cAMP protein kinase phosphorylates the Mos1 transposase and regulates its activity: evidences from mass spectrometry and biochemical analyses.

Genomic plasticity mediated by transposable elements can have a dramatic impact on genome integrity. To minimize its genotoxic effects, it is tightly regulated either by intrinsic mechanisms (linked to the element itself) or by host-mediated mechanisms. Using mass spectrometry, we show here for the first time that MOS1, the transposase driving the mobility of the mariner Mos1 element, is phosphorylated. We also show that the transposition activity of MOS1 is downregulated by protein kinase AMP cyclic-dependent phosphorylation at S170, which renders the transposase unable to promote Mos1 transposition. One step in the transposition cycle, the assembly of the paired-end complex, is specifically inhibited. At the cellular level, we provide evidence that phosphorylation at S170 prevents the active transport of the transposase into the nucleus. Our data suggest that protein kinase AMP cyclic-dependent phosphorylation may play a double role in the early stages of genome invasion by mariner elements.

New selective peptidyl di(chlorophenyl) phosphonate esters for visualizing and blocking neutrophil proteinase 3 in human diseases.

The function of neutrophil protease 3 (PR3) is poorly understood despite of its role in autoimmune vasculitides and its possible involvement in cell apoptosis. This makes it different from its structural homologue neutrophil elastase (HNE). Endogenous inhibitors of human neutrophil serine proteases preferentially inhibit HNE and to a lesser extent, PR3. We constructed a single-residue mutant PR3 (I217R) to investigate the S4 subsite preferences of PR3 and HNE and used the best peptide substrate sequences to develop selective phosphonate inhibitors with the structure Ac-peptidyl(P)(O-C6H4-4-Cl)2. The combination of a prolyl residue at P4 and an aspartyl residue at P2 was totally selective for PR3. We then synthesized N-terminally biotinylated peptidyl phosphonates to identify the PR3 in complex biological samples. These inhibitors resisted proteolytic degradation and rapidly inactivated PR3 in biological fluids such as inflammatory lung secretions and the urine of patients with bladder cancer. One of these inhibitors revealed intracellular PR3 in permeabilized neutrophils and on the surface of activated cells. They hardly inhibited PR3 bound to the surface of stimulated neutrophils despite their low molecular mass, suggesting that the conformation and reactivity of membrane-bound PR3 is altered. This finding is relevant for autoantibody binding and the subsequent activation of neutrophils in granulomatosis with polyangiitis (formerly Wegener disease). These are the first inhibitors that can be used as probes to monitor, detect, and control PR3 activity in a variety of inflammatory diseases.

Cotyledonary somatic embryos of Pinus pinaster Ait. most closely resemble fresh, maturing cotyledonary zygotic embryos: biological, carbohydrate and proteomic analyses.

Cotyledonary somatic embryos (SEs) of maritime pine are routinely matured for 12 weeks before being germinated and converted to plantlets. Although regeneration success is highly dependent on SEs quality, the date of harvesting is currently determined mainly on the basis of morphological features. This empirical method does not provide any accurate information about embryo quality with respect to storage compounds (proteins, carbohydrates). We first analyzed SEs matured for 10, 12 and 14 weeks by carrying out biological (dry weight, water content) and biochemical measurements (total protein and carbohydrate contents). No difference could be found between collection dates, suggesting that harvesting SEs after 12 weeks is appropriate. Cotyledonary SEs were then compared to various stages, from fresh to fully desiccated, in the development of cotyledonary zygotic embryos (ZEs). We identified profiles that were similar using hierarchical ascendant cluster analysis (HCA). Fresh and dehydrated ZEs could be distinguished, and SEs clustered with fresh ZEs. Both types of embryo exhibited similar carbohydrate and protein contents and signatures. This high level of similarity (94.5 %) was further supported by proteome profiling. Highly expressed proteins included storage, stress-related, late embryogenesis abundant and energy metabolism proteins. By comparing overexpressed proteins in developing and cotyledonary SEs or ZEs, some (23 proteins) could be identified as candidate biomarkers for the late, cotyledonary stage. This is the first report of useful generic protein markers for monitoring embryo development in maritime pine. Our results also suggest that improvements of SEs quality may be achieved if the current maturation conditions are refined.

In search of markers for somatic embryo maturation in hybrid larch (Larix × eurolepis): global DNA methylation and proteomic analyses.

A global DNA methylation and proteomics approach was used to investigate somatic embryo maturation in hybrid larch. Each developmental step during somatic embryogenesis was associated with a distinct and significantly different global DNA methylation level: from 45.8% mC for undifferentiated somatic embryos (1-week proliferation) to 61.5% mC for immature somatic embryos (1-week maturation), while maturation was associated with a decrease in DNA methylation to 53.4% for mature cotyledonary somatic embryos (8-weeks maturation). The presence of 5-azacytidine (hypo-methylating agent) or hydroxyurea (hyper-methylating agent) in the maturation medium altered the global DNA methylation status of the embryogenic cultures, and significantly reduced both their relative growth rate and embryogenic potential, suggesting an important role for DNA methylation in embryogenesis. Maturation was also assessed by examining changes in the total protein profile. Storage proteins, identified as legumin- and vicilin-like, appeared at the precotyledonary stage. In the proteomic study, total soluble proteins were extracted from embryos after 1 and 8 weeks of maturation, and separated by two-dimensional gel electrophoresis. There were 147 spots which showed significant differences between the stages of maturation; they were found to be involved mainly in primary metabolism and the stabilization of the resulting metabolites. This indicated that the somatic embryo was still metabolically active at 8 weeks of maturation. This is the first report of analyses of global DNA methylation (including the effects of hyper- and hypo-treatments) and proteome during somatic embryogenesis in hybrid larch, and thus provides novel insights into maturation of conifer somatic embryos.

Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water availability: transcriptomic and proteomic analyses.

Maritime pine somatic embryos (SEs) require a reduction in water availability (high gellan gum concentration in the maturation medium) to reach the cotyledonary stage. This key switch, reported specifically for pine species, is not yet well understood. To facilitate the use of somatic embryogenesis for mass propagation of conifers, we need a better understanding of embryo development. Comparison of both transcriptome (Illumina RNA sequencing) and proteome [two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with mass spectrometry (MS) identification] of immature SEs, cultured on either high (9G) or low (4G) gellan gum concentration, was performed, together with analysis of water content, fresh and dry mass, endogenous abscisic acid (ABA; gas chromatography-MS), soluble sugars (high-pressure liquid chromatography), starch and confocal laser microscope observations. This multiscale, integrated analysis was used to unravel early molecular and physiological events involved in SE development. Under unfavorable conditions (4G), the glycolytic pathway was enhanced, possibly in relation to cell proliferation that may be antagonistic to SE development. Under favorable conditions (9G), SEs adapted to culture constraint by activating specific protective pathways, and ABA-mediated molecular and physiological responses promoting embryo development. Our results suggest that on 9G, germin-like protein and ubiquitin-protein ligase could be used as predictive markers of SE development, whereas protein phosphatase 2C could be a biomarker for culture adaptive responses. This is the first characterization of early molecular mechanisms involved in the development of pine SEs following an increase in gellan gum concentration in the maturation medium, and it is also the first report on somatic embryogenesis in conifers combining transcriptomic and proteomic datasets.

Initial insights into structure-activity relationships of avian defensins.

Numerous ß-defensins have been identified in birds, and the potential use of these peptides as alternatives to antibiotics has been proposed, in particular to fight antibiotic-resistant and zoonotic bacterial species. Little is known about the mechanism of antibacterial activity of avian ß-defensins, and this study was carried out to obtain initial insights into the involvement of structural features or specific residues in the antimicrobial activity of chicken AvBD2. Chicken AvBD2 and its enantiomeric counterpart were chemically synthesized. Peptide elongation and oxidative folding were both optimized. The similar antimicrobial activity measured for both L- and D-proteins clearly indicates that there is no chiral partner. Therefore, the bacterial membrane is in all likelihood the primary target. Moreover, this work indicates that the three-dimensional fold is required for an optimal antimicrobial activity, in particular for gram-positive bacterial strains. The three-dimensional NMR structure of chicken AvBD2 defensin displays the structural three-stranded antiparallel ß-sheet characteristic of ß-defensins. The surface of the molecule does not display any amphipathic character. In light of this new structure and of the king penguin AvBD103b defensin structure, the consensus sequence of the avian ß-defensin family was analyzed. Well conserved residues were highlighted, and the potential strategic role of the lysine 31 residue of AvBD2 was emphasized. The synthetic AvBD2-K31A variant displayed substantial N-terminal structural modifications and a dramatic decrease in activity. Taken together, these results demonstrate the structural as well as the functional role of the critical lysine 31 residue in antimicrobial activity.

A sDOE (Simple Design-of-Experiment) Approach for Parameter Optimization in Mass Spectrometry. Part 1. Parameter Selection and Interference Effects in Top-Down ETD Fragmentation of Proteins in a UHR-QTOF Instrument.

Design-of-experiment (DOE) approaches, originally conceived by Fischer, are widely applied in industry, particularly in the context of production for which they have been greatly expended. In a research and development context, DOE can be of great use for method development. Specifically, DOE can greatly speed up instrument parameter optimization by first identifying parameters that are critical to a given outcome, showing parameter interdependency where it occurs and accelerating optimization of said parameters using matrices of experimental conditions. While DOE approaches have been applied in mass spectrometry experiments, they have so far failed to gain widespread adoption. This could be attributed to the fact that DOE can get quite complex and daunting to the everyday user. Here we make the case that a subset of DOE tools, hereafter called SimpleDOE (sDOE), can make DOE accessible and useful to the Mass Spectrometry community at large. We illustrate the progressive gains from a purely manual approach to sDOE through a stepwise optimization of parameters affecting the efficiency of top-down ETD fragmentation of proteins on a high-resolution Q-TOF mass spectrometer, where the aim is to maximize sequence coverage of fragmentation events.

Reproductives signature revealed by protein profiling and behavioral bioassays in termite.

Proteins are known to be social interaction signals in many species in the animal kingdom. Common mediators in mammals and aquatic species, they have seldom been identified as such in insects' behaviors. Yet, they could represent an important component to support social signals in social insects, as the numerous physical contacts between individuals would tend to favor the use of contact compounds in their interactions. However, their role in social interactions is largely unexplored: are they rare or simply underestimated? In this preliminary study, we show that, in the termite Reticulitermes flavipes, polar extracts from reproductives trigger body-shaking of workers (a vibratory behavior involved in reproductives recognition) while extracts from workers do not. Molecular profiling of these cuticular extracts using MALDI-TOF mass spectrometry reveals higher protein diversity in reproductives than in workers and a sex-specific composition exclusive to reproductives. While the effects observed with extracts are not as strong as with live termites, these results open up the intriguing possibility that social signaling may not be limited to cuticular hydrocarbons or other non-polar, volatile chemicals as classically accepted. Our results suggest that polar compounds, in particular some of the Cuticular Protein Compounds (CPCs) shown here by MALDI to be specific to reproductives, could play a significant role in insect societies. While this study is preliminary and further comprehensive molecular characterization is needed to correlate the body-shaking triggering effects with a given set of polar compounds, this exploratory study opens new perspectives for understanding the role of polar compounds such as proteins in caste discrimination, fertility signaling, or interspecific insect communication.

Interaction proteomics suggests a new role for the Tfs1 protein in yeast.

The PEBP (phosphatidylethanolamine-binding protein) family is a large group of proteins whose human member, hPEBP1, has been shown to play multiple functions, influencing intracellular signaling cascades, cell cycle regulation, neurodegenerative processes, and reproduction. It also acts, by an unknown mechanism, as a metastasis suppressor in a number of cancers. A more complete understanding of its biological role is thus necessary. As the yeast Saccharomyces cerevisiae is a powerful and easy to handle model organism, we focused on Tfs1p, the yeast ortholog of hPEBP1. In a previous study based on a two-hybrid approach, we showed that Tfs1p interacts and inhibits Ira2p, a GTPase Activating Protein (GAP) of the small GTPase Ras. To further characterize the molecular functions of Tfs1p, we undertook the identification of protein complexes formed around Tfs1p using a targeted proteomics approach. Complexed proteins were purified by tandem-affinity, cleaved with trypsin, and identified by nanoflow liquid chromatography coupled with tandem mass spectrometry. Overall, 14 new interactors were identified, including several proteins involved in intermediate metabolism. We confirmed by co-immunoprecipitation that Tfs1p interacts with Glo3p, a GAP for Arf GTPases belonging to the Ras superfamily of small GTPases, indicating that Tfs1p may be involved in the regulation of another GAP. We similarly confirmed the binding of Tfs1p with the metabolic enzymes Idp1p and Pro1p. Integration of these results with known functional partners of Tfs1p shows that two subnetworks meet through the Tfs1p node, suggesting that it may act as a bridge between cell signaling and intermediate metabolism in yeast.

Human proteinase 3 resistance to inhibition extends to alpha-2 macroglobulin.

Polymorphonuclear neutrophils contain at least four serine endopeptidases, namely neutrophil elastase (NE), proteinase 3 (PR3), cathepsin G (CatG), and NSP4, which contribute to the regulation of infection and of inflammatory processes. In physiological conditions, endogenous inhibitors including α2-macroglobulin (α2-M), serpins [α1-proteinase inhibitor (α1-PI)], monocyte neutrophil elastase inhibitor (MNEI), α1-antichymotrypsin, and locally produced chelonianins (elafin, SLPI) control excessive proteolytic activity of neutrophilic serine proteinases. In contrast to human NE (hNE), hPR3 is weakly inhibited by α1-PI and MNEI but not by SLPI. α2-M is a large spectrum inhibitor that traps a variety of proteinases in response to cleavage(s) in its bait region. We report here that α2-M was more rapidly processed by hNE than hPR3 or hCatG. This was confirmed by the observation that the association between α2-M and hPR3 is governed by a kass in the ≤ 105  m-1 ·s-1 range. Since α2-M-trapped proteinases retain peptidase activity, we first predicted the putative cleavage sites within the α2-M bait region (residues 690-728) using kinetic and molecular modeling approaches. We then identified by mass spectrum analysis the cleavage sites of hPR3 in a synthetic peptide spanning the 39-residue bait region of α2-M (39pep-α2-M). Since the 39pep-α2-M peptide and the corresponding bait area in the whole protein do not contain sequences with a high probability of specific cleavage by hPR3 and were indeed only slowly cleaved by hPR3, it can be concluded that α2-M is a poor inhibitor of hPR3. The resistance of hPR3 to inhibition by endogenous inhibitors explains at least in part its role in tissue injury during chronic inflammatory diseases and its well-recognized function of major target autoantigen in granulomatosis with polyangiitis.

Mass spectrometry of full-length integral membrane proteins to define functionally relevant structural features.

The crystallization and structure determination of integral membrane proteins remains a difficult task relying on a good understanding of the behavior of the protein for success. To date, membrane protein structures are still far outnumbered by soluble protein structures. Mass spectrometry is a powerful and versatile tool offering deep insights into the state of the integral membrane protein the structuralist intends to crystallize. With appropriate sample preparation methods, it provides information that can sometimes prove critical at various stages of the structure determination process, from protein expression to model building. Moreover, valuable knowledge is gained when the identified structural features underlie important functional aspects. Electrospray and matrix assisted laser desorption ionization (MALDI) methods, however, face a particular challenge when dealing with integral membrane proteins. A MALDI method specifically optimized for membrane protein analysis is presented here, with detailed information on the sample preparation and deposition, as well as guidelines for domain determination by limited proteolysis. MALDI-time of flight mass spectrometry can be used to do a proper inventory of initiation sites, to tailor a protein to a stable, well-folded form, and to evaluate selenomethionine replacement. These approaches are illustrated with a few examples drawn from the structural biology of ion channels.

Radiation-induced oxidative damage to the DNA-binding domain of the lactose repressor.

Understanding the cellular effects of radiation-induced oxidation requires the unravelling of key molecular events, particularly damage to proteins with important cellular functions. The Escherichia coli lactose operon is a classical model of gene regulation systems. Its functional mechanism involves the specific binding of a protein, the repressor, to a specific DNA sequence, the operator. We have shown previously that upon irradiation with gamma-rays in solution, the repressor loses its ability to bind the operator. Water radiolysis generates hydroxyl radicals (OH* radicals) which attack the protein. Damage of the repressor DNA-binding domain, called the headpiece, is most likely to be responsible of this loss of function. Using CD, fluorescence spectroscopy and a combination of proteolytic cleavage with MS, we have examined the state of the irradiated headpiece. CD measurements revealed a dose-dependent conformational change involving metastable intermediate states. Fluorescence measurements showed a gradual degradation of tyrosine residues. MS was used to count the number of oxidations in different regions of the headpiece and to narrow down the parts of the sequence bearing oxidized residues. By calculating the relative probabilities of reaction of each amino acid with OH. radicals, we can predict the most probable oxidation targets. By comparing the experimental results with the predictions we conclude that Tyr7, Tyr12, Tyr17, Met42 and Tyr47 are the most likely hotspots of oxidation. The loss of repressor function is thus correlated with chemical modifications and conformational changes of the headpiece.

Liquid Native MALDI Mass Spectrometry for the Detection of Protein-Protein Complexes.

Native mass spectrometry (MS) encompasses methods to keep noncovalent interactions of biomolecular complexes intact in the gas phase throughout the instrument and to measure the mass-to-charge ratios of supramolecular complexes directly in the mass spectrometer. Electrospray ionization (ESI) in nondenaturing conditions is now an established method to characterize noncovalent systems. Matrix-assisted laser desorption/ionization (MALDI), on the other hand, consumes low quantities of samples and largely tolerates contaminants, making it a priori attractive for native MS. However, so-called native MALDI approaches have so far been based on solid deposits, where the rapid transition of the sample through a solid state can engender the loss of native conformations. Here we present a new method for native MS based on liquid deposits and MALDI ionization, unambiguously detecting intact noncovalent protein complexes by direct desorption from a liquid spot for the first time. To control for aggregation, we worked with HUαß, a heterodimer that does not spontaneously rearrange into homodimers in solution. Screening through numerous matrix solutions to observe first the monomeric protein, then the dimer complex, we settled on a nondenaturing binary matrix solution composed of acidic and basic organic matrices in glycerol, which is stable in vacuo. The role of temporal and spatial laser irradiation patterns was found to be critical. Both a protein-protein and a protein-ligand complex could be observed free of aggregation. To minimize gas-phase dissociation, source parameters were optimized to achieve a conservation of complexes above 50% for both systems. Graphical Abstract ᅟ.

The Interaction between the Drosophila EAG Potassium Channel and the Protein Kinase CaMKII Involves an Extensive Interface at the Active Site of the Kinase.

The Drosophila EAG (dEAG) potassium channel is the founding member of the superfamily of KNCH channels, which are involved in cardiac repolarization, neuronal excitability and cellular proliferation. In flies, dEAG is involved in regulation of neuron firing and assembles with CaMKII to form a complex implicated in memory formation. We have characterized the interaction between the kinase domain of CaMKII and a 53-residue fragment of the dEAG channel that includes a canonical CaMKII recognition sequence. Crystal structures together with biochemical/biophysical analysis show a substrate-kinase complex with an unusually tight and extensive interface that appears to be strengthened by phosphorylation of the channel fragment. Electrophysiological recordings show that catalytically active CaMKII is required to observe active dEAG channels. A previously identified phosphorylation site in the recognition sequence is not the substrate for this crucial kinase activity, but rather contributes importantly to the tight interaction of the kinase with the channel. The available data suggest that the dEAG channel is a docking platform for the kinase and that phosphorylation of the channel's kinase recognition sequence modulates the strength of the interaction between the channel and the kinase.

SSPaQ: A Subtractive Segmentation Approach for the Exhaustive Parallel Quantification of the Extent of Protein Modification at Every Possible Site.

Protein modifications, whether chemically induced or post-translational (PTMs), play an essential role for the biological activity of proteins. Understanding biological processes and alterations thereof will rely on the quantification of these modifications on individual residues. Here we present SSPaQ, a subtractive method for the parallel quantification of the extent of modification at each possible site of a protein. The method combines uniform isotopic labeling and proteolysis with MS, followed by a segmentation approach, a powerful tool to refine the quantification of the degree of modification of a peptide to a segment containing a single modifiable amino acid. The strength of this strategy resides in: (1) quantification of all modifiable sites in a protein without prior knowledge of the type(s) of modified residues; (2) insensitivity to changes in the solubility and ionization efficiency of peptides upon modification; and (3) detection of missed cleavages caused by the modification for mitigation. The SSPaQ method was applied to quantify modifications resulting from the interaction of human phosphatidyl ethanolamine binding protein 1 (hPEBP1), a metastasis suppressor gene product, with locostatin, a covalent ligand and antimigratory compound with demonstrated activity towards hPEBP1. Locostatin is shown to react with several residues of the protein. SSPaQ can more generally be applied to induced modification in the context of drugs that covalently bind their target protein. With an alternate front-end protocol, it could also be applied to the quantification of protein PTMs, provided a removal tool is available for that PTM. Graphical Abstract ᅟ.

Discovery and characterisation of a novel toxin from Dendroaspis angusticeps, named Tx7335, that activates the potassium channel KcsA.

Due to their central role in essential physiological processes, potassium channels are common targets for animal toxins. These toxins in turn are of great value as tools for studying channel function and as lead compounds for drug development. Here, we used a direct toxin pull-down assay with immobilised KcsA potassium channel to isolate a novel KcsA-binding toxin (called Tx7335) from eastern green mamba snake (Dendroaspis angusticeps) venom. Sequencing of the toxin by Edman degradation and mass spectrometry revealed a 63 amino acid residue peptide with 4 disulphide bonds that belongs to the three-finger toxin family, but with a unique modification of its disulphide-bridge scaffold. The toxin induces a dose-dependent increase in both open probabilities and mean open times on KcsA in artificial bilayers. Thus, it unexpectedly behaves as a channel activator rather than an inhibitor. A charybdotoxin-sensitive mutant of KcsA exhibits similar susceptibility to Tx7335 as wild-type, indicating that the binding site for Tx7335 is distinct from that of canonical pore-blocker toxins. Based on the extracellular location of the toxin binding site (far away from the intracellular pH gate), we propose that Tx7335 increases potassium flow through KcsA by allosterically reducing inactivation of the channel.

Revealing Higher Order Protein Structure Using Mass Spectrometry.

The development of rapid, sensitive, and accurate mass spectrometric methods for measuring peptides, proteins, and even intact protein assemblies has made mass spectrometry (MS) an extraordinarily enabling tool for structural biology. Here, we provide a personal perspective of the increasingly useful role that mass spectrometric techniques are exerting during the elucidation of higher order protein structures. Areas covered in this brief perspective include MS as an enabling tool for the high resolution structural biologist, for compositional analysis of endogenous protein complexes, for stoichiometry determination, as well as for integrated approaches for the structural elucidation of protein complexes. We conclude with a vision for the future role of MS-based techniques in the development of a multi-scale molecular microscope. Graphical Abstract ᅟ.

Molecular Insights into the Mechanism of Calmodulin Inhibition of the EAG1 Potassium Channel.

The human EAG1 potassium channel belongs to the superfamily of KCNH voltage-gated potassium channels that have roles in cardiac repolarization and neuronal excitability. EAG1 is strongly inhibited by Ca2+/calmodulin (CaM) through a mechanism that is not understood. We determined the binding properties of CaM with each one of three previously identified binding sites (BDN, BDC1, and BDC2), analyzed binding to protein stretches that include more than one site, and determined the effect of neighboring globular domains on the binding properties. The determination of the crystal structure of CaM bound to BDC2 shows the channel fragment interacting with only the C lobe of calmodulin and adopting an unusual bent conformation. Based on this structure and on a functional and biochemical analysis of mutants, we propose a model for the mechanism of inhibition whereby the local conformational change induced by CaM binding at BDC2 lies at the basis of channel modulation.

Improved accuracy of low affinity protein-ligand equilibrium dissociation constants directly determined by electrospray ionization mass spectrometry.

There is continued interest in the determination by ESI-MS of equilibrium dissociation constants (K(D)) that accurately reflect the affinity of a protein-ligand complex in solution. Issues in the measurement of K(D) are compounded in the case of low affinity complexes. Here we present a K(D) measurement method and corresponding mathematical model dealing with both gas-phase dissociation (GPD) and aggregation. To this end, a rational mathematical correction of GPD (f(sat)) is combined with the development of an experimental protocol to deal with gas-phase aggregation. A guide to apply the method to noncovalent protein-ligand systems according to their kinetic behavior is provided. The approach is validated by comparing the K(D) values determined by this method with in-solution K(D) literature values. The influence of the type of molecular interactions and instrumental setup on f(sat) is examined as a first step towards a fine dissection of factors affecting GPD. The method can be reliably applied to a wide array of low affinity systems without the need for a reference ligand or protein.

Crystal structure of greglin, a novel non-classical Kazal inhibitor, in complex with subtilisin.

Greglin is an 83-residue serine protease inhibitor purified from the ovaries of the locust Schistocerca gregaria. Greglin is a strong inhibitor of subtilisin and human neutrophil elastase, acting at sub-nanomolar and nanomolar concentrations, respectively; it also inhibits neutrophil cathepsin G, α-chymotrypsin and porcine pancreatic elastase, but to a lesser extent. In the present study, we show that greglin resists denaturation at high temperature (95 °C) and after exposure to acetonitrile and acidic or basic pH. Greglin is composed of two domains consisting of residues 1-20 and 21-83. Mass spectrometry indicates that the N-terminal domain (1-20) is post-translationally modified by phosphorylations at three sites and probably contains a glycosylation site. The crystal structure of the region of greglin comprising residues 21-78 in complex with subtilisin was determined at 1.75 Å resolution. Greglin represents a novel member of the non-classical Kazal inhibitors, as it has a unique additional C-terminal region (70-83) connected to the core of the molecule via a supplementary disulfide bond. The stability of greglin was compared with that of an ovomucoid inhibitor. The thermostability and inhibitory specificity of greglin are discussed in light of its structure. In particular, we propose that the C-terminal region is responsible for non-favourable interactions with the autolysis loop (140-loop) of serine proteases of the chymotrypsin family, and thus governs specificity. DATABASE: The atomic coordinates and structure factors for the greglin-subtilisin complex have been deposited with the RCSB Protein Data Bank under accession number 4GI3. STRUCTURED DIGITAL ABSTRACT: Greglin and Subtilisin Carlsberg bind by X-ray crystallography (View interaction).