Cysteine Connectivity Research Articles

Isolation and structural identification of a new T1-conotoxin with unique disulfide connectivities derived from Conus bandanus.

Background:Conopeptides are neuropharmacological peptides derived from the venomous salivary glands of cone snails. Among 29 superfamilies based on conserved signal sequences, T-superfamily conotoxins, which belong to the smallest group, include four different frameworks that contain four cysteines denominated I, V, X and XVI. In this work, the primary structure and the cysteine connectivity of novel conotoxin of Conus bandanus were determined by tandem mass spectrometry using collision-induced dissociation.Methods:The venom glands of C. bandanus snails were dissected, pooled, and extracted with 0.1% trifluoroacetic acid in three steps and lyophilized. The venom was fractionated and purified in an HPLC system with an analytical reversed-phase C18 column. The primary peptide structure was analyzed by MALDI TOF MS/MS using collision-induced dissociation and confirmed by Edman's degradation. The peptide’s cysteine connectivity was determined by rapid partial reduction-alkylation technique.Results:The novel conotoxin, NGC1C2(I/L)VREC3C4, was firstly derived from de novo sequencing by MS/MS. The presence of isoleucine residues in this conotoxin was confirmed by the Edman degradation method. The conotoxin, denominated Bn5a, belongs to the T1-subfamily of conotoxins. However, the disulfide bonds (C1-C4/C2-C3) of Bn5a were not the same as found in other T1-subfamily conopeptides but shared common connectivities with T2-subfamily conotoxins. The T1-conotoxin of C. bandanus proved the complexity of the disulfide bond pattern of conopeptides. The homological analysis revealed that the novel conotoxin could serve as a valuable probe compound for the human-nervous-system norepinephrine transporter.Conclusion:We identified the first T1-conotoxin, denominated Bn5a, isolated from C. bandanus venom. However, Bn5a conotoxin exhibited unique C1-C4/C2-C3 disulfide connectivity, unlike other T1-conotoxins (C1-C3/C2-C4). The structural and homological analyses herein have evidenced novel conotoxin Bn5a that may require further investigation.

Combination of Capillary Zone Electrophoresis-Mass Spectrometry, Ion Mobility-Mass Spectrometry, and Theoretical Calculations for Cysteine Connectivity Identification in Peptides Bearing Two Intramolecular Disulfide Bonds.

Disulfide bonds between cysteine residues are commonly involved in the stability of numerous peptides and proteins and are crucial for providing biological activities. In such peptides, the appropriate cysteine connectivity ensures the proper conformation allowing an efficient binding to their molecular targets. Disulfide bond connectivity characterization is still challenging and is a critical issue in the analysis of structured peptides/proteins targeting pharmaceutical or pharmacological utilizations. This study describes the development of new and fast gas-phase and in-solution electrophoretic methods coupled to mass spectrometry to characterize the cysteine connectivity of disulfide bonds. For this purpose, disulfide isomers of three peptides bearing two intramolecular disulfide bonds but different cysteine connectivity have been investigated. Capillary zone electrophoresis and ion mobility both coupled to mass spectrometry were used to perform the separation in both aqueous and gas phases, respectively. The separation efficiency of each technique has been critically evaluated and compared. Finally, theoretical calculations were performed to support and explain the experimental data based on the predicted physicochemical properties of the different peptides.

The three-dimensional structure of an H-superfamily conotoxin reveals a granulin fold arising from a common ICK cysteine framework

Venomous marine cone snails produce peptide toxins (conotoxins) that bind ion channels and receptors with high specificity and therefore are important pharmacological tools. Conotoxins contain conserved cysteine residues that form disulfide bonds that stabilize their structures. To gain structural insight into the large, yet poorly characterized conotoxin H-superfamily, we used NMR and CD spectroscopy along with MS-based analyses to investigate H-Vc7.2 from Conus victoriae, a peptide with a VI/VII cysteine framework. This framework has CysI-CysIV/CysII-CysV/CysIII-CysVI connectivities, which have invariably been associated with the inhibitor cystine knot (ICK) fold. However, the solution structure of recombinantly expressed and purified H-Vc7.2 revealed that although it displays the expected cysteine connectivities, H-Vc7.2 adopts a different fold consisting of two stacked β-hairpins with opposing β-strands connected by two parallel disulfide bonds, a structure homologous to the N-terminal region of the human granulin protein. Using structural comparisons, we subsequently identified several toxins and nontoxin proteins with this "mini-granulin" fold. These findings raise fundamental questions concerning sequence-structure relationships within peptides and proteins and the key determinants that specify a given fold.

Local Similarity Matrix for Cysteine Disulfide Connectivity Prediction from Protein Sequences.

Accurately predicting three dimensional protein structures from sequences would present us with targets for drugs via molecular dynamics that would treat cancer, viral infections, and neurological diseases. These treatments would have a far reaching impact to our economy, quality of life, and society. The goal of this research was to build a data mining framework to predict cysteine connectivity in proteins from the sequence and oxidation state of cysteines. Accurately predicting the cysteine bonding configuration improves the TM-Score, a quantitative measurement of protein structure prediction accuracy. We provided state of the art Qp and Qc on the PDBCYS and IVD-54 Datasets. Furthermore, we have produced a Local Similarity Matrix that compares favorably to the default PSSMs generated from PSI-Blast in a statistically significant way. Our Qp for SP39, PDBCYS, and IVD-54 were 90.6, 80.6, and 68.5, respectively.

Corona discharge electrospray ionization of formate-containing solutions enables in-source reduction of disulfide bonds.

Disulfide bonds are critical linkages for maintaining protein structure and enzyme activity. These linkages, however, can limit peptide sequencing efforts by mass spectrometry (MS) and often require chemical reduction and alkylation. Under such conditions, information regarding cysteine connectivity is lost. Online partial disulfide reduction within the electrospray (ESI) source has recently been established as a means to identify complex cysteine linkage patterns in a liquid chromatography-MS experiment without the need for sample pre-treatment. Corona discharge (CD) is invoked as the causative factor of this in-source reduction (ISR); however, evidence remains largely circumstantial. In this study, we demonstrate that instrumental factors-nebulizing gas, ESI capillary material, organic solvent content, ESI spray needle-to-MS distance-all modulate the degree of reduction observed for the single disulfide in oxytocin, further implicating CD in ISR. Rigorous analysis of solution conditions, however, reveals that corona discharge alone can induce only minor disulfide reduction. We establish that CD-ESI of peptide solutions containing formic acid or its conjugate base results in a dramatic increase in disulfide reduction. It is also determined that ISR is exacerbated at low pH for complex peptides containing multiple disulfide bonds and possessing higher-order structure, as well as for a small protein. Overall, our results demonstrate that ESI of formate/formic acid-containing solutions under corona discharge conditions facilitates disulfide ISR, likely by a similar reduction pathway measured in γ-radiolysis studies nearly three decades ago.

Disulfide Connectivity Analysis of Peptides Bearing Two Intramolecular Disulfide Bonds Using MALDI In-Source Decay.

Disulfide connectivity in peptides bearing at least two intramolecular disulfide bonds is highly important for the structure and the biological activity of the peptides. In that context, analytical strategies allowing a characterization of the cysteine pairing are of prime interest for chemists, biochemists, and biologists. For that purpose, this study evaluates the potential of MALDI in-source decay (ISD) for characterizing cysteine pairs through the systematic analysis of identical peptides bearing two disulfide bonds, but not the same cysteine connectivity. Three different matrices have been tested in positive and/or in negative mode (1,5-DAN, 2-AB and 2-AA). As MALDI-ISD is known to partially reduce disulfide bonds, the data analysis of this study rests firstly on the deconvolution of the isotope pattern of the parent ions. Moreover, data analysis is also based on the formed fragment ions and their signal intensities. Results from MS/MS-experiments (MALDI-ISD-MS/MS) constitute the last reference for data interpretation. Owing to the combined use of different ISD-promoting matrices, cysteine connectivity identification could be performed on the considered peptides. Graphical Abstract ᅟ.

Generic Workflow for Mapping of Complex Disulfide Bonds Using In-Source Reduction and Extracted Ion Chromatograms from Data-Dependent Mass Spectrometry.

Disulfide bond mapping is a critical task in protein characterization as protein stability, structure, and function is dependent on correct cysteine connectivities. Mass spectrometry (MS) is the method of choice for this, providing fast and accurate characterization of simple disulfide bonds. Disulfide mapping by liquid chromatography tandem mass spectrometry (LC-MS/MS) is performed by identifying disulfide-bonded partner peptides following proteolytic digestion. With the recently introduced ability to assign complex disulfide patterns by online postcolumn partial disulfide reduction by in-source reduction (ISR) in a LC-ISR-MS/MS methodology, the main challenge is data analysis to ensure detection of both expected and unexpected disulfide species. In this study, we introduced a workflow for confident and unbiased mapping of complex disulfide bonds using the powerful combination of extracted ion chromatograms (XICs) of LC-ISR-MS/MS data. With postcolumn partial reduction, identical LC retention times of intact disulfide-bonded species, their constituting free peptides, and partially reduced variants were observed. Subsequent selective MS/MS fragmentation of all reduction products allowed confident identification of free cysteine-containing peptides using a classical shotgun proteomics database search. Matching XICs of the identified cysteine-containing peptides allowed identification of both predicted and unpredicted disulfide species, including unforeseen proteolytic specificities, missed cleavage sites, scrambled disulfide variants, and the presence of disulfide-entangled complexes. Applying this workflow, we successfully mapped the complex disulfide bonds of tertiapin and the epidermal growth factor (EGF) family members transforming growth factor α (TGFα) and EGF. In addition, we were able to characterize the disulfide patterns of the special disulfide fold of the TGFβ superfamily in an all-online methodology.

Expression, Purification and Characterization of GMZ2'.10C, a Complex Disulphide-Bonded Fusion Protein Vaccine Candidate against the Asexual and Sexual Life-Stages of the Malaria-Causing Plasmodium falciparum Parasite.

Production and characterization of a chimeric fusion protein (GMZ2'.10C) which combines epitopes of key malaria parasite antigens: glutamate-rich protein (GLURP), merozoite surface protein 3 (MSP3), and the highly disulphide bonded Pfs48/45 (10C). GMZ2'.10C is a potential candidate for a multi-stage malaria vaccine that targets both transmission and asexual life-cycle stages of the parasite. GMZ2'.10C was produced in Lactococcus lactis and purified using either an immunoaffinity purification (IP) or a conventional purification (CP) method. Protein purity and stability was analysed by RP-HPLC, SEC-HPLC, 2-site ELISA, gel-electrophoresis and Western blotting. Structural characterization (mass analysis, peptide mapping and cysteine connectivity mapping) was performed by LC-MS/MS. CP-GMZ2'.10C resulted in similar purity, yield, structure and stability as compared to IP-GMZ2'.10C. CP-GMZ2'.10C and IP-GMZ2'.10C both elicited a high titer of transmission blocking (TB) antibodies in rodents. The intricate disulphide-bond connectivity of C-terminus Pfs48/45 was analysed by tandem mass spectrometry and was established for GMZ2'.10C and two reference fusion proteins encompassing similar parts of Pfs48/45. GMZ2'.10C, combining GMZ2' and correctly-folded Pfs48/45 can be produced by the Lactoccus lactis P170 based expression system in purity and quality for pharmaceutical development and elicit high level of TB antibodies. The cysteine connectivity for the 10C region of Pfs48/45 was revealed experimentally, providing an important guideline for employing the Pfs48/45 antigen in vaccine design.

Ion Mobility-Mass Spectrometry as a Tool for the Structural Characterization of Peptides Bearing Intramolecular Disulfide Bond(s).

Disulfide bonds are post-translationnal modifications that can be crucial for the stability and the biological activities of natural peptides. Considering the importance of these disulfide bond-containing peptides, the development of new techniques in order to characterize these modifications is of great interest. For this purpose, collision cross cections (CCS) of a large data set of 118 peptides (displaying various sequences) bearing zero, one, two, or three disulfide bond(s) have been measured in this study at different charge states using ion mobility-mass spectrometry. From an experimental point of view, CCS differences (ΔCCS) between peptides bearing various numbers of disulfide bonds and peptides having no disulfide bonds have been calculated. The ΔCCS calculations have also been applied to peptides bearing two disulfide bonds but different cysteine connectivities (Cys1-Cys2/Cys3-Cys4; Cys1-Cys3/Cys2-Cys4; Cys1-Cys4/Cys2-Cys3). The effect of the replacement of a proton by a potassium adduct on a peptidic structure has also been investigated. Graphical Abstract ᅟ.

Disulfide Linkage Characterization of Disulfide Bond-Containing Proteins and Peptides by Reducing Electrochemistry and Mass Spectrometry

Unravelling of disulfide linkage patterns is a crucial part of protein characterization, whether it is for a previously uncharacterized protein in basic research or a recombinant pharmaceutical protein. In the biopharmaceutical industry, elucidation of the cysteine connectivities is a necessity to avoid disulfide scrambled and incorrectly folded forms in the final product. Mass spectrometry (MS) is a highly utilized analytical tool for this due to fast and accurate characterization. However, disulfide bonds being an additional covalent bond in the protein structure represent a challenge in protein sequencing by tandem MS (MS/MS). Electrochemical (EC) reduction of disulfide bonds has recently been demonstrated to provide efficient reduction efficiencies, significantly enhancing sequence coverages in online coupling with MS characterization. In this study, the potential use of EC disulfide reduction in combination with MS characterization for disulfide mapping was assessed. We employed two approaches based on (1) the high flexibility and instant information about the degree of reduction in infusion EC-MS to generate partially reduced species on the intact protein level and (2) the preserved link between parent disulfide-linked fragments and free reduced peptides in an LC-EC-MS platform of nonreduced proteolytic protein digestions. Here we report the successful use of EC as a partial reduction approach in mapping of disulfide bonds of intact human insulin (HI) and lysozyme. In addition, we established a LC-EC-MS platform advantageous in disulfide characterization of complex and highly disulfide-bonded proteins such as human serum albumin (HSA) by online EC reduction of nonreduced proteolytic digestions.

Characterization of cysteine related variants in an IgG2 antibody by LC-MS with an automated data analysis approach.

In this communication, a high-throughput method for automated data analysis of cysteine-related product quality attributes (PQAs) in IgG2 antibodies is reported. This method leverages recent advances in the relative quantification of PQAs to facilitate the characterization of disulfide variants and free sulfhydryls (SHs) in IgG2 antibodies. The method uses samples labeled with a mass tag (N-ethyl maleimide [NEM]) followed by enzymatic digestion under non-reducing conditions to maintain the cysteine connectivity. The digested IgG2 samples are separated and detected by mass spectrometry (MS) and the resulting peptide map is analyzed in an automated fashion using Pinpoint software (Thermo Scientific). Previous knowledge of IgG2 disulfide structures can be fed into the Pinpoint software to create workbooks for various disulfide linkages and hinge disulfide variants. In addition, the NEM mass tag can be added to the workbooks for targeted analysis of labeled cysteine-containing peptides. The established Pinpoint workbooks are a high-throughput approach to quantify relative abundances of unpaired cysteines and disulfide linkages, including complicated hinge disulfide variants. This approach is especially efficient for comparing large sets of similar samples such as those created in comparability and stability studies or chromatographic fractions. Here, the high throughput method is applied to quantify the relative abundance of hinge disulfide variants and unpaired cysteines in the IgG2 fractions from non-reduced reversed-phase high-performance liquid chromatography (nrRP-HPLC). The LC-MS data analyzed by the Pinpoint workbook suggests that the nrRP-HPLC separated peaks contain hinge disulfide isoforms and free cysteine pairs for each major disulfide isoform structure.

Combined use of ion mobility and collision-induced dissociation to investigate the opening of disulfide bridges by electron-transfer dissociation in peptides bearing two disulfide bonds.

Disulfide bonds are post-translational modifications (PTMs) often found in peptides and proteins. They increase their stability toward enzymatic degradations and provide the structure and (consequently) the activity of such folded proteins. The characterization of disulfide patterns, i.e., the cysteine connectivity, is crucial to achieve a global picture of the active conformation of the protein of interest. Electron-transfer dissociation (ETD) constitutes a valuable tool to cleave the disulfide bonds in the gas phase, avoiding chemical reduction/alkylation in solution. To characterize the cysteine pairing, the present work proposes (i) to reduce by ETD one of the two disulfide bridges of model peptides, resulting in the opening of the cyclic structures, (ii) to separate the generated species by ion mobility, and (iii) to characterize the species using collision-induced dissociation (CID). Results of this strategy applied to several peptides show different behaviors depending on the connectivity. The loss of SH· radical species, observed for all the peptides, confirms the cleavage of the disulfides during the ETD process.

Isolation, purification and functional characterization of alpha-BnIA from Conus bandanus venom

We report the isolation and characterization by proteomic approach of a native conopeptide, named BnIA, from the crude venom of Conus bandanus, a molluscivorous cone snail species, collected in the South central coast of Vietnam. Its primary sequence was determined by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry using collision-induced dissociation and confirmed by Edman's degradation of the pure native fraction. BnIA was present in high amounts in the crude venom and the complete sequence of the 16 amino acid peptide was the following GCCSHPACSVNNPDIC*, with C-terminal amidation deduced from Edman's degradation and theoretical monoisotopic mass calculation. Sequence alignment revealed that its –C1C2X4C3X7C4– pattern belongs to the A-superfamily of conopeptides. The cysteine connectivity of BnIA was 1–3/2–4 as determined by partial-reduction technique, like other α4/7-conotoxins, reported previously on other Conus species. Additionally, we found that native α-BnIA shared the same sequence alignment as Mr1.1, from the closely related molluscivorous Conus marmoreus venom, in specimens collected in the same coastal region of Vietnam. Functional studies revealed that native α-BnIA inhibited acetylcholine-evoked currents reversibly in oocytes expressing the human α7 nicotinic acetylcholine receptors, and blocked nerve-evoked skeletal muscle contractions in isolated mouse neuromuscular preparations, but with ∼200-times less potency.

Ion Mobility Mass Spectrometry as a Potential Tool To Assign Disulfide Bonds Arrangements in Peptides with Multiple Disulfide Bridges

Disulfide bridges play a major role in defining the structural properties of peptides and proteins. However, the determination of the cysteine pairing is still challenging. Peptide sequences are usually achieved using tandem mass spectrometry (MS/MS) spectra of the totally reduced unfolded species, but the cysteine pairing information is lost. On the other hand, MS/MS experiments performed on native folded species show complex spectra composed of nonclassical ions. MS/MS alone does not allow either the cysteine pairing or the full sequence of an unknown peptide to be determined. The major goal of this work is to set up a strategy for the full structural characterization of peptides including disulfide bridges annotation in the sequence. This strategy was developed by combining ion mobility spectrometry (IMS) and collision-induced dissociation (CID). It is assumed that the opening of one S-S bridge in a peptide leads to a structural evolution which results in a modification of IMS drift time. In the presence of multiple S-S bridges, the shift in arrival time will depend on which disulfide(s) has (have) been reduced and on the shape adopted by the generated species. Due to specific fragmentations observed for each species, CID experiments performed after the mobility separation could provide not only information on peptide sequence but also on the localization of the disulfide bridges. To achieve this goal, synthetic peptides containing two disulfides were studied. The openings of the bridges were carried out following different experimental conditions such as reduction, reduction/alkylation, or oxidation. Due to disulfide scrambling highlighted with the reduction approaches, oxidation of S-S bonds into cysteic acids appeared to be the best strategy. Cysteine connectivity was then unambiguously determined for the two peptides, without any disulfide scrambling interference.

Conopeptide ρ-TIA Defines a New Allosteric Site on the Extracellular Surface of the α1B-Adrenoceptor

The G protein-coupled receptor (GPCR) superfamily is an important drug target that includes over 1000 membrane receptors that functionally couple extracellular stimuli to intracellular effectors. Despite the potential of extracellular surface (ECS) residues in GPCRs to interact with subtype-specific allosteric modulators, few ECS pharmacophores for class A receptors have been identified. Using the turkey β(1)-adrenergic receptor crystal structure, we modeled the α(1B)-adrenoceptor (α(1B)-AR) to help identify the allosteric site for ρ-conopeptide TIA, an inverse agonist at this receptor. Combining mutational radioligand binding and inositol 1-phosphate signaling studies, together with molecular docking simulations using a refined NMR structure of ρ-TIA, we identified 14 residues on the ECS of the α(1B)-AR that influenced ρ-TIA binding. Double mutant cycle analysis and docking confirmed that ρ-TIA binding was dominated by a salt bridge and cation-π between Arg-4-ρ-TIA and Asp-327 and Phe-330, respectively, and a T-stacking-π interaction between Trp-3-ρ-TIA and Phe-330. Water-bridging hydrogen bonds between Asn-2-ρ-TIA and Val-197, Trp-3-ρ-TIA and Ser-318, and the positively charged N terminus and Glu-186, were also identified. These interactions reveal that peptide binding to the ECS on transmembrane helix 6 (TMH6) and TMH7 at the base of extracellular loop 3 (ECL3) is sufficient to allosterically inhibit agonist signaling at a GPCR. The ligand-accessible ECS residues identified provide the first view of an allosteric inhibitor pharmacophore for α(1)-adrenoceptors and mechanistic insight and a new set of structural constraints for the design of allosteric antagonists at related GPCRs.

A novel disulphide switch mechanism in Ero1α balances ER oxidation in human cells

Oxidative maturation of secretory and membrane proteins in the endoplasmic reticulum (ER) is powered by Ero1 oxidases. To prevent cellular hyperoxidation, Ero1 activity can be regulated by intramolecular disulphide switches. Here, we determine the redox-driven shutdown mechanism of Ero1alpha, the housekeeping Ero1 enzyme in human cells. We show that functional silencing of Ero1alpha in cells arises from the formation of a disulphide bond-identified by mass spectrometry--between the active-site Cys(94) (connected to Cys(99) in the active enzyme) and Cys(131). Competition between substrate thiols and Cys(131) creates a feedback loop where activation of Ero1alpha is linked to the availability of its substrate, reduced protein disulphide isomerase (PDI). Overexpression of Ero1alpha-Cys131Ala or the isoform Ero1beta, which does not have an equivalent disulphide switch, leads to augmented ER oxidation. These data reveal a novel regulatory feedback system where PDI emerges as a central regulator of ER redox homoeostasis.

Structure and folding of disulfide‐rich miniproteins: Insights from molecular dynamics simulations and MM‐PBSA free energy calculations

The fold of small disulfide-rich proteins largely relies on two or more disulfide bridges that are main components of the hydrophobic core. Because of the small size of these proteins and their high cystine content, the cysteine connectivity has been difficult to ascertain in some cases, leading to uncertainties and debates in the literature. Here, we use molecular dynamics simulations and MM-PBSA free energy calculations to compare similar folds with different disulfide pairings in two disulfide-rich miniprotein families, namely the knottins and the short-chain scorpion toxins, for which the connectivity has been discussed. We first show that the MM-PBSA approach is able to discriminate the correct knotted topology of knottins from the laddered one. Interestingly, a comparison of the free energy components for kalata B1 and MCoTI-II suggests that cyclotides and squash inhibitors, although sharing the same scaffold, are stabilized through different interactions. Application to short-chain scorpion toxins suggests that the conventional cysteine pairing found in many homologous toxins is significantly more stable than the unconventional pairing reported for maurotoxin and for spinoxin. This would mean that native maurotoxin and spinoxin are not at the lowest free energy minimum and might result from kinetically rather than thermodynamically driven oxidative folding processes. For both knottins and toxins, the correct or conventional disulfide connectivities provide lower flexibilities and smaller deviations from the initial conformations. Overall, our work suggests that molecular dynamics simulations and the MM-PBSA approach to estimate free energies are useful tools to analyze and compare disulfide bridge connectivities in miniproteins.

Synaptic arrangement of the neuroligin/beta-neurexin complex revealed by X-ray and neutron scattering.

Neuroligins are postsynaptic cell-adhesion proteins that associate with their presynaptic partners, the neurexins. Using small-angle X-ray scattering, we determined the shapes of the extracellular region of several neuroligin isoforms in solution. We conclude that the neuroligins dimerize via the characteristic four-helix bundle observed in cholinesterases, and that the connecting sequence between the globular lobes of the dimer and the cell membrane is elongated, projecting away from the dimer interface. X-ray scattering and neutron contrast variation data show that two neurexin monomers, separated by 107 A, bind at symmetric locations on opposite sides of the long axis of the neuroligin dimer. Using these data, we developed structural models that delineate the spatial arrangements of different neuroligin domains and their partnering molecules. As mutations of neurexin and neuroligin genes appear to be linked to autism, these models provide a structural framework for understanding altered recognition by these proteins in neurodevelopmental disorders.

The Crystal Structure of the Costimulatory OX40-OX40L Complex

OX40 is a T cell costimulator activated by OX40L. Blockade of the OX40L-OX40 interaction has ameliorative effects in animal models of T cell pathologies. In order to better understand the interaction between OX40 and OX40L, we have determined the crystal structure of murine OX40L and of the human OX40-OX40L complex at 1.45 and 2.4 A, respectively. These structures show that OX40L is an unusually small member of the tumor necrosis factor superfamily (TNFSF). The arrangement of the OX40L protomers forming the functional trimer is atypical and differs from that of other members by a 15 degrees rotation of each protomer with respect to the trimer axis, resulting in an open assembly. Site-directed changes of the interfacial residues of OX40L suggest this interface lacks a single "hot spot" and that instead, binding energy is dispersed over at least two distinct areas. These structures demonstrate the structural plasticity of TNFSF members and their interactions with receptors.

Post-translational Modifications of Human Thrombin-Activatable Fibrinolysis Inhibitor (TAFI): Evidence for a Large Shift in the Isoelectric Point and Reduced Solubility upon Activation

Thrombin-activable fibrinolysis inhibitor (TAFI) is distinct from pancreatic procarboxypeptidase B in several ways. The enzymatic activity of TAFIa is unstable and decays with a half-life of a few minutes. During this study, we observed that (i) the isoelectric point (pI) of TAFI shifts dramatically from pH 5 toward pH 8 upon activation and (ii) TAFIa is significantly less soluble than TAFI. The structural bases for these observations were investigated by characterizing all post-translational modifications, including attached glycans and disulfide connectivity. The analyses revealed that all five potential N-glycosylation sites were utilized including Asn22, Asn51, Asn63, Asn86 (located in the activation peptide), and Asn219 (located in the catalytic domain). Asn219 was also found in an unglycosylated variant. Four of the glycans, Asn51, Asn63, Asn86, and Asn219 displayed microheterogeneity, while the glycan attached to Asn22 appeared to be homogeneous. In addition, bisecting GlcNAc attached to the trimannose core was detected, suggesting an origin other than the liver. Monosaccharide composition and LC-MS/MS analyses did not produce evidence for O glycosylation. TAFI contains eight cysteine residues, of which two, Cys69 and Cys383, are not involved in disulfides and contain free sulfhydryl groups. The remaining six cystines form disulfides, including Cys156-Cys169, Cys228-Cys252, and Cys243-Cys257. This pattern is homologous to pancreatic procarboxypeptidase B, and it is therefore unlikely that permutations in the cysteine connectivity are responsible for the enzymatic instability. LC-MS/MS analyses covering more than 90% of the TAFI amino acid sequence revealed no additional modifications. When these results are taken together, they suggest that the inherent instability of TAFIa is not caused by post-translational modifications. However, after activation, TAFIa loses 80% of the attached glycans, generating a large shift in pI and a propensity to precipitate. These changes are likely to significantly affect the properties of TAFIa as compared to TAFI.