Ching-Ju Tsai

Backbone NMR reveals allosteric signal transduction networks in the β1-adrenergic receptor

G protein-coupled receptors (GPCRs) are physiologically important transmembrane signalling proteins that trigger intracellular responses upon binding of extracellular ligands. Despite recent breakthroughs in GPCR crystallography, the details of ligand-induced signal transduction are not well understood owing to missing dynamical information. In principle, such information can be provided by NMR, but so far only limited data of functional relevance on few side-chain sites of eukaryotic GPCRs have been obtained. Here we show that receptor motions can be followed at virtually any backbone site in a thermostabilized mutant of the turkey β1-adrenergic receptor (β1AR). Labelling with [15N]valine in a eukaryotic expression system provides over twenty resolved resonances that report on structure and dynamics in six ligand complexes and the apo form. The response to the various ligands is heterogeneous in the vicinity of the binding pocket, but gets transformed into a homogeneous readout at the intracellular side of helix 5 (TM5), which correlates linearly with ligand efficacy for the G protein pathway. The effect of several pertinent, thermostabilizing point mutations was assessed by reverting them to the native sequence. Whereas the response to ligands remains largely unchanged, binding of the G protein mimetic nanobody NB80 and G protein activation are only observed when two conserved tyrosines (Y227 and Y343) are restored. Binding of NB80 leads to very strong spectral changes throughout the receptor, including the extracellular ligand entrance pocket. This indicates that even the fully thermostabilized receptor undergoes activating motions in TM5, but that the fully active state is only reached in presence of Y227 and Y343 by stabilization with a G protein-like partner. The combined analysis of chemical shift changes from the point mutations and ligand responses identifies crucial connections in the allosteric activation pathway, and presents a general experimental method to delineate signal transmission networks at high resolution in GPCRs.

Femtosecond X-ray diffraction from two-dimensional protein crystals

Bragg diffraction achieved from two-dimensional protein crystals using femtosecond X-ray laser snapshots is presented.

Structural interactions between inhibitor and substrate docking sites give insight into mechanisms of human PS1 complexes.

Summary Presenilin-mediated endoproteolysis of transmembrane proteins plays a key role in physiological signaling and in the pathogenesis of Alzheimer disease and some cancers. Numerous inhibitors have been found via library screens, but their structural mechanisms remain unknown. We used several biophysical techniques to investigate the structure of human presenilin complexes and the effects of peptidomimetic γ-secretase inhibitors. The complexes are bilobed. The head contains nicastrin ectodomain. The membrane-embedded base has a central channel and a lateral cleft, which may represent the initial substrate docking site. Inhibitor binding induces widespread structural changes, including rotation of the head and closure of the lateral cleft. These changes block substrate access to the catalytic pocket and inhibit the enzyme. Intriguingly, peptide substrate docking has reciprocal effects on the inhibitor binding site. Similar reciprocal shifts may underlie the mechanisms of other inhibitors and of the “lateral gate” through which substrates access to the catalytic site.

Two Alternative Conformations of a Voltage-Gated Sodium Channel.

Activation and inactivation of voltage-gated sodium channels (Navs) are well studied, yet the molecular mechanisms governing channel gating in the membrane remain unknown. We present two conformations of a Nav from Caldalkalibacillus thermarum reconstituted into lipid bilayers in one crystal at 9Å resolution based on electron crystallography. Despite a voltage sensor arrangement identical with that in the activated form, we observed two distinct pore domain structures: a prominent form with a relatively open inner gate and a closed inner-gate conformation similar to the first prokaryotic Nav structure. Structural differences, together with mutational and electrophysiological analyses, indicated that widening of the inner gate was dependent on interactions among the S4-S5 linker, the N-terminal part of S5 and its adjoining part in S6, and on interhelical repulsion by a negatively charged C-terminal region subsequent to S6. Our findings suggest that these specific interactions result in two conformational structures.

7 Å resolution in protein two-dimensional-crystal X-ray diffraction at Linac Coherent Light Source

Membrane proteins arranged as two-dimensional crystals in the lipid environment provide close-to-physiological structural information, which is essential for understanding the molecular mechanisms of protein function. Previously, X-ray diffraction from individual two-dimensional crystals did not represent a suitable investigational tool because of radiation damage. The recent availability of ultrashort pulses from X-ray free-electron lasers (XFELs) has now provided a means to outrun the damage. Here, we report on measurements performed at the Linac Coherent Light Source XFEL on bacteriorhodopsin two-dimensional crystals mounted on a solid support and kept at room temperature. By merging data from about a dozen single crystal diffraction images, we unambiguously identified the diffraction peaks to a resolution of 7 Å, thus improving the observable resolution with respect to that achievable from a single pattern alone. This indicates that a larger dataset will allow for reliable quantification of peak intensities, and in turn a corresponding increase in the resolution. The presented results pave the way for further XFEL studies on two-dimensional crystals, which may include pump–probe experiments at subpicosecond time resolution.

Time-resolved structural studies with serial crystallography: A new light on retinal proteins

Structural information of the different conformational states of the two prototypical light-sensitive membrane proteins, bacteriorhodopsin and rhodopsin, has been obtained in the past by X-ray cryo-crystallography and cryo-electron microscopy. However, these methods do not allow for the structure determination of most intermediate conformations. Recently, the potential of X-Ray Free Electron Lasers (X-FELs) for tracking the dynamics of light-triggered processes by pump-probe serial femtosecond crystallography has been demonstrated using 3D-micron-sized crystals. In addition, X-FELs provide new opportunities for protein 2D-crystal diffraction, which would allow to observe the course of conformational changes of membrane proteins in a close-to-physiological lipid bilayer environment. Here, we describe the strategies towards structural dynamic studies of retinal proteins at room temperature, using injector or fixed-target based serial femtosecond crystallography at X-FELs. Thanks to recent progress especially in sample delivery methods, serial crystallography is now also feasible at synchrotron X-ray sources, thus expanding the possibilities for time-resolved structure determination.

Structure determination of secondary transport proteins by electron crystallography: two-dimensional crystallization of the betaine uptake system BetP.

Structure determination at high resolution is still a challenge for membrane proteins in general, but in particular for secondary transporters due to their highly dynamic nature. X-ray structures of ten secondary transporters have recently been determined, but a thorough understanding of transport mechanisms necessitates structures at different functional states. Electron cryo-microscopy of two-dimensional (2D) crystals offers an alternative to obtain structural information at intermediate resolution. Electron crystallography is a sophisticated way to study proteins in a natural membrane environment and to track conformational changes in situ. Furthermore, basic interactions between protein and lipids can be investigated. Projection and 3-dimensional maps of six secondary transporters from different families have been determined by electron crystallography of 2D crystals at a resolution of 8 Å and better. In this review, we give an overview about the principles of 2D crystallization, in particular of secondary transporters, and summarize the important steps successfully applied to establish and improve the 2D crystallization of the high-affinity glycine betaine uptake system from Corynebacterium glutamicum, BetP.

Coupling electron cryomicroscopy and X-ray crystallography to understand secondary active transport

In the past few years we have seen an amazing increase in the number of high-resolution structures for secondary transporters determined by X-ray crystallography, while 3D data obtained by electron cryomicroscopy (cryo-EM) from two-dimensional (2D) crystals are only available at medium resolutions of about 6-10A. Despite their superior resolution, it turned out that the description of a molecular mechanism of secondary transport could not solely rely on high-resolution X-ray structures and have to be supplemented with biochemical and spectroscopic data. Moreover, the comparison of X-ray structures and 3D EM maps has proved to be an important tool for validating native conformations of several membrane proteins, especially when functional data contradicted predictions based on a crystal structure. In addition, 3D EM maps are better suited to investigate transporter activation because of the lipidic environment.

Structure of β-adrenergic receptors.

β-Adrenergic receptors (βARs) control key physiological functions by transducing signals encoded in catecholamine hormones and neurotransmitters to activate intracellular signaling pathways. As members of the large family of G protein-coupled receptors (GPCRs), βARs have a seven-transmembrane helix topology and signal via G protein- and arrestin-dependent pathways. Until 2007, three-dimensional structural information of GPCRs activated by diffusible ligands, including βARs, was limited to homology models that used the related photoreceptor rhodopsin as a template. Over many years, several labs have developed strategies that have finally allowed the structures of the turkey β(1)AR and the human β(2)AR to be determined experimentally. The challenges to overcome included heterologous receptor overexpression, design of stabilized and crystallizable modified receptor constructs, ligand-affinity purification of active receptor and the development of novel techniques in crystallization and microcrystallography. The structures of βARs in complex with inverse agonists, antagonists, and agonists have revealed the binding mode of ligands with different efficacies, have allowed to obtain insights into ligand selectivity, and have provided better templates for drug design. Also, the structures of β(2)AR in complex with a G protein and a G protein-mimicking nanobody have provided important insights into the mechanism of receptor activation and G protein coupling. This chapter summarizes the strategies and methods that have been successfully applied to the structural studies of βARs. These are exemplified with detailed protocols toward the structure determination of stabilized turkey β(1)AR-ligand complexes. We also discuss the spectacular insights into adrenergic receptor function that were obtained from the structures.

Resolution extension by image summing in serial femtosecond crystallography of two-dimensional membrane-protein crystals

The resolution limit of serial diffraction from two-dimensional crystals at a free-electron laser was extended to the detector edge (4 Å) by exploiting the large redundancy of the data set.