Computational and biochemical docking of the irreversible cocaine analog RTI 82 directly demonstrates ligand positioning in the dopamine transporter central substrate-binding site.

The dopamine transporter (DAT) functions as a key regulator of dopaminergic neurotransmission via re-uptake of synaptic dopamine (DA). Cocaine binding to DAT blocks this activity and elevates extracellular DA, leading to psychomotor stimulation and addiction, but the mechanisms by which cocaine interacts with DAT and inhibits transport remain incompletely understood. Here, we addressed these questions using computational and biochemical methodologies to localize the binding and adduction sites of the photoactivatable irreversible cocaine analog 3ß-(p-chlorophenyl)tropane-2ß-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([(125)I]RTI 82). Comparative modeling and small molecule docking indicated that the tropane pharmacophore of RTI 82 was positioned in the central DA active site with an orientation that juxtaposed the aryliodoazide group for cross-linking to rat DAT Phe-319. This prediction was verified by focused methionine substitution of residues flanking this site followed by cyanogen bromide mapping of the [(125)I]RTI 82-labeled mutants and by the substituted cysteine accessibility method protection analyses. These findings provide positive functional evidence linking tropane pharmacophore interaction with the core substrate-binding site and support a competitive mechanism for transport inhibition. This synergistic application of computational and biochemical methodologies overcomes many uncertainties inherent in other approaches and furnishes a schematic framework for elucidating the ligand-protein interactions of other classes of DA transport inhibitors.

Daily bursts of biogenic cyanogen bromide (BrCN) control biofilm formation around a marine benthic diatom.

The spatial organization of biofilms is strongly regulated by chemical cues released by settling organisms. However, the exact nature of these interactions and the repertoire of chemical cues and signals that micro-organisms produce and exude in response to the presence of competitors remain largely unexplored. Biofilms dominated by microalgae often show remarkable, yet unexplained fine-scale patchy variation in species composition. Because this occurs even in absence of abiotic heterogeneity, antagonistic interactions might play a key role. Here we show that a marine benthic diatom produces chemical cues that cause chloroplast bleaching, a reduced photosynthetic efficiency, growth inhibition and massive cell death in naturally co-occurring competing microalgae. Using headspace solid phase microextraction (HS-SPME)-GC-MS, we demonstrate that this diatom exudes a diverse mixture of volatile iodinated and brominated metabolites including the natural product cyanogen bromide (BrCN), which exhibits pronounced allelopathic activity. Toxin production is light-dependent with a short BrCN burst after sunrise. BrCN acts as a short-term signal, leading to daily "cleaning" events around the algae. We show that allelopathic effects are H(2)O(2) dependent and link BrCN production to haloperoxidase activity. This strategy is a highly effective means of biofilm control and may provide an explanation for the poorly understood role of volatile halocarbons from marine algae, which contribute significantly to the atmospheric halocarbon budget.

Hypothiocyanous acid oxidation of tubulin cysteines inhibits microtubule polymerization.

Thiol oxidation is a probable outcome of cellular oxidative stress and is linked to degenerative disease progression. In addition, protein thiol redox reactions are increasingly identified as a mechanism to regulate protein structure and function. We assessed the effect of hypothiocyanous acid on the cytoskeletal protein tubulin. Total cysteine oxidation by hypothiocyanous and hypochlorous acids was monitored by labeling tubulin with 5-iodoacetamidofluorescein and by detecting higher molecular weight inter-chain tubulin disulfides by Western blot under nonreducing conditions. Hypothiocyanous acid induced nearly stoichiometric oxidation of tubulin cysteines (1.9 mol cysteine/mol oxidant) and no methionine oxidation was observed. Because disulfide reducing agents restored all the polymerization activity that was lost due to oxidant treatment, we conclude that cysteine oxidation of tubulin inhibits microtubule polymerization. Hypothiocyanous acid oxidation of tubulin cysteines was markedly decreased in the presence of 4% glycerol, a component of the tubulin purification buffer. Due to its instability and buffer- and pH-dependent reactivity, hypothiocyanous acid studies require careful consideration of reaction conditions.

A PagP fusion protein system for the expression of intrinsically disordered proteins in Escherichia coli.

PagP, a beta-barrel membrane protein found in Gram-negative bacteria, expresses robustly in inclusion bodies when its signal sequence is removed. We have developed a new fusion protein expression system based on PagP and demonstrated its utility in the expression of the unstructured N-terminal region of human cardiac troponin I (residues 1-71). A yield of 100mg fusion protein per liter M9 minimal media was obtained. The troponin I fragment was removed from PagP using cyanogen bromide cleavage at methionine residues followed by nickel affinity chromatography. We further demonstrate that optimal cleavage requires complete reduction of methionine residues prior to cyanogen bromide treatment, and this is effectively accomplished using potassium iodide under acidic conditions. The PagP-based fusion protein system is more effective at targeting proteins into inclusion bodies than a commercially available system that uses ketosteroid isomerase; it thus represents an important advance for producing large quantities of unfolded peptides or proteins in Escherichia coli.

MudPIT analysis: application to human heart tissue.

Although two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has been used as the standard proteomic approach for separating proteins in a complex mixture, this technique has many drawbacks. These include a limited molecular mass range, poor separation of highly acidic or basic proteins, and exclusion of the majority of membrane proteins from analysis. Considering the important functions of many membrane proteins, such as receptors, ion transporters, signal transducers, and cell adhesion proteins, it is increasingly important that these proteins are not excluded during the global proteomic analysis of cellular systems. Multidimensional Protein Identification Technology (MudPIT) offers a gel-free alternative to 2D-PAGE for the analysis of both membrane and soluble proteins.The goal of this chapter is to provide detailed methods for using MudPIT to profile both membrane and soluble proteins in complex unfractionated samples. Methods discussed will include tissue homogenization, sample preparation, MudPIT, data analysis, and an application for the analysis of unfractionated total tissue homogenate from human heart.

Probing the steroid binding domain-like I (SBDLI) of the sigma-1 receptor binding site using N-substituted photoaffinity labels.

Radioiodinated photoactivatable photoprobes can provide valuable insights regarding protein structure. Previous work in our laboratory showed that the cocaine derivative and photoprobe 3-[ (125)I]iodo-4-azidococaine ([ (125)I]IACoc) binds to the sigma-1 receptor with 2-3 orders of magnitude higher affinity than cocaine [Kahoun, J. R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1393-1397]. Using this photoprobe, we demonstrated the insertion site for [ (125)I]IACoc to be Asp188 [Chen, Y. (2007) Biochemistry 46, 3532-3542], which resides in the proposed steroid binding domain-like II (SBDLII) region (amino acids 176-194) [Pal, A. (2007) Mol. Pharmacol. 72, 921-933]. An additional photoprobe based on the sigma-1 receptor ligand fenpropimorph, 1- N-(2-3-[ (125)I]iodophenyl)propane ([ (125)I]IAF), was found to label a peptide in both the SBDLII and steroid binding domain-like I (SBDLI) (amino acids 91-109) [Pal, A. (2007) Mol. Pharmacol. 72, 921-933]. In this report, we describe two novel strategically positioned carrier-free, radioiodinated photoaffinity labels specifically designed to probe the putative "nitrogen interacting region" of sigma-1 receptor ligands. These two novel photoprobes are (-)-methyl 3-(benzoyloxy)-8-2-(4-azido-3-[ (125)I]iodobenzene)-1-ethyl-8-azabicyclo[3.2.1]octane-2-carboxylate ([ (125)I]-N-IACoc) and N-propyl- N-(4-azido-3-iodophenylethyl)-3-(4-fluorophenyl)propylamine ([ (125)I]IAC44). In addition to reporting their binding affinities to the sigma-1 and sigma-2 receptors, we show that both photoaffinity labels specifically and covalently derivatized the pure guinea pig sigma-1 receptor (26.1 kDa) [Ramachandran, S. (2007) Protein Expression Purif. 51, 283-292]. Cleavage of the photolabeled sigma-1 receptor using Endo Lys C and cyanogen bromide (CNBr) revealed that the [ (125)I]-N-IACoc label was located primarily in the N-terminus and SBDLI-containing peptides of the sigma-1 receptor, while [ (125)I]IAC44 was found in peptide fragments consistent with labeling of both SBDLI and SBDLII.

Labeling of dopamine transporter transmembrane domain 1 with the tropane ligand N-[4-(4-azido-3-[125I]iodophenyl)butyl]-2beta-carbomethoxy-3beta-(4-chlorophenyl)tropane implicates proximity of cocaine and substrate active sites.

The novel photoaffinity ligand N-[4-(4-azido-3-(125)I-iodophenyl)-butyl]-2-beta-carbomethoxy-3beta-(4-chlorophenyl) tropane ([(125)I]MFZ 2-24) was used to investigate the site for cocaine binding on the dopamine transporter (DAT). [(125)I]MFZ 2-24 irreversibly labeled both rat striatal and expressed human DAT with high affinity and appropriate pharmacological specificity. Tryptic proteolysis of [(125)I]MFZ 2-24 labeled DAT followed by epitope-specific immunoprecipitation demonstrated that the ligand becomes adducted almost exclusively to transmembrane domains (TMs) 1-2. Further localization of [(125)I]MFZ 2-24 incorporation achieved by proteolyzing labeled wild-type and methionine mutant DATs with cyanogen bromide identified the sequence between residues 68 and 80 in TM1 as the ligand adduction site. This is in marked contrast to the previously identified attachment of the photoaffinity label [(125)I]RTI 82 in TM6. Because [(125)I]MFZ 2-24 and [(125)I]RTI 82 possess identical tropane pharmacophores and differ only in the placement of the reactive azido moieties, their distinct incorporation profiles identify the regions of the protein adjacent to different aspects of the cocaine molecule. These findings thus strongly support the direct interaction of cocaine on DAT with TM1 and TM6, both of which have been implicated by mutagenesis and homology to a bacterial leucine transporter as active sites for substrates. These results directly establish the proximity of TMs 1 and 6 in DAT and suggest that the mechanism of transport inhibition by cocaine involves close interactions with multiple regions of the substrate permeation pathway.

Hydrophobic interaction chromatography of soluble interleukin I receptor type II to reveal chemical degradations resulting in loss of potency.

A hydrophobic interaction chromatography method was developed to analyze recombinant soluble Interleukin 1 receptor type II (sIL-1R type II) drug substance and assess the stability of the drug under accelerated degradation studies. HIC resolved the degraded molecules into three peaks. A combination of several analytical techniques, including cyanogen bromide cleavage, reversed-phase chromatography, mass spectrometry, and N-terminal sequencing, were used to identify the origins of these peaks. We found that accelerated degradation resulted from three different events, deamidation and isomerization at asparagine 317 (Asn317), C-terminal cleavage, and aggregation. The iso-aspartate 317 (iso-Asp317)-containing species were shown to elute in HIC peak I and the Asp317-containing species in HIC peak II, respectively. Deamidation-isomerization to iso-Asp317, but not deamidation to Asp317, resulted in altered retention time on HIC companied by loss of potency, presumably by introducing a significant conformational change. CNBr C-terminal analysis showed that the inactive HIC peak I consisted of sIL-1R type II with "large" C-terminal truncations of 13 or 14 amino acids, whereas the active HIC peak II contained C-terminally full length and "small" C-terminal clips of two amino acids. Molecular modeling indicates that the short loop D317-S320, in the third domain of IL-1R type II, has a crucial impact on the stability of the molecule.

The role of the S-S bridge in retroviral protease function and virion maturation.

Retroviral proteases are translated as a part of Gag-related polyproteins, and are released and activated during particle release. Mason-Pfizer monkey virus (M-PMV) Gag polyproteins assemble into immature capsids within the cytoplasm of the host cells; however, their processing occurs only after transport to the plasma membrane and subsequent release. Thus, the activity of M-PMV protease is expected to be highly regulated during the replication cycle. It has been proposed that reversible oxidation of protease cysteine residues might be responsible for such regulation. We show that cysteine residues in M-PMV protease can form an intramolecular S-S bridge. The disulfide bridge shifts the monomer/dimer equilibrium in favor of the dimer, and increases the proteolytic activity significantly. To investigate the role of this disulfide bridge in virus maturation and replication, we engineered an M-PMV clone in which both protease cysteine residues were replaced by alanine (M-PMV(PRC7A/C106A)). Surprisingly, the cysteine residues were dispensable for Gag polyprotein processing within the virus, indicating that even low levels of protease activity are sufficient for polyprotein processing during maturation. However, the long-term infectivity of M-PMV(PRC7A/C106A) was noticeably compromised. These results show clearly that the proposed redox mechanism does not rely solely on the formation of the stabilizing S-S bridge in the protease. Thus, in addition to the protease disulfide bridge, reversible oxidation of cysteine and/or methionine residues in other domains of the Gag polyprotein or in related cellular proteins must be involved in the regulation of maturation.

Identification of carriers of a variant plasma prealbumin (transthyretin) associated with familial amyloidotic polyneuropathy type I.

A method is described for detecting carriers of a variant plasma prealbumin that is associated with familial amyloidotic polyneuropathy (FAP) type I. It is based on the finding of an extra methionine in the variant prealbumin, at position 30 from the amino terminals. Since normal prealbumin has only one methionine (position 13), treatment with cyanogen bromide (CNBr), which cleaves only at methionines, results in two peptides. CNBr treatment of the variant prealbumin gives three peptides. The extra can then be detected in two ways: by HPLC using a reverse phase C18 column, and by sequential Edman degradation. Each method can detect as little as 1% variant prealbumin in isolated plasma prealbumin, and therefore, can identify carriers of the gene for the variant protein. Since FAP type I usually is not manifest until after the childbearing years, this method to identify carriers of the gene offers a new approach for genetic counseling of families with this disease. To date, kindreds with hereditary amyloidosis that could benefit from these studies include those with FAP type I of Swedish, Japanese, and Portuguese origins.

A C-terminal region of RAG1 contacts the coding DNA during V(D)J recombination.

The site-specific DNA rearrangement process, called V(D)J recombination, creates much of the diversity of immune receptor molecules in the adaptive immune system. Central to this reaction is the organization of the protein-DNA complex containing the proteins RAG1 and RAG2 and their DNA targets. A long-term goal is to appreciate the three-dimensional relationships between the proteins and DNA that allow the assembly of the appropriate reaction intermediates, resulting in concerted cleavage and directed rejoining of the DNA ends. Previous cross-linking approaches have mapped RAG1 contacts on the DNA. RAG1 protein contacts the DNA at the conserved heptamer and nonamer sequences as well as at the coding DNA adjacent to the heptamer. Here we subject RAG1, covalently cross-linked to DNA substrates, to partial cyanogen bromide degradation or trypsin proteolysis in order to map contacts on the protein. We find that coding-sequence contacts occur near the C terminus of RAG1, while contacts made within the recombination signal sequence occur nearer the N terminus of the core region of RAG1. A deletion protein lacking the C-terminal DNA-contacting region is still capable of making the N-terminal contacts. This suggests that the two binding interactions may exist on two separate domains of the protein. A trypsin cleavage pattern of the native protein supports this conclusion. A two-domain model for RAG1 is evaluated with respect to the larger recombination complex.

Posttranslational modification of tubulin by palmitoylation: II. Identification of sites of palmitoylation.

As shown in the companion article, tubulin is posttranslationally modified in vivo by palmitoylation. Our goal in this study was to identify the palmitoylation sites by protein structure analysis. To obtain quantities of palmitoylated tubulin required for this analysis, a cell-free system for enzymatic [3H]palmitoylation was developed and characterized in our companion article. We then developed a methodology to examine directly the palmitoylation of all 451 amino acids of alpha-tubulin. 3H-labeled palmitoylated alpha-tubulin was cleaved with cyanogen bromide (CNBr). The CNBr digest was resolved according to peptide size by gel filtration on Sephadex LH60 in formic acid:ethanol. The position of 3H-labeled palmitoylated amino acids in peptides could not be identified by analysis of the Edman degradation sequencer product because the palmitoylated sequencer products were lost during the final derivatization step to phenylthiohydantoin derivatives. Modification of the gas/liquid-phase sequencer to deliver the intermediate anilinothiozolinone derivative, rather than the phenylthiohydantoin derivative, identified the cycle containing the 3H-labeled palmitoylated residue. Therefore, structure analysis of peptides obtained from gel filtration necessitated dual sequencer runs of radioactive peptides, one for sequence analysis and one to identify 3H-labeled palmitoylated amino acids. Further cleavage of the CNBr peptides by trypsin and Lys-C protease, followed by gel filtration on Sephadex LH60 and dual sequencer runs, positioned the 3H-labeled palmitoylated amino acid residues in peptides. Integration of all the available structural information led to the assignment of the palmitoyl moiety to specific residues in alpha-tubulin. The palmitoylated residues in alpha-tubulin were confined to cysteine residues only. The major site for palmitoylation was cysteine residue 376.

Preparation and properties of glucagon analogs prepared by semi-synthesis from CNBr-glucagon.

The semi-synthetic approach has been used to obtain new analogs of the peptide hormone glucagon. Using the highly purified 27 amino acid fragment of cyanogen bromide-treated glucagon, we have prepared, by nucleophilic addition to the lactone ring, the following derivatives: CNBr-Gly28-glucagon, CNBr-glucagon hydrazide, CNBr-glucagon n-butylamide and CNBr-glucagon biotinamide. Direct aminolysis of the lactone was successful only with sterically unhindered primary amines. Addition of an amino acid could be accomplished by formation of the peptide hydrazide followed by azide coupling. All these analogs were full agonists with decreased potency relative to the native hormone. Examination of the structure-function relationships of these new C-terminal glucagon derivatives suggests that the hydrophobic side-chain of methionine is important to the binding of glucagon to its receptor and that the C-terminal portion of glucagon is only involved in the binding of the hormone to the receptor and not in the transduction process.

Identification of the creatine binding domain of creatine kinase by photoaffinity labeling.

A new photoaffinity probe with a benzophenone group, N-dibenzylphospho-N'-(4-benzoyl)-benzylguanidine (BzPG), has been synthesized on the basis on our previously described creatine kinase bisubstrate analog. BzPG is also a bisubstrate type analog whose photoinsertion is inhibited by the natural substrates of creatine kinase. When rabbit CK-MM is irradiated in the presence of BzPG then cleaved by CNBr, one labeled peptide can be purified by reverse phase HPLC and sequenced. This sequence of 31 amino acids (Ala30-Val60) contains a region which could be responsible for isoenzyme selectivity and another one just preceding the 11 amino acid peptide (Asp61-Thr70) very recently described as a putative creatine binding site. This second peptide was deduced from the comparison of 18 amino acid sequence alignments. We proposed the creatine binding site to be essentially a peptide from Lys39 to Val71.

Collagen in the ageing human intervertebral disc: an increase in covalently bound fluorophores and chromophores.

Human disc tissue gradually changes in colour from white in the young to yellow brown in the elderly. It was investigated to what degree this colouration and an associated fluorescence (which are characteristic of the non-enzymic reaction products of sugars or oxidized lipids with proteins), were the result of covalent derivatives on the collagen and other extracellular matrix proteins. Annulus fibrosus was obtained from four subjects aged 19 to 92 years. Papain-solubilized samples of tissue showed increasing yellow colour and the glycation-related fluorescence with age. On chromatography of CNBr-digests of tissue, the yellow colour and fluorescence remained bound to the CNBr-peptide fragments of the collagen and other matrix proteins. One peptide, alpha 1(II)CB12 (from type II collagen), was selected for purification and shown to contain increasing amounts of the characteristic fluorescence with age. Sequencing by Edman degradation over 24 cycles confirmed the identity of the peptide, and by analysis of a portion of the PTH-derivatives showed fluorescence at cycle 11, a lysine residue. The results imply that much of the yellow colour and characteristic fluorescence that accumulate in ageing human discs are contributed by covalent adducts (possibly derived from non-enzymic reactions with carbohydrates or lipids) linked to the collagen and probably to other long-lived matrix proteins. The disc is perhaps particularly susceptible to such protein modifications because, being large and avascular, of its tendency to a low oxygen tension. Such modifications to structural proteins may contribute to the commonly observed degeneration and impaired material function of ageing disc tissue.

Antigenic determinants and reactive sites of a trypsin/chymotrypsin double-headed inhibitor from horse gram (Dolichos biflorus).

The major proteinase inhibitor in horse gram (Dolichos biflorus) is a low molecular weight (approximately 8500) Bowman-Birk inhibitor (BBI), HGI-III, that inhibits both trypsin and chymotrypsin simultaneously. Analysis of the reactivity of the polyclonal antibodies raised against native HGI-III, with tryptic, lysylendoproteinase-C and CNBr peptides, in dot-blot assays, revealed the presence of three sequential epitopes (Asp1-Lys14 (I), Leu37-Lys63 (II) and Asp64-Lys71 (III)). Of these, epitope II and III occur consecutively in the sequence of HGI-III. The reactive site peptide bonds were identified by cleavage with catalytic quantities of either trypsin or chymotrypsin at acidic pH. The reactive site peptide bond for trypsin was found to be Lys24-Ser25, whereas for chymotrypsin it was Phe51-Ser52. The highly conserved reactive site loop residues of the Bowman-Birk inhibitors are also conserved in HGI-III. The less immunogenic peptide sequence, Leu37-Lys63, also contains the chymotrypsin reactive site. The recognition of this polypeptide by the immune system provides for a new strategy in the design of ideal, smaller proteinase inhibitors as cancer preventive agents.

Antibodies to a fragment of the Bothrops moojenil-amino acid oxidase cross-react with snake venom components unrelated to the parent protein.

It is widely accepted that immunological cross-reactivity of snake venoms is mediated by antibodies that recognize venom components bearing either amino acid sequence homology or similar biological functions. However, here we demonstrate that polyspecific Bothrops antivenom is a source of cross-reactive antibodies that interact with venom proteins of distinctive primary structures and biological functions. The homoserine lactone derivative of the undecapeptide IQRWSLDKYAM (Ile1-Hse11), excised from the l-amino acid oxidase (LAAO) of the Bothrops moojeni venom, was the ligand of an affinity resin used to isolate specific anti-Ile1-Hse11 antibodies which were instrumental in revealing immunological cross-reactivity among unrelated venom proteins. We examined the extent of the cross-reactivity of these antibodies by probing electroblots of venoms from representative snakes of genera Bothrops, Lachesis, Crotalus and Micrurus, and by unambiguous structural characterization of the affinity-purified proteins of B. moojeni venom recovered from an agarose-anti-Ile1-Hse11 column. Our results indicate that all venoms tested had at least three reactive components toward anti-Ile1-Hse11 antibodies, among which we identified two serine proteases, one phospholipase A2 homologue, and LAAO. We hypothesize that the cross-reactivity of the anti-Ile1-Hse11 antibodies to unrelated venom proteins derives from their mechanism of antigen recognition, whereby complementarity is achieved through reciprocal conformational adaptation of the reacting molecules. Also, we believe these findings have implications both in the development of improved antivenoms and the preparation of immunochemical reagents for diagnostic and scientific investigation purposes in the field of snake venoms.

Identification of the growth hormone-releasing peptide binding site in CD36: a photoaffinity cross-linking study.

The GHRPs (growth hormone-releasing peptides) are a class of small synthetic peptides known to stimulate GH release through binding of a G-protein-coupled receptor (designated GHS-R). We have found that hexarelin, a hexapeptide member of the GHRPs, binds to another protein identified as CD36, a scavenger receptor that is expressed in various tissues, including monocytes/macrophages and the endothelial microvasculature. CD36 is involved in the endocytosis of oxLDL (oxidized low-density lipoprotein) by macrophages, and in the modulation of angiogenesis elicited by thrombospondin-1 through binding to endothelial cells. To define the binding domain for hexarelin on CD36, covalent photolabelling of CD36 followed by enzymic and chemical degradation of the photoligand-receptor complex was performed. A 8 kDa photolabelled fragment corresponding to the CD36-(Asn132-Glu177) sequence has been identified as the hexarelin-binding site. Chemical cleavage of this fragment with CNBr resulted in the release of the free ligand, suggesting that Met169 is the contact point for the ligand within the receptor binding pocket. We conclude that the binding domain for hexarelin on CD36 overlaps with that for oxLDL, which corresponds to residues Gln155-Lys183 of CD36. Hence hexarelin might interfere with the CD36-mediated uptake of modified lipoproteins by macrophages. This may contribute, at least in part, to the anti-atherosclerotic effect of GHRPs in apolipoprotein E-deficient mice.

Structural/functional properties of the Glu1-HSer57 N-terminal fragment of human plasminogen: conformational characterization and interaction with kringle domains.

The Glu1-Val79 N-terminal peptide (NTP) domain of human plasminogen (Pgn) is followed by a tandem array of five kringle (K) structures of approximately 9 kDa each. K1, K2, K4, and K5 contain each a lysine-binding site (LBS). Pgn was cleaved with CNBr and the Glul-HSer57 N-terminal fragment (CB-NTP) isolated. In addition, the Ile27-Ile56 peptide (L-NTP) that spans the doubly S-S bridged loop segment of NTP was synthesized. Pgn kringles were generated either by proteolytic fragmentation of Pgn (K4, K5) or via recombinant gene expression (rK1, rK2, and rK3). Interactions of CB-NTP with each of the Pgn kringles were monitored by 1H-NMR at 500 MHz and values for the equilibrium association constants (Ka) determined: rK1, Ka approximately 4.6 mM(-1); rK2, Ka approximately 3.3 mM(-1); K4, Ka approximately 6.2 mM-'; K5, K, 2.3 mM(-1). Thus, the lysine-binding kringles interact with CB-NTP more strongly than with Nalpha-acetyl-L-lysine methyl ester (Ka < 0.6 mM(-l), which reveals specificity for the NTP. In contrast, CB-NTP does not measurably interact with rK3. which is devoid of a LBS. CB-NTP and L-NTP 1H-NMR spectra were assigned and interproton distances estimated from 1H-1H Overhauser (NOESY) experiments. Structures of L-NTP and the Glul-Ile27 segment of CB-NTP were computed via restrained dynamic simulated annealing/energy minimization (SA/EM) protocols. Conformational models of CB-NTP were generated by joining the two (sub)structures followed by a round of constrained SA/EM. Helical turns are indicated for segments 6-9, 12-16, 28-30, and 45-48. Within the Cys34-Cys42 loop of L-NTP, the structure of the Glu-Glu-Asp-Glu-Glu39 segment appears to be relatively less defined, as is the case for the stretch containing Lys5O within the Cys42-Cys54 segment, consistent with the latter possibly interacting with kringle domains in intact Glul-Pgn. Overall, the CB-NTP and L-NTP fragments are of low regular secondary structure content-as indicated by UV-CD spectra- and exhibit fast amide 1H-2H exchange in 2H2O, suggestive of high flexibility.

Lysine-50 is a likely site for anchoring the plasminogen N-terminal peptide to lysine-binding kringles.

Interactions between the kringle 4 (K4) domain of human plasminogen (Pgn) and segments of the N-terminal Glu1-Lys77 peptide (NTP) have been investigated via 1H-NMR at 500 MHz. NTP peptide stretches devoid of Lys residues but carrying an internal Arg residue show negligible affinity toward K4 (equilibrium association constant Ka < 0.05 mM(-1)). In contrast, while most fragments containing an internal Lys residue exhibit affinities comparable to that shown by the blocked Lys derivative Nalpha-acetyl-L-lysine-methyl ester (Ka approximately 0.2 mM(-1), peptides encompassing Lys50O consistently show higher Ka values. Among the investigated linear peptides, Nalpha-acetyl-Ala-Phe-Tyr-His-Ser-Ser-Lys5O-Glu-Gln-NH2 (AcAFYHSK5OEQ-NH2) exhibits the strongest interaction with K4 (Ka approximately 1.4 mM(-1)), followed by AcYHSK50EQ-NH2 (Ka approximately 0.9 mM(-1)). Relative to the wild-type sequence, mutated hexapeptides exhibit lesser affinity for K4. When a Lys50 --> Ser mutation was introduced (==> AcYHSS50EQ-NH2), binding was abolished. The Ile27-lle56 construct (L-NTP) contains the Lys50 site within a loop constrained by two cystine bridges. The propensity of recombinant Pgn K1 (rK1) and K2 (rK2) modules, and of Pgn fragments encompassing the intact K4 and K5 domains, for binding L-NTP, was investigated. We find that L-NTP interacts with rK1, rK2, K4, and K5-all lysine-binding kringles-in a fashion that closely mimics what has been observed for the Glul-HSer57 N-terminal fragment of Pgn (CB-NTP). Thus, both the constellation of kringle lysine binding site (LBS) aromatic residues that are perturbed upon complexation of L-NTP and magnitudes of kringle-L-NTP binding affinities (rK1, Ka approximately 4.3 mM(-1); rK2, Ka approximately 3.7 mM(-1; K4, Ka approximately 6.4 mM(1); and K5, Ka approximately 2.1 mM(-1)) are essentially the same as for the corresponding kringle-CB-NTP pairs. Molecular modeling studies suggest that the Glu39-Lys50 stretch in NTP generates an area that complements, both topologically and electrostatically, the solvent-exposed kringle LBS surface.