improving-high-viscosity-extrusion-microcrystals-for-time-resolved

Improving High Viscosity Extrusion of Microcrystals for Time-resolved Serial Femtosecond Crystallography at X-ray Lasers

High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray ...

We present active-state structures of the G protein-coupled receptor (GPCRs) rhodopsin carrying the disease-causing mutation G90D. Mutations of G90 cause either retinitis pigmentosa (RP) or congenital stationary night blindness (CSNB), a milder, non-progressive form of RP. Our analysis shows that the CSNB-causing G90D mutation introduces a salt bridge with K296. The mutant thus interferes with the E113Q-K296 activation switch and the covalent binding of the inverse agonist 11-cis-retinal, two interactions that are crucial for the deactivation of rhodopsin. Other mutations, including G90V causing RP, cannot promote similar interactions. We discuss our findings in context of a model in which CSNB is caused by constitutive activation of the visual signalling cascade.

Insights into congenital stationary night blindness based on the structure of G90D rhodopsin.

The effects of activating mutations associated with night blindness on the stoichiometry of rhodopsin interactions with G protein-coupled receptor kinase 1 (GRK1) and arrestin-1 have not been reported. Here we show that the monomeric form of WT rhodopsin and its constitutively active mutants M257Y, G90D, and T94I, reconstituted into HDL particles are effectively phosphorylated by GRK1, as well as two more ubiquitously expressed subtypes, GRK2 and GRK5. All versions of arrestin-1 tested (WT, pre-activated, and constitutively monomeric mutants) bind to monomeric rhodopsin and show the same selectivity for different functional forms of rhodopsin as in native disc membranes. Rhodopsin phosphorylation by GRK1 and GRK2 promotes arrestin-1 binding to a comparable extent, whereas similar phosphorylation by GRK5 is less effective, suggesting that not all phosphorylation sites on rhodopsin are equivalent in promoting arrestin-1 binding. The binding of WT arrestin-1 to phospho-opsin is comparable to the binding to its preferred target, P-Rh*, suggesting that in photoreceptors arrestin-1 only dissociates after opsin regeneration with 11-cis-retinal, which converts phospho-opsin into inactive phospho-rhodopsin that has lower affinity for arrestin-1. Reduced binding of arrestin-1 to the phospho-opsin form of G90D mutant likely contributes to night blindness caused by this mutation in humans.

Constitutively active rhodopsin mutants causing night blindness are effectively phosphorylated by GRKs but differ in arrestin-1 binding.

Scanning mutagenesis is a powerful protein engineering technique used to study protein structure-function relationship, map binding sites and design more stable proteins or proteins with altered properties. One of the time-consuming tasks encountered in application of this technique is the design of primers for site-directed mutagenesis. Here we present an open-source multi-platform software AAscan developed to design primers for this task according to a set of empirical rules such as melting temperature, overall length, length of overlap regions, and presence of GC clamps at the 3' end, for any desired substitution. We also describe additional software tools which are used to analyse a large number of sequencing results for the presence of desired mutations, as well as related software to design primers for ligation independent cloning. We have used AAscan software to design primers to make over 700 mutants, with a success rate of over 80%. We hope that the open-source nature of our software and ready availability of freeware tools used for its development will facilitate its adaptation and further development. The software is distributed under GPLv3 licence and is available at http://www.psi.ch/lbr/aascan. 

AAscan, PCRdesign and MutantChecker: a suite of programs for primer design and sequence analysis for high-throughput scanning mutagenesis.

The activation of the G-protein transducin (Gt) by rhodopsin (Rho) has been intensively studied for several decades. It is the best understood example of GPCR activation mechanism and serves as a template for other GPCRs. The structure of the Rho/G protein complex, which is transiently formed during the signaling reaction, is of particular interest. It can help understanding the molecular details of how retinal isomerization leads to the G protein activation, as well as shed some light on how GPCR recognizes its cognate G protein. The native Rho/Gt complex isolated from bovine retina suffers from low stability and loss of the retinal ligand. Recently, we reported that constitutively active mutant of rhodopsin E113Q forms a Rho/Gt complex that is stable in detergent solution. Here, we introduce methods for a large scale preparation of the complex formed by the thermo-stabilized and constitutively active rhodopsin mutant N2C/M257Y/D282C(RhoM257Y) and the native Gt purified from bovine retinas. We demonstrate that the light-activated rhodopsin in this complex contains a covalently bound unprotonated retinal and therefore corresponds to the active metarhodopin II state; that the isolated complex is active and dissociates upon addition of GTPγS; and that the stoichiometry corresponds to a 1∶1 molar ratio of rhodopsin to the heterotrimeric G-protein. And finally, we show that the rhodopsin also forms stable complex with Gi. This complex has significantly higher thermostability than RhoM257Y/Gt complex and is resistant to a variety of detergents. Overall, our data suggest that the RhoM257Y/Gi complex is an ideal target for future structural and mechanistic studies of signaling in the visual system. 

Crystallization scale preparation of a stable GPCR signaling complex between constitutively active rhodopsin and G-protein.

The past years have seen tremendous progress towards understanding how arrestins recognize phosphorylated G protein-coupled receptors (GPCRs). Two arrestin crystal structures, one of a pre-activated splice variant and one bound to a GPCR phosphopeptide, provided insights into the conformational changes upon phosphate recognition. Scanning mutagenesis and spectroscopic studies complete the picture of arrestin activation and receptor binding. Most perspicuous is the C-tail exchange mechanism, by which the C-tail of arrestin is released from its basal conformation and replaced by the phosphorylated GPCR C-terminus. Three positively charged clusters could act as conserved arrestin phosphosensors. Variations in the pattern of phosphorylation in a GPCR and variations within the C-terminus of different GPCRs may encode specificity to arrestin subtypes and particular physiological responses. 

Molecular mechanism of phosphorylation-dependent arrestin activation.

Structural biology. Viral chemokine mimicry.

Congenital stationary night blindness (CSNB) is an inherited and non-progressive retinal dysfunction. Here, we present the crystal structure of CSNB-causing T94I rhodopsin in the active conformation at 2.3 Å resolution. The introduced hydrophobic side chain prolongs the lifetime of the G protein activating metarhodopsin-II state by establishing a direct van der Waals contact with K296, the site of retinal attachment. This is in stark contrast to the light-activated state of the CSNB-causing G90D mutation, where the charged mutation forms a salt bridge with K296 To find the common denominator between these two functional modifications, we combined our structural data with a kinetic biochemical analysis and molecular dynamics simulations. Our results indicate that both the charged G90D and the hydrophobic T94I mutation alter the dark state by weakening the interaction between the Schiff base (SB) and its counterion E113 We propose that this interference with the tight regulation of the dim light photoreceptor rhodopsin increases background noise in the visual system and causes the loss of night vision characteristic for CSNB patients.

Structural role of the T94I rhodopsin mutation in congenital stationary night blindness.

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) is a powerful method to determine high-resolution structures of pharmaceutically relevant membrane proteins. Recently, the technology has been adapted to carry out serial millisecond crystallography (SMX) at synchrotron sources, where beamtime is more abundant. In an injector-based approach, crystals grown in lipidic cubic phase (LCP) or embedded in viscous medium are delivered directly into the unattenuated beam of a microfocus beamline. Pilot experiments show the application of microjet-based SMX for solving the structure of a membrane protein and compatibility of the method with de novo phasing. Planned synchrotron upgrades, faster detectors and software developments will go hand-in-hand with developments at free-electron lasers to provide a powerful methodology for solving structures from microcrystals at room temperature, ligand screening or crystal optimization for time-resolved studies with minimal or no radiation damage.

Serial Millisecond Crystallography of Membrane Proteins.

Historically, room-temperature structure determination was succeeded by cryo-crystallography to mitigate radiation damage. Here, we demonstrate that serial millisecond crystallography at a synchrotron beamline equipped with high-viscosity injector and high frame-rate detector allows typical crystallographic experiments to be performed at room-temperature. Using a crystal scanning approach, we determine the high-resolution structure of the radiation sensitive molybdenum storage protein, demonstrate soaking of the drug colchicine into tubulin and native sulfur phasing of the human G protein-coupled adenosine receptor. Serial crystallographic data for molecular replacement already converges in 1,000-10,000 diffraction patterns, which we collected in 3 to maximally 82 minutes. Compared with serial data we collected at a free-electron laser, the synchrotron data are of slightly lower resolution, however fewer diffraction patterns are needed for de novo phasing. Overall, the data we collected by room-temperature serial crystallography are of comparable quality to cryo-crystallographic data and can be routinely collected at synchrotrons.Serial crystallography was developed for protein crystal data collection with X-ray free-electron lasers. Here the authors present several examples which show that serial crystallography using high-viscosity injectors can also be routinely employed for room-temperature data collection at synchrotrons.

Serial millisecond crystallography for routine room-temperature structure determination at synchrotrons.

In the degenerative eye disease retinitis pigmentosa (RP), protein misfolding leads to fatal consequences for cell metabolism and rod and cone cell survival. To stop disease progression, a therapeutic approach focuses on stabilizing inherited protein mutants of the G protein-coupled receptor (GPCR) rhodopsin using pharmacological chaperones (PC) that improve receptor folding and trafficking. In this study, we discovered stabilizing nonretinal small molecules by virtual and thermofluor screening and determined the crystal structure of pharmacologically stabilized opsin at 2.4 Å resolution using one of the stabilizing hits (S-RS1). Chemical modification of S-RS1 and further structural analysis revealed the core binding motif of this class of rhodopsin stabilizers bound at the orthosteric binding site. Furthermore, previously unobserved conformational changes are visible at the intradiscal side of the seven-transmembrane helix bundle. A hallmark of this conformation is an open channel connecting the ligand binding site with the membrane and the intradiscal lumen of rod outer segments. Sufficient in size, the passage permits the exchange of hydrophobic ligands such as retinal. The results broaden our understanding of rhodopsin's conformational flexibility and enable therapeutic drug intervention against rhodopsin-related retinitis pigmentosa.

Ligand channel in pharmacologically stabilized rhodopsin.

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.

Article	Year
Insights into congenital stationary night blindness based on the structure of G90D rhodopsin. EMBO reports\| PubMed ID: 23579341	2013
Constitutively active rhodopsin mutants causing night blindness are effectively phosphorylated by GRKs but differ in arrestin-1 binding. Cellular signalling\| PubMed ID: 23872075	2013
AAscan, PCRdesign and MutantChecker: a suite of programs for primer design and sequence analysis for high-throughput scanning mutagenesis. PloS one\| PubMed ID: 24205336	2013
Crystallization scale preparation of a stable GPCR signaling complex between constitutively active rhodopsin and G-protein. PloS one\| PubMed ID: 24979345	2014
Molecular mechanism of phosphorylation-dependent arrestin activation. Current opinion in structural biology\| PubMed ID: 25484000	2014
Structural biology. Viral chemokine mimicry. Science (New York, N.Y.)\| PubMed ID: 25745149	2015
Structural role of the T94I rhodopsin mutation in congenital stationary night blindness. EMBO reports\| PubMed ID: 27458239	2016
Serial Millisecond Crystallography of Membrane Proteins. Advances in experimental medicine and biology\| PubMed ID: 27553240	2016
Serial millisecond crystallography for routine room-temperature structure determination at synchrotrons. Nature communications\| PubMed ID: 28912485	2017
Ligand channel in pharmacologically stabilized rhodopsin. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 29555765	2018
Retinal isomerization in bacteriorhodopsin captured by a femtosecond x-ray laser. Science (New York, N.Y.)\| PubMed ID: 29903883	2018

Joerg Standfuss

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