Cross-linking mass spectrometry uncovers protein interactions and functional assemblies in synaptic vesicle membranes.

Synaptic vesicles are storage organelles for neurotransmitters. They pass through a trafficking cycle and fuse with the pre-synaptic membrane when an action potential arrives at the nerve terminal. While molecular components and biophysical parameters of synaptic vesicles have been determined, our knowledge on the protein interactions in their membranes is limited. Here, we apply cross-linking mass spectrometry to study interactions of synaptic vesicle proteins in an unbiased approach without the need for specific antibodies or detergent-solubilisation. Our large-scale analysis delivers a protein network of vesicle sub-populations and functional assemblies including an active and an inactive conformation of the vesicular ATPase complex as well as non-conventional arrangements of the luminal loops of SV2A, Synaptophysin and structurally related proteins. Based on this network, we specifically target Synaptobrevin-2, which connects with many proteins, in different approaches. Our results allow distinction of interactions caused by 'crowding' in the vesicle membrane from stable interaction modules.

Formation and Stoichiometry of CRISPR-Cascade Complexes with Varying Spacer Lengths Revealed by Native Mass Spectrometry.

The adaptive immune system of bacteria and archaea against viral DNA is based on clustered, regularly interspaced, short palindromic repeats (CRISPRs) which are encoded in the host genome and translated into CRISPR RNAs (crRNAs) containing single spacer sequences complementary to foreign DNA. crRNAs assemble with CRISPR-associated (Cas) proteins forming surveillance complexes that base-pair with viral DNA and mediate its degradation. As specificity of degradation is provided by the crRNA spacer sequence, genetic engineering of the CRISPR system has emerged as a popular molecular tool, for instance, in gene silencing and programmed DNA degradation. Elongating or shortening the crRNA spacer sequence are therefore promising ventures to modify specificity toward the target DNA. However, even though the stoichiometry of wild-type complexes is well established, it is unknown how variations in crRNA spacer length affect their stoichiometry. The CRISPR-associated antiviral defense surveillance complexes of Streptococcus thermophilus (StCascade complexes) contain crRNA and five protein subunits. Using native mass spectrometry, we studied the formation and stoichiometry of StCascade complexes assembled on a set of crRNAs with different spacer lengths. We assigned all relevant complexes and gained insights into the stoichiometry of the complexes as well as their preferred assembly. We found that stable complexes, which incorporate or lose a (Cas7)2(Cse2)1-module, assemble on crRNA varied in length by 12-nucleotide units, while varying crRNA length in six-nucleotide units results in heterogeneous mixtures of complexes. Combining our results from the various variants, we generated an assembly pathway revealing general features of I-E type Cascade complex formation.

Decision-Making in Cascade Complexes Harboring crRNAs of Altered Length.

The multi-subunit type I CRISPR-Cas surveillance complex Cascade uses its crRNA to recognize dsDNA targets. Recognition involves DNA unwinding and base-pairing between the crRNA spacer region and a complementary DNA strand, resulting in formation of an R-loop structure. The modular Cascade architecture allows assembly of complexes containing crRNAs with altered spacer lengths that promise increased target specificity in emerging biotechnological applications. Here we produce type I-E Cascade complexes containing crRNAs with up to 57-nt-long spacers. We show that these complexes form R-loops corresponding to the designed target length, even for the longest spacers tested. Furthermore, the complexes can bind their targets with much higher affinity compared with the wild-type form. However, target recognition and the subsequent Cas3-mediated DNA cleavage do not require extended R-loops but already occur for wild-type-sized R-loops. These findings set important limits for specificity improvements of type I CRISPR-Cas systems.

Structural and Functional Analyses of the Human PDH Complex Suggest a "Division-of-Labor" Mechanism by Local E1 and E3 Clusters.

The pseudo-atomic structural model of human pyruvate dehydrogenase complex (PDHc) core composed of full-length E2 and E3BP components, calculated from our cryoelectron microscopy-derived density maps at 6-Å resolution, is similar to those of prokaryotic E2 structures. The spatial organization of human PDHc components as evidenced by negative-staining electron microscopy and native mass spectrometry is not homogeneous, and entails the unanticipated formation of local clusters of E1:E2 and E3BP:E3 complexes. Such uneven, clustered organization translates into specific duties for E1-E2 clusters (oxidative decarboxylation and acetyl transfer) and E3BP-E3 clusters (regeneration of reduced lipoamide) corresponding to half-reactions of the PDHc catalytic cycle. The addition of substrate coenzyme A modulates the conformational landscape of PDHc, in particular of the lipoyl domains, extending the postulated multiple random coupling mechanism. The conformational and associated chemical landscapes of PDHc are thus not determined entirely stochastically, but are restrained and channeled through an asymmetric architecture and further modulated by substrate binding.

Oligomerisation of Synaptobrevin-2 Studied by Native Mass Spectrometry and Chemical Cross-Linking.

Synaptobrevin-2 is a key player in signal transmission in neurons. It forms, together with SNAP25 and Syntaxin-1A, the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex and mediates exocytosis of synaptic vesicles with the pre-synaptic membrane. While Synaptobrevin-2 is part of a four-helix bundle in this SNARE complex, it is natively unstructured in the absence of lipids or other SNARE proteins. Partially folded segments, presumably SNARE complex formation intermediates, as well as formation of Synaptobrevin-2 dimers and oligomers, were identified in previous studies. Here, we employ three Synaptobrevin-2 variants-the full-length protein Syb(1-116), the soluble, cytosolic variant Syb(1-96) as well as a shorter version Syb(49-96) containing structured segments but omitting a trigger site for SNARE complex formation-to study oligomerisation in the absence of interaction partners or when incorporated into the lipid bilayer of liposomes. Combining native mass spectrometry with chemical cross-linking, we find that the truncated versions show increased oligomerisation. Our findings from both techniques agree well and confirm the presence of oligomers in solution while membrane-bound Synaptobrevin-2 is mostly monomeric. Using ion mobility mass spectrometry, we could further show that lower charge states of Syb(49-96) oligomers, which most likely represent solution structures, follow an isotropic growth curve suggesting that they are intrinsically disordered. From a technical point of view, we show that the combination of native ion mobility mass spectrometry with chemical cross-linking is well-suited for the analysis of protein homo-oligomers. Graphical Abstract ᅟ.

Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of Multi-subunit Protein Assemblies.

Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is therefore fundamental for the understanding of cellular processes. However, many protein complexes are dynamic assemblies and are not accessible by conventional structural techniques. Mass spectrometry contributes to the structural investigation of these assemblies, and particularly the combination of various mass spectrometric techniques delivers valuable insights into their structural arrangement. In this article, we describe the application and combination of two complementary mass spectrometric techniques, namely chemical cross-linking coupled with mass spectrometry and native mass spectrometry. Chemical cross-linking involves the covalent linkage of amino acids in close proximity by using chemical reagents. After digestion with proteases, cross-linked di-peptides are identified by mass spectrometry and protein interactions sites are uncovered. Native mass spectrometry on the other hand is the analysis of intact protein assemblies in the gas phase of a mass spectrometer. It reveals protein stoichiometries as well as protein and ligand interactions. Both techniques therefore deliver complementary information on the structure of protein-ligand assemblies and their combination proved powerful in previous studies.

Characterization of the osseointegration of Algipore and Algipore modified with mineralized collagen type I.

OBJECTIVE: Algipore is a clinically established bone substitute. The present study evaluated the osseoconductive and resorptive characteristics of Algipore modified with collagen type I (ACI). STUDY DESIGN: Three defects of 10 × 3 mm were set in the frontal bone of 10 adult female minipigs. One cavity was filled with commercially available Algipore, and the second with ACI. The third cavity was left unfilled and served as reference. After 4 months of healing, the animals were humanely killed. Bone formation and resorption characteristics of the substitutes were evaluated histomorphologically and histomorphometrically using Donath's sawing and grinding technique. RESULTS: Neither material caused inflammatory reactions. Compared with controls, both substitutes showed significantly higher fractions of trabecular bone (control: 42.2%; Algipore: 58.7%, [P < .001]; ACI: 53.6%, [P = .013]). After 4 months, the remaining fraction of Algipore was 42.2% and the fraction of ACI was 47.9% (P = .016). CONCLUSIONS: The present study demonstrates that the modification of Algipore with collagen I does not show any benefits compared with pure Algipore in small calvarial bone defects in minipigs.