NMR Experiments Provide Insights into Ligand-Binding to the SARS-CoV-2 Spike Protein Receptor-Binding Domain.

We have used chemical shift perturbation (CSP) and saturation transfer difference (STD) NMR experiments to identify and characterize the binding of selected ligands to the receptor-binding domain (RBD) of the spike glycoprotein (S-protein) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We also subjected full-length S-protein to STD NMR experiments, allowing correlations with RBD-based results. CSPs reveal the binding sites for heparin and fondaparinux, and affinities were measured using CSP titrations. We then show that α-2,3-sialyllactose binds to the S-protein but not to the RBD. Finally, combined CSP and STD NMR experiments show that lifitegrast, a compound used for the treatment of dry eye, binds to the linoleic acid (LA) binding pocket with a dissociation constant in the µM range. This is an interesting finding, as lifitegrast lends itself well as a blueprint for medicinal chemistry, eventually furnishing novel entry inhibitors targeting the highly conserved LA binding site.

Further insights from structural mass spectrometry into endocytosis adaptor protein assemblies.

As a fundament in many biologically relevant processes, endocytosis in its different guises has been arousing interest for decades and still does so. This is true for the actual transport and its initiation alike. In clathrin-mediated endocytosis, a comparatively well understood endocytic pathway, a set of adaptor proteins bind specific lipids in the plasma membrane, subsequently assemble and thus form a crucial bridge from clathrin to actin for the ongoing process. These adaptor proteins are highly interesting themselves and the subject of this manuscript. Using many of the instruments that are available now in the mass spectrometry toolbox, we added some facets to the picture of how these minimal assemblies may look, how they form, and what influences the structure. Especially, lipids in the adaptor protein complexes result in reduced charging of a normal sized complex due to their specific binding position. The results further support our structural model of a double ring structure with interfacial lipids.

Conformational Shift of a ß-Hairpin Peptide upon Complex Formation with an Oligo-proline Peptide Studied by Mass Spectrometry.

So-called super-secondary structures such as the ß-hairpin, studied here, form an intermediate hierarchy between secondary and tertiary structures of proteins. Their sequence-derived 'pure' peptide backbone conformation is combined with 'remote' interstrand or interresidue contacts reminiscent of the 3D-structure of full-length proteins. This renders them ideally suited for studying potential nucleation sites of protein folding reactions as well as intermolecular interactions. But ß-hairpins do not merely serve as model systems; their unique structure characteristics warrant a central role in structural studies on their own. In this study we applied photo cross-linking in combination with high-resolution mass spectrometry and computational modeling as well as with ion mobility-mass spectrometry to elucidate these structural properties. Using variants of a known ß-hairpin representative, the so-called trpzip peptide and its ligands, we found evidence for a conformational transition of the ß-hairpin and its impact on ligand binding.

Native state dynamics affects the folding transition of porcine pancreatic phospholipase A2.

Porcine pancreatic phospholipase A2, a small and disulfide rich protein, is extremely resistant against chemically or thermally induced unfolding. Despite this marked resistance, the protein displays broad unfolding transitions resulting in comparatively low apparent thermodynamic stability. Broad unfolding transitions may result from undetected folding intermediates, residual structures in the unfolded state or an inhomogeneity of the native state. Using circular dichroism, fluorescence, and NMR spectroscopy, we ruled out the existence of stably populated folding intermediates, whereas UV absorbance measurements hinted at stable residual structures in the unfolded state. These residual structures proved, however, to have no impact on the folding parameters. Studies by limited proteolysis, CD, and NMR spectroscopy under non-denaturing conditions suggested pronounced dynamics of the protein in the native state, which as long as unrestrained by acidic pH or bound Ca(2+) ions exert considerable influence on the unfolding transition.

Analysis of nidogen-1/laminin γ1 interaction by cross-linking, mass spectrometry, and computational modeling reveals multiple binding modes.

We describe the detailed structural investigation of nidogen-1/laminin γ1 complexes using full-length nidogen-1 and a number of laminin γ1 variants. The interactions of nidogen-1 with laminin variants γ1 LEb2-4, γ1 LEb2-4 N836D, γ1 short arm, and γ1 short arm N836D were investigated by applying a combination of (photo-)chemical cross-linking, high-resolution mass spectrometry, and computational modeling. In addition, surface plasmon resonance and ELISA studies were used to determine kinetic constants of the nidogen-1/laminin γ1 interaction. Two complementary cross-linking strategies were pursued to analyze solution structures of laminin γ1 variants and nidogen-1. The majority of distance information was obtained with the homobifunctional amine-reactive cross-linker bis(sulfosuccinimidyl)glutarate. In a second approach, UV-induced cross-linking was performed after incorporation of the diazirine-containing unnatural amino acids photo-leucine and photo-methionine into laminin γ1 LEb2-4, laminin γ1 short arm, and nidogen-1. Our results indicate that Asn-836 within laminin γ1 LEb3 domain is not essential for complex formation. Cross-links between laminin γ1 short arm and nidogen-1 were found in all protein regions, evidencing several additional contact regions apart from the known interaction site. Computational modeling based on the cross-linking constraints indicates the existence of a conformational ensemble of both the individual proteins and the nidogen-1/laminin γ1 complex. This finding implies different modes of interaction resulting in several distinct protein-protein interfaces.

Monitoring conformational changes in peroxisome proliferator-activated receptor α by a genetically encoded photoamino acid, cross-linking, and mass spectrometry.

Chemical cross-linking combined with an enzymatic digestion and mass spectrometric analysis of the reaction products has evolved into an alternative strategy to structurally resolve protein complexes. We investigated conformational changes in peroxisome proliferator-activated receptor α (PPARα) upon ligand binding. Using E. coli cells with a special tRNA/aminoacyl-tRNA synthetase pair, two PPARα variants were prepared in which Leu-258 or Phe-273 were site-specifically replaced by the genetically encoded photoreactive amino acid p-benzoylphenylalanine (Bpa). PPARα variants were subjected to UV-induced cross-linking, both in the absence and in the presence of ligands. After the photo-cross-linking reaction, reaction mixtures were enzymatically digested and peptides were analyzed by mass spectrometry. The inter-residue distances disclosed by the photochemical cross-links served to monitor conformational changes in PPARα upon agonist and antagonist binding. The data obtained with our strategy emphasize the potential of genetically encoded internal photo-cross-linkers in combination with mass spectrometry as an alternative method to monitor in-solution 3D-protein structures.

Analysis of peptide secondary structures by photoactivatable amino acid analogues.

Photochemical cross-linking was applied to trap intramolecular interactions in peptides. The incorporation of diazirine-labeled amino acid analogues in combination with high-resolution mass spectrometry made it possible to catch reverse-turn conformations within peptides, exactly map their self-interacting surfaces, and discriminate between stable and transient interactions.

Nonconserved Ca(2+)/calmodulin binding sites in Munc13s differentially control synaptic short-term plasticity.

Munc13s are presynaptic proteins that mediate synaptic vesicle priming and thereby control the size of the readily releasable pool of vesicles. During high synaptic activity, Munc13-1 and its closely related homolog, ubMunc13-2, bind Ca(2+)/calmodulin, resulting in enhanced priming activity and in changes of short-term synaptic plasticity characteristics. Here, we studied whether bMunc13-2 and Munc13-3, two remote isoforms of Munc13-1 with a neuronal subtype-specific expression pattern, mediate synaptic vesicle priming and regulate short-term synaptic plasticity in a Ca(2+)/calmodulin-dependent manner. We identified a single functional Ca(2+)/calmodulin binding site in these isoforms and provide structural evidence that all Munc13s employ a common mode of interaction with calmodulin despite the lack of sequence homology between their Ca(2+)/calmodulin binding sites. Electrophysiological analysis showed that, during high-frequency activity, Ca(2+)/calmodulin binding positively regulates the priming activity of bMunc13-2 and Munc13-3, resulting in an increase in the size of the readily releasable pool of vesicles and subsequently in strong short-term synaptic enhancement of neurotransmission. We conclude that Ca(2+)/calmodulin-dependent regulation of priming activity is structurally and functionally conserved in all Munc13 proteins, and that the composition of Munc13 isoforms in a neuron differentially controls its short-term synaptic plasticity characteristics.

Peptide backbone conformation affects the substrate preference of protein arginine methyltransferase I.

Asymmetric dimethylation of arginine side chains is a common post-translational modification of eukaryotic proteins, which serves mostly to regulate protein-protein interactions. The modification is catalyzed by type I protein arginine methyltransferases, PRMT1 being the predominant member of the family. Determinants of substrate specificity of these enzymes are poorly understood. The Nuclear poly(A) binding protein 1 (PABPN1) is methylated by PRMT1 at 13 arginine residues located in RXR sequences in the protein's C-terminal domain. We have identified a preferred site for PRMT1-catalyzed methylation in PABPN1 and in a corresponding synthetic peptide. Variants of these substrates were analyzed by steady-state kinetic analysis and mass spectrometry. The data indicate that initial methylation is directed toward the preferred arginine residue by an N-terminally adjacent proline. Enhanced methylation upon peptide cyclization suggests that induction of a reverse turn structure is the basis for the ability of the respective proline residue to enable preferred methylation of the neighboring arginine residue, and this notion is supported by far-UV circular dichroism spectroscopy. We suggest that the formation of a reverse turn facilitates the access of arginine side chains to the active sites of PRMT1, which are located in the central cavity of a doughnut-shaped PRMT1 homodimer.

Structural Insights into retinal guanylylcyclase-GCAP-2 interaction determined by cross-linking and mass spectrometry.

The retinal guanylylcyclases ROS-GC 1 and 2 are regulated via the intracellular site by guanylylcyclase-activating proteins (GCAPs). The mechanisms of how GCAPs activate their target proteins remain elusive as exclusively structures of nonactivating calcium-bound GCAP-1 and -2 are available. In this work, we apply a combination of chemical cross-linking with amine-reactive cross-linkers and photoaffinity labeling followed by a mass spectrometric analysis of the created cross-linked products to study the interaction between N-terminally myristoylated GCAP-2 and a peptide derived from the catalytic domain of full-length ROS-GC 1. In our studies, only a few cross-linked products were obtained for calcium-bound GCAP-2, pointing to a well-defined structure of the GCAP-2-GC peptide complex. A much larger number of cross-links were detected in the absence of calcium, indicating a high flexibility of calcium-free GCAP-2 in the complex with the GC peptide. On the basis of the distance constraints imposed by the cross-links, we were able to create a structural model of the calcium-loaded complex between myristoylated GCAP-2 and the GC peptide.

Type I Arginine Methyltransferases PRMT1 and PRMT-3 Act Distributively.

Asymmetric dimethylation of arginine residues is a common posttranslational modification of proteins carried out by type I protein arginine methyltransferases, including PRMT1 and -3. We report that the consecutive transfer of two methyl groups to a single arginine side chain by PRMT1 and -3 occurs in a distributive manner, i.e. with intermittent release of the monomethylated intermediate. The oligomeric state of PRMTs together with the clustering of methylated arginine residues in most proteins carrying this type of modification suggests that multiple methyl transfers to a single polypeptide chain might proceed in a processive manner by cooperation of multiple active sites. However, three different types of experiments provide evidence that the reaction is distributive even with substrates containing multiple methyl-accepting arginines, including one with 13 such residues. PRMT1 also does not prefer substrates already containing one or more singly or doubly methylated arginine residues. Even though the reaction is distributive, the efficiency of methylation of one particular protein strongly depends on the number of methyl-accepting arginine residues it contains.

Promiscuous modification of the nuclear poly(A)-binding protein by multiple protein-arginine methyltransferases does not affect the aggregation behavior.

The mammalian nuclear poly(A)-binding protein, PABPN1, carries 13 asymmetrically dimethylated arginine residues in its C-terminal domain. By fractionation of cell extracts, we found that protein-arginine methyltransferases (PRMTs)-1, -3, and -6 are responsible for the modification of PABPN1. Recombinant PRMT1, -3, and -6 also methylated PABPN1. Our data suggest that these enzymes act on their own, and additional polypeptides are not involved in recognizing PABPN1 as a substrate. PRMT1 is the predominant methyltransferase acting on PABPN1. Nevertheless, PABPN1 was almost fully methylated in a Prmt1(-/-) cell line; thus, PRMT3 and -6 suffice for methylation. In contrast to PABPN1, the heterogeneous nuclear ribonucleoprotein (hnRNP) K is selectively methylated only by PRMT1. Efficient methylation of synthetic peptides derived from PABPN1 or hnRNP K suggested that PRMT1, -3, and -6 recognize their substrates by interacting with local amino acid sequences and not with additional domains of the substrates. However, the use of fusion proteins suggested that the inability of PRMT3 and -6 to modify hnRNP K is because of structural masking of the methyl-accepting amino acid sequences by neighboring domains. Mutations leading to intracellular aggregation of PABPN1 cause the disease oculopharyngeal muscular dystrophy. The C-terminal domain containing the methylated arginine residues is known to promote PAPBN1 self-association, and arginine methylation has been reported to inhibit self-association of an orthologous protein. Thus, arginine methylation might be relevant for oculopharyngeal muscular dystrophy. However, in two different types of assays we have been unable to detect any effect of arginine methylation on the aggregation of bovine PABPN1.