biological studies involving proteomics. He is the lead inventor of the SEQUEST software for correlating tandem mass spectrometry data to sequences in the database and developer of the shotgun proteomics technique for the analysis of protein mixtures. His laboratory has developed the use of proteomic techniques to analyze protein complexes, posttranslational modifications, organelles and quantitative analysis of protein expression for the discovery of new biology. Many proteomic approaches developed by Yates have become a national and international resource to many investigators in the scientific community. He has received the American Society for Mass Spectrometry research award, the Pehr Edman Award in Protein Chemistry, the American Society for Mass Spectrometry Biemann Medal, the HUPO Distinguished Achievement Award in Proteomics, Herbert Sober Award from the ASBMB, and the Christian Anfinsen Award from The Protein Society, the 2015 ACS’s Analytical Chemistry award, 2015 The Ralph N. Adams Award in Bioanalytical Chemistry, the 2018 Thomson Medal from the International Mass Spectrometry Society, and the 2019 John B. Fenn Distinguished Contribution to Mass Spectrometry award from the ASMS. He was ranked by Citation Impact, Science Watch as one of the Top 100 Chemists for the decade, 2000-2010. He was #1 on a List of Most Influential in Analytical Chemistry compiled by The Analytical Scientist 10/30/2013 and is on the List Of Most Highly Influential Biomedical Researchers, 1996-2011, European J. Clinical Investigation 2013, 43, 1339-1365 and the Thomson Reuters 2015 List of Highly Cited Scientists. He has published over 950 scientific articles with >125,000 citations, and an H index of 174 (Google Scholar). Dr. Yates served as an Associate Editor at Analytical Chemistry for 15 years and is currently the Editor in Chief at the Journal of Proteome Research.

Abstract

Protein conformation is dynamic as it is influenced by post-translational modifications (PTMs) and interactions with other proteins, small molecules or RNA, for example. However, in vivo characterization of protein structures and protein structural changes after perturbation is a major challenge. Therefore, experiments to characterize protein structures are typically performed in vitro and with highly purified proteins or protein complexes, revealing a static picture of the protein. To identify the true conformational space occupied by proteins in vivo, we developed a novel low-resolution method named Covalent Protein Painting (CPP) that allows the characterization of protein conformations in vivo. Here, we report how an ion channel, the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), is conformationally changed during biogenesis and channel opening in the cell. Our study led to the identification of a novel opening mechanism for CFTR by revealing that the interaction of the intracellular loop 2 (ICL2) with the nucleotide binding domain 2 (NDB2) of CFTR is needed for channel gating, and this interaction occurs concomitantly with changes to the narrow part of the pore and the walker A lysine in NBD1 for wt CFTR. However, the ICL2:NBD2 interface, which forms a “ball-in-a-socket” motif, is uncoupled during biogenesis, likely to prevent inadvertent channel activation during transport. Mutation of K273 in the ICL2 loop severely impaired CFTR biogenesis and led to accumulation of CFTR in the Golgi and TGN. CPP further revealed that, even upon treatment with current approved drugs such as Trikafta or at permissive temperature, the uncoupled state of ICL2 is a prominent feature of the misfolded CFTR mutants ∆F508 and N1303K that cause Cystic Fibrosis (CF). Although Trikafta treatment reduced the amount of uncoupled ICL2:NBD2 interfaces, more than 75% of F508 CFTR remained in the uncoupled state, suggesting that stabilization of this interface could produce a more efficient CF drug. CPP can characterize a protein in its native environment and measure the effect of complex PTMs and protein interactions on protein structure, making it broadly applicable and valuable for the development of new therapies.

Financial Disclosure

Description	Y/N	Source
Grants	no
Salary	yes	Cambridge Isotope Laboratories, 908 Devices, Chemical Abstract Services, American Chemical Society
Board Member	yes	US HUPO President, Partnership for Clean Competition
Stock	no	Yatiri Biotherapeutics, Integrated Proteomic Applications
Expenses	no
IP Royalty	yes	University of Washington, Bruker

Planning to mention or discuss specific products or technology of the company(ies) listed above:
no