Ultrafast isomerization of retinal is the primary step in photoresponsive biological functions including vision in humans and ion transport across bacterial membranes. We used an x-ray laser to study ...the subpicosecond structural dynamics of retinal isomerization in the light-driven proton pump bacteriorhodopsin. A series of structural snapshots with near-atomic spatial resolution and temporal resolution in the femtosecond regime show how the excited all-trans retinal samples conformational states within the protein binding pocket before passing through a twisted geometry and emerging in the 13-cis conformation. Our findings suggest ultrafast collective motions of aspartic acid residues and functional water molecules in the proximity of the retinal Schiff base as a key facet of this stereoselective and efficient photochemical reaction.
Directional transport of protons across an energy transducing membrane-proton pumping-is ubiquitous in biology. Bacteriorhodopsin (bR) is a light-driven proton pump that is activated by a buried all-
...trans
retinal chromophore being photoisomerized to a 13-
cis
conformation. The mechanism by which photoisomerization initiates directional proton transport against a proton concentration gradient has been studied by a myriad of biochemical, biophysical, and structural techniques. X-ray free electron lasers (XFELs) have created new opportunities to probe the structural dynamics of bR at room temperature on timescales from femtoseconds to milliseconds using time-resolved serial femtosecond crystallography (TR-SFX). Wereview these recent developments and highlight where XFEL studies reveal new details concerning the structural mechanism of retinal photoisomerization and proton pumping. We also discuss the extent to which these insights were anticipated by earlier intermediate trapping studies using synchrotron radiation. TR-SFX will open up the field for dynamical studies of other proteins that are not naturally light-sensitive.
Abstract
In addition to the serotonin 5-HT
2A
receptor (5-HT
2A
R), the dopamine D
2
receptor (D
2
R) is a key therapeutic target of antipsychotics for the treatment of schizophrenia. The inactive ...state structures of D
2
R have been described in complex with the inverse agonists risperidone (D
2
R
ris
) and haloperidol (D
2
R
hal
). Here we describe the structure of human D
2
R in complex with spiperone (D
2
R
spi
). In D
2
R
spi
, the conformation of the extracellular loop (ECL) 2, which composes the ligand-binding pocket, was substantially different from those in D
2
R
ris
and D
2
R
hal
, demonstrating that ECL2 in D
2
R is highly dynamic. Moreover, D
2
R
spi
exhibited an extended binding pocket to accommodate spiperone’s phenyl ring, which probably contributes to the selectivity of spiperone to D
2
R and 5-HT
2A
R. Together with D
2
R
ris
and D
2
R
hal
, the structural information of D
2
R
spi
should be of value for designing novel antipsychotics with improved safety and efficacy.
The taste receptor type 1 (T1r) family perceives 'palatable' tastes. These receptors function as T1r2-T1r3 and T1r1-T1r3 heterodimers to recognize a wide array of sweet and umami (savory) tastes in ...sugars and amino acids. Nonetheless, it is unclear how diverse tastes are recognized by so few receptors. Here we present crystal structures of the extracellular ligand-binding domains (LBDs), the taste recognition regions of the fish T1r2-T1r3 heterodimer, bound to different amino acids. The ligand-binding pocket in T1r2LBD is rich in aromatic residues, spacious and accommodates hydrated percepts. Biophysical studies show that this binding site is characterized by a broad yet discriminating chemical recognition, contributing for the particular trait of taste perception. In contrast, the analogous pocket in T1r3LBD is occupied by a rather loosely bound amino acid, suggesting that the T1r3 has an auxiliary role. Overall, we provide a structural basis for understanding the chemical perception of taste receptors.
A data processing pipeline for serial femtosecond crystallography at SACLA was developed, based on Cheetah Barty et al. (2014). J. Appl. Cryst.47, 1118–1131 and CrystFEL White et al. (2016). J. Appl. ...Cryst.49, 680–689. The original programs were adapted for data acquisition through the SACLA API, thread and inter‐node parallelization, and efficient image handling. The pipeline consists of two stages: The first, online stage can analyse all images in real time, with a latency of less than a few seconds, to provide feedback on hit rate and detector saturation. The second, offline stage converts hit images into HDF5 files and runs CrystFEL for indexing and integration. The size of the filtered compressed output is comparable to that of a synchrotron data set. The pipeline enables real‐time feedback and rapid structure solution during beamtime.
A data processing pipeline for SACLA was developed, based on Cheetah and CrystFEL. Real‐time analysis and rapid structure solution were enabled.
Serial femtosecond X-ray crystallography (SFX) has revolutionized atomic-resolution structural investigation by expanding applicability to micrometer-sized protein crystals, even at room temperature, ...and by enabling dynamics studies. However, reliable crystal-carrying media for SFX are lacking. Here we introduce a grease-matrix carrier for protein microcrystals and obtain the structures of lysozyme, glucose isomerase, thaumatin and fatty acid-binding protein type 3 under ambient conditions at a resolution of or finer than 2 Å.
Phytochrome proteins control the growth, reproduction, and photosynthesis of plants, fungi, and bacteria. Light is detected by a bilin cofactor, but it remains elusive how this leads to activation of ...the protein through structural changes. We present serial femtosecond X-ray crystallographic data of the chromophore-binding domains of a bacterial phytochrome at delay times of 1 ps and 10 ps after photoexcitation. The data reveal a twist of the D-ring, which leads to partial detachment of the chromophore from the protein. Unexpectedly, the conserved so-called pyrrole water is photodissociated from the chromophore, concomitant with movement of the A-ring and a key signaling aspartate. The changes are wired together by ultrafast backbone and water movements around the chromophore, channeling them into signal transduction towards the output domains. We suggest that the observed collective changes are important for the phytochrome photoresponse, explaining the earliest steps of how plants, fungi and bacteria sense red light.
Abstract
In meso crystallization of membrane proteins relies on the use of lipids capable of forming a lipidic cubic phase (LCP). However, almost all previous crystallization trials have used ...monoacylglycerols, with 1-(
cis
-9-octadecanoyl)-
rac
-glycerol (MO) being the most widely used lipid. We now report that EROCOC
17+4
mixed with 10% (w/w) cholesterol (Fig. 1) serves as a new matrix for crystallization and a crystal delivery medium in the serial femtosecond crystallography of Adenosine A
2A
receptor (A
2A
R). The structures of EROCOC
17+4
-matrix grown A
2A
R crystals were determined at 2.0 Å resolution by serial synchrotron rotation crystallography at a cryogenic temperature, and at 1.8 Å by LCP-serial femtosecond crystallography, using an X-ray free-electron laser at 4 and 20 °C sample temperatures, and are comparable to the structure of the MO-matrix grown A
2A
R crystal (PDB ID: 4EIY). Moreover, X-ray scattering measurements indicated that the EROCOC
17+4
/water system did not form the crystalline L
C
phase at least down to − 20 °C, in marked contrast to the equilibrium MO/water system, which transforms into the crystalline L
C
phase below about 17 °C. As the L
C
phase formation within the LCP-matrix causes difficulties in protein crystallography experiments in meso, this feature of EROCOC
17+4
will expand the utility of the in meso method.
Phytochromes are red/far-red light photoreceptors in bacteria to plants, which elicit a variety of important physiological responses. They display a reversible photocycle between the resting Pr state ...and the light-activated Pfr state. Light signals are transduced as structural change through the entire protein to modulate its activity. It is unknown how the Pr-to-Pfr interconversion occurs, as the structure of intermediates remains notoriously elusive. Here, we present short-lived crystal structures of the photosensory core modules of the bacteriophytochrome from myxobacterium Stigmatella aurantiaca captured by an X-ray free electron laser 5 ns and 33 ms after light illumination of the Pr state. We observe large structural displacements of the covalently bound bilin chromophore, which trigger a bifurcated signaling pathway that extends through the entire protein. The snapshots show with atomic precision how the signal progresses from the chromophore, explaining how plants, bacteria, and fungi sense red light.
Display omitted
•Successful TR-SFX experiments were conducted on a myxobacterial phytochrome at SACLA•The Z-to-E isomerization of the biliverdin chromophore is observed at 5 ns and 33 ms•Structural changes extend through the entire phytochrome at both time delays
Phytochromes are the molecular eyes of plants and bacteria that regulate cellular response to light, an essential environmental signal. Carrillo, Pandey et al. successfully investigate these structural changes in real time and with extreme magnification at one of the strongest X-ray sources, the free electron laser SACLA in Japan.
Many of the extracellular proteins or extracellular domains of plasma membrane proteins exist or function as homo‐ or heteromeric multimer protein complexes. Successful recombinant production of such ...proteins is often achieved by co‐expression of the components using eukaryotic cells via the secretory pathway. Here we report a strategy addressing large‐scale expression of hetero‐multimeric extracellular domains of plasma membrane proteins and its application to the extracellular domains of a taste receptor. The target receptor consists of a heterodimer of T1r2 and T1r3 proteins, and their extracellular ligand binding domains (LBDs) are responsible for the perception of major taste substances. However, despite the functional importance, recombinant production of the heterodimeric proteins has so far been unsuccessful. We achieved the successful preparation of the heterodimeric LBD by use of Drosophila S2 cells, which have a high secretory capacity, and by the establishment of a stable high‐expression clone producing both subunits at a comparable level. The method overcame the problems encountered in the conventional transient expression of the receptor protein in insect cells using baculovirus or vector lipofection, which failed in the proper heterodimer production because of the biased expression of T1r3LBD over T1r2LBD. The large‐scale expression methodology reported here may serve as one of the considerable strategies for the preparation of multimeric extracellular protein complexes.
PDF
