In the Lerch lab, we explore the molecular mechanisms of membrane protein function, particularly with respect to the functional role of protein conformational heterogeneity. Proteins are inherently flexible structures and exhibit functional dynamic modes across a wide range of timescales, from picoseconds to milliseconds, and experimental techniques that can characterize the ensemble of conformations and identify their functional role are required for a detailed description of the molecular mechanisms of protein function. Site-directed spin-labeling (SDSL) in combination with electron paramagnetic resonance (EPR) is uniquely suited to this task, particularly in large membrane-bound proteins and protein complexes. The wide array of EPR technologies that are available span the entire picosecond to millisecond timescale in their ability to characterize protein motion. EPR also provides detailed structural information about proteins, from helical rocking modes and loop fluctuations to large-scale tertiary rearrangements and protein-protein interactions. Much of my work has focused on developing instrumentation and methodologies that enable SDSL-EPR spectroscopy to be performed on proteins at high hydrostatic pressure, which populates rare conformational states to allow characterization of their structure and dynamics and definition of their functional roles. Currently, these and other methodologies are used in the Lerch lab to explore the mechanisms of signal transduction in G-protein-coupled receptors (GPCRs), although the principles derived from these studies are expected to be applicable to membrane proteins outside the GPCR family.
Endogenous modulation of beta2-adrenergic receptor signaling
GPCRs are a large and diverse class of cell surface receptors responsible for regulating nearly every physiological process in the human body and are therefore important targets for drug development. Upon binding to the extracellular surface of GPCRs, ligands trigger conformational changes that lead to binding and activating signaling proteins at the cytoplasmic surface of the receptor. Many GPCRs signal through several pathways that include different heterotrimeric G protein subtypes as well as arrestins, and “biased” ligands differentially modulate receptor signaling through these various pathways. GPCRs rely on a high degree of conformational flexibility to achieve this signaling complexity, and one of the major goals in the field of structure-based drug design is to identify the conformations that generate a particular signaling profile. Significant progress toward this goal has been made, yet relatively little is known about the effects of endogenous modulators including protein-lipid interactions, post-translational modifications, and accessory proteins on GPCR structure and dynamics. To achieve the goals of rational drug design, the effect of endogenous modulators on the conformational landscape and the interplay of endogenous modulators with potential drugs need to be characterized, and this is a major long-term goal of this lab.
Current investigations include the molecular basis for modulation of beta2-adrenergic receptor signaling by two post-translational modifications: glycosylation and palmitoylation. We use a complementary combination of continuous-wave and pulsed EPR techniques and functional assays to reveal the molecular mechanisms by which these endogenous modulators regulate receptor activity. Additionally, in collaboration with researchers from across the country, we characterize other aspects of signal transduction through GPCRs, and in particular the effects of biased and allosteric ligands on GPCR structure and dynamics. In a recent study of ligand-mediated signaling, we revealed the structural basis for agonism of the beta2-adrenergic receptor using the pulsed EPR technique double electron-electron resonance, which is the only technique capable of defining the amplitude of structural changes and the full structural heterogeneity in a complex conformational ensemble for large membrane proteins such as GPCRs.
Development and application of high-pressure EPR technology
The application of high pressure to study biomedically relevant proteins and enzymes by EPR spectroscopy is emerging as an unparalleled approach to characterizing rare conformational states. Functionally relevant conformational states may have relative energies of only a few kilocalories per mole (Mittag, T. et al. J Molecular Recognition 2010, 23 (2), 105-116; Baldwin, A. J. et al. Nature Chemical Biology 2009, 5 (11), 808-814), yet this results in equilibrium populations too low to be detected by most spectroscopic methods. Pressurization reversibly increases the population of these low-lying excited states, but also reveals regions of elevated compressibility, and thus flexibility, within individual conformational states (Akasaka, K., Chemical Reviews 2006, 106 (5), 1814-1835; Kalbitzer, H., In High Pressure Bioscience, Akasaka, K.; Matsuki, H., Eds. Springer Netherlands: 2015, 72, 179-197). Cutting edge high-pressure EPR technology, much of which I developed, provides unique mechanistic insights by mapping sparsely populated regions of the conformational landscape. Early applications of these techniques demonstrated their ability to identify compressible (flexible) regions of a protein, populate and characterize excited conformational states of a protein undetected at atmospheric pressure, and reveal structural heterogeneity within the conformational ensemble. Technology development and application is a major component of my research, and we will continue to develop methods and instrumentation to expand the suite of EPR techniques that may be utilized to investigate proteins under pressure. Our current work on GPCRs is among the first comprehensive applications of high-pressure EPR technologies in proteins.
Anjana Adhikari, Research Technologist I
Patrick Brennan, Graduate Student
Alex Garces, Graduate Student
(Lerch MT, Matt RA, Masureel M, Elgeti M, Kumar KK, Hilger D, Foys B, Kobilka BK, Hubbell WL.) Proc Natl Acad Sci U S A. 2020 12 15;117(50):31824-31831 PMID: 33257561 PMCID: PMC7749303 12/02/2020
(Hilger D, Kumar KK, Hu H, Pedersen MF, O'Brien ES, Giehm L, Jennings C, Eskici G, Inoue A, Lerch M, Mathiesen JM, Skiniotis G, Kobilka BK.) Science. 2020 07 31;369(6503) PMID: 32732395 PMCID: PMC7954662 08/01/2020
(Wingler LM, Elgeti M, Hilger D, Latorraca NR, Lerch MT, Staus DP, Dror RO, Kobilka BK, Hubbell WL, Lefkowitz RJ.) Cell. 2019 01 24;176(3):468-478.e11 PMID: 30639099 PMCID: PMC6475118 01/15/2019
(Stadtmueller BM, Bridges MD, Dam KM, Lerch MT, Huey-Tubman KE, Hubbell WL, Bjorkman PJ.) Immunity. 2018 08 21;49(2):235-246.e4 PMID: 30076100 PMCID: PMC6104740 08/05/2018
(Bergdoll LA, Lerch MT, Patrick JW, Belardo K, Altenbach C, Bisignano P, Laganowsky A, Grabe M, Hubbell WL, Abramson J.) Proc Natl Acad Sci U S A. 2018 01 09;115(2):E172-E179 PMID: 29279396 PMCID: PMC5777057 12/28/2017
(Dror RO, Mildorf TJ, Hilger D, Manglik A, Borhani DW, Arlow DH, Philippsen A, Villanueva N, Yang Z, Lerch MT, Hubbell WL, Kobilka BK, Sunahara RK, Shaw DE.) Science. 2015 Jun 19;348(6241):1361-5 PMID: 26089515 PMCID: PMC4968074 06/20/2015
(Manglik A, Kim TH, Masureel M, Altenbach C, Yang Z, Hilger D, Lerch MT, Kobilka TS, Thian FS, Hubbell WL, Prosser RS, Kobilka BK.) Cell. 2015 May 21;161(5):1101-1111 PMID: 25981665 PMCID: PMC4441853 05/20/2015
(Lerch MT, López CJ, Yang Z, Kreitman MJ, Horwitz J, Hubbell WL.) Proc Natl Acad Sci U S A. 2015 May 12;112(19):E2437-46 PMID: 25918400 PMCID: PMC4434698 04/29/2015
(Lerch MT, Yang Z, Altenbach C, Hubbell WL.) Methods Enzymol. 2015;564:29-57 PMID: 26477247 10/20/2015
(Yang Z, Bridges M, Lerch MT, Altenbach C, Hubbell WL.) Methods Enzymol. 2015;564:3-27 PMID: 26477246 10/20/2015
(Lerch MT, Yang Z, Brooks EK, Hubbell WL.) Proc Natl Acad Sci U S A. 2014 Apr 01;111(13):E1201-10 PMID: 24707053 PMCID: PMC3977274 04/08/2014
(Lerch MT, Horwitz J, McCoy J, Hubbell WL.) Proc Natl Acad Sci U S A. 2013 Dec 03;110(49):E4714-22 PMID: 24248390 PMCID: PMC3856799 11/20/2013