First 3D-Structural Data of Full-Length Guanylyl Cyclase 1 in Rod-Outer-Segment Preparations of Bovine Retina by Cross-Linking/Mass Spectrometry.

The rod-outer-segment guanylyl cyclase 1 (ROS-GC1) is a key transmembrane protein for retinal phototransduction. Mutations of ROS-GC1 correlate with different retinal diseases that often lead to blindness. No structural data are available for ROS-GC1 so far. We performed a 3D-structural analysis of native ROS-GC1 from bovine retina by cross-linking/mass spectrometry (XL-MS) and computational modeling. Absolute quantification and activity measurements of native ROS-GC1 were performed by MS-based assays directly in bovine retina samples. Our data present the first 3D-structural analysis of active, full-length ROS-GC1 derived from bovine retina. We propose a novel domain organization for the intracellular domain ROS-GC1. Our XL-MS data of native ROS-GC1 from rod-outer-segment preparations of bovine retina agree with a dimeric architecture. Our integrated approach can serve as a blueprint for conducting 3D-structural studies of membrane proteins in their native environment.

Oligomeric state, hydrodynamic properties and target recognition of human Calcium and Integrin Binding protein 2 (CIB2).

Calcium- and Integrin-Binding protein 2 (CIB2) is a small and ubiquitously expressed protein with largely unknown biological function but ascertained role in hearing physiology and disease. Recent studies found that CIB2 binds Ca2+ with moderate affinity and dimerizes under conditions mimicking the physiological ones. Here we provided new lines of evidence on CIB2 oligomeric state and the mechanism of interaction with the α7B integrin target. Based on a combination of native mass spectrometry, chemical cross-linking/mass spectrometry, analytical gel filtration, dynamic light scattering and molecular dynamics simulations we conclude that CIB2 is monomeric under all tested conditions and presents uncommon hydrodynamic properties, most likely due to the high content of hydrophobic solvent accessible surface. Surface plasmon resonance shows that the interaction with α7B occurs with relatively low affinity and is limited to the cytosolic region proximal to the membrane, being kinetically favored in the presence of physiological Mg2+ and in the absence of Ca2+. Although CIB2 binds to an α7B peptide in a 1:1 stoichiometry, the formation of the complex might induce binding of another CIB2 molecule.

The First MS-Cleavable, Photo-Thiol-Reactive Cross-Linker for Protein Structural Studies.

Cleavable cross-linkers are gaining increasing importance for chemical cross-linking/mass spectrometry (MS) as they permit a reliable and automated data analysis in structural studies of proteins and protein assemblies. Here, we introduce 1,3-diallylurea (DAU) as the first CID-MS/MS-cleavable, photo-thiol-reactive cross-linker. DAU is a commercially available, inexpensive reagent that efficiently undergoes an anti-Markovnikov hydrothiolation with cysteine residues in the presence of a radical initiator upon UV-A irradiation. Radical cysteine cross-linking proceeds via an orthogonal "click reaction" and yields stable alkyl sulfide products. DAU reacts at physiological pH and cross-linking reactions with peptides, and proteins can be performed at temperatures as low as 4 °C. The central urea bond is efficiently cleaved upon collisional activation during tandem MS experiments generating characteristic product ions. This improves the reliability of automated cross-link identification. Different radical initiators have been screened for the cross-linking reaction of DAU using the thiol-containing compounds cysteine and glutathione. Our concept has also been exemplified for the biologically relevant proteins bMunc13-2 and retinal guanylyl cyclase-activating protein-2. Graphical abstract ᅟ.

Molecular Details of Retinal Guanylyl Cyclase 1/GCAP-2 Interaction.

The rod outer segment guanylyl cyclase 1 (ROS-GC1) is an essential component of photo-transduction in the retina. In the light-induced signal cascade, membrane-bound ROS-GC1 restores cGMP levels in the dark in a calcium-dependent manner. With decreasing calcium concentration in the intracellular compartment, ROS-GC1 is activated via the intracellular site by guanylyl cyclase-activating proteins (GCAP-1/-2). Presently, the exact activation mechanism is elusive. To obtain structural insights into the ROS-GC1 regulation by GCAP-2, chemical cross-linking/mass spectrometry studies using GCAP-2 and three ROS-GC1 peptides were performed in the presence and absence of calcium. The majority of cross-links were identified with the C-terminal lobe of GCAP-2 and a peptide comprising parts of ROS-GC1's catalytic domain and C-terminal extension. Consistently with the cross-linking results, surface plasmon resonance and fluorescence measurements confirmed specific binding of this ROS-GC peptide to GCAP-2 with a dissociation constant in the low micromolar range. These results imply that a region of the catalytic domain of ROS-GC1 can participate in the interaction with GCAP-2. Additional binding surfaces upstream of the catalytic domain, in particular the juxtamembrane domain, can currently not be excluded.

Carboxyl-Photo-Reactive MS-Cleavable Cross-Linkers: Unveiling a Hidden Aspect of Diazirine-Based Reagents.

A major challenge in cross-linking/mass spectrometry (MS) is targeting carboxyl functions in proteins under physiological conditions that do not disturb the protein's conformation. Cross-linking of glutamic acid and aspartic acid residues in proteins will greatly expand the scope of structural mass spectrometry. We discovered that carboxyl-reactive cross-linkers have already been employed for many years in cross-linking/MS studies, yet in a completely different context. Diazirine-based cross-linkers, such as photomethionine and succinimidyldiazirine cross-linkers, are currently considered to react nonspecifically upon UV-A photoactivation with all 20 proteinogenic amino acids through a reactive carbene that inserts mainly into C-H bonds. We discovered that the cross-linking capability of diazirines based on X-H (X = C, N, O) insertion is in fact only the tip of the iceberg. Diazirines isomerize to linear diazo compounds that can react with carboxylic acids to yield esters. On top of that, the resulting cross-linked products are MS-cleavable allowing an automated analysis of cross-links via customized software tools. Therefore, diazirines open an entirely new route for photo-cross-linking of carboxylic acids. Previous cross-linking studies using diazirines have to be revisited in the light of these findings.

The First Zero-Length Mass Spectrometry-Cleavable Cross-Linker for Protein Structure Analysis.

Combining the properties of a zero-length cross-linker with cleavability by tandem mass spectrometry (MS/MS) poses great advantages for protein structure analysis using the cross-linking/MS approach. These include a reliable, automated data analysis and the possibility to obtain short-distance information of protein 3D-structures. We introduce 1,1'-carbonyldiimidazole (CDI) as an easy-to-use and commercially available, low-cost reagent that ideally fulfils these features. CDI bridges primary amines and hydroxy groups in proteins with the lowest possible spacer length of one carbonyl unit (ca. 2.6 Å). The cross-linking reaction can be conducted under physiological conditions in the pH range between 7.2 and 8. Urea and carbamate cross-linked products are cleaved upon collisional activation during MS/MS experiments generating characteristic product ions, greatly improving the unambiguous identification of cross-links. Our innovative analytical concept is exemplified and applied for bovine serum albumin (BSA), wild-type tumor suppressor p53, an intrinsically disordered protein, and retinal guanylyl cyclase activating protein-2 (GCAP-2).