The structural basis for molecular recognition
Disparate proteins form transient complexes across the membrane during signal propagation, yet these complexes likely share common mechanisms of signaling mediated by changes in conformation, electrostatics, or dynamics. While challenging, the determination of the architecture of these complexes is required for understanding how biological signals are transmitted across a membrane. The research in the Iverson laboratory uses several model systems to investigate these transmembrane signaling processes, and is concurrently working to develop methods for membrane protein crystallography.
Mechanism of G protein signaling
GPCRs bind a diverse set of ligands to elicit cellular responses and represent >50% of pharmacological therapeutic targets. A major challenge in the determination of GPCR structures is that these receptors sample the activated and inactivated states in the absence of cognate ligand. This conformational heterogeneity results in basal activity observed in GPCRs. We are developing methods for the expression and stabilization of GPCRs for structure determination.
The rate-determining step of GPCR signaling is nucleotide exchange of GDP for GTP in the Gα subunit of the G protein. Physiologically, a complex between GDP-bound Gαβγ heterotrimer and activated GPCR catalyzes this exchange. In collaboration with Prof. Heidi Hamm (Vanderbilt) we have developed methods for the stabilization of G proteins that represent this activated state as well as stabilization of the GPCR-Gαβγ complex itself. Our initial finding suggests that helix dipole movement in the Gα subunit of the G protein contributes to the release of GDP during receptor-catalyzed nucleotide exchange.
|The Gαβγ heterotrimer shown in a view highlighting the receptor binding site. The α5 helix of the Gα subunit may contribute to receptor catalyzed nucleotide exchange.|
The quinol:fumarate reductase respiratory complex – a bioenergetic enzyme and a signaling scaffold
The structure of the E. coli complex II homolog quinol:fumarate reductase (QFR) was the 11th published membrane protein structure. With our long-term collaborator Prof. Gary Cecchini (UCSF VA Medical Center), this research continues to investigate the structure-activity relationships in the QFR enzyme and how this bioenergetic protein works as a signaling scaffold to affect flagellar switching.
|Structure of the E. coli QFR.Each of the 4 subunit of the heterotetramer is highlighted with a different color. We are investigating the importance of movement of the capping domain (circled), and three catalytically-important residues (magenta). Electron transfer cofactors and intercofactor distances are labeled to the left.|
Recognition of pathogens by human hosts
Recognition of pathogens by toll-like receptors (TLRs) is the first step in the activation of the inflammatory responses of innate immunity. TLRs bind to components of pathogens, but do not undergo affinity maturation. TLR1 and TLR2 may be involved in the recognition of outer membrane proteins (OMPs). The basis for this recognition is difficult to envision since OMPs vary in sequence, structure, diameter, and conductance. Importantly, mis-activation of innate immunity underlies a variety of autoimmune diseases and can contribute to cardiovascular disease. Understanding how TLRs recognize pathogens may shed light on these disease processes.
As a model system, we are using the interaction between the TLR1/2 heterodimer and the OMP PorB from Neisseria meningitidis. We have recently determined the de novo x-ray crystal structure of the N. meningitidis PorB; the structure of the chimeric TLR1/TLR2 heterodimer has been reported in the literature. From analysis of these structures, we speculate that electrostatics contribute to complex formation.
|Structure of N. meningitidis PorB. a) PorB trimer viewed from the top of the membrane b) PorB monomer viewed through the membrane normal. c) Model for the binding mode between TLR ectodomains and outer membrane porins.|
Gating and selectivity in ion channels
The electrophysiology of PorB suggests that it allows promiscuous solute translocation through a large pore; however the crystal structure suggests that PorB and other related OMPs may instead have multiple transloation pathways with differing selectivities. In contrast to PorB and OMP channels, most ion channels are exquisitely selective for their permeant ion. The molecular basis for this selectivity is best understood in potassium channels. Using the Streptomyces lividans minimalistic potassium channel KcsA as a model system, we have proposed how these K+ channels prevent conduction of Na+ and Li+ through the pore.
|Location of Li+ binding site inferred from water molecules in the selectivity filter. Composite omit electron density maps are contoured at 1.25σ.|
Structure determination of membrane proteins by any method is still challenging. We have worked to improve the methods for stabilization and crystallization of these proteins. Our efforts have resulted in the development of a screen for b-barrel membrane proteins that is currently marketed by Emerald Biosystems. We are further working to identify alternative media for the solubilization of membrane proteins.