Cyclic thrombospondin-1 mimetics: grafting of a thrombospondin sequence into circular disulfide-rich frameworks to inhibit endothelial cell migration

Tumour formation is dependent on nutrient and oxygen supply from adjacent blood vessels. Angiogenesis inhibitors can play a vital role in controlling blood vessel formation and consequently tumour progression by inhibiting endothelial cell proliferation, sprouting and migration. The primary aim of the present study was to design cyclic thrombospondin-1 (TSP-1) mimetics using disulfide-rich frameworks for anti-angiogenesis therapies and to determine whether these peptides have better potency than the linear parent peptide. A short anti-angiogenic heptapeptide fragment from TSP-1 (GVITRIR) was incorporated into two cyclic disulfide-rich frameworks, namely MCoTI-II (Momordica cochinchinensis trypsin inhibitor-II) and SFTI-1 (sunflower trypsin inhibitor-1). The cyclic peptides were chemically synthesized and folded in oxidation buffers, before being tested in a series of in vitro evaluations. Incorporation of the bioactive heptapeptide fragment into the cyclic frameworks resulted in peptides that inhibited microvascular endothelial cell migration, and had no toxicity against normal primary human endothelial cells or cancer cells. Importantly, all of the designed cyclic TSP-1 mimetics were far more stable than the linear heptapeptide in human serum. The present study has demonstrated a novel approach to stabilize the active region of TSP-1. The anti-angiogenic activity of the native TSP-1 active fragment was maintained in the new TSP-1 mimetics and the results provide a new chemical approach for the design of TSP-1 mimetics.     

CD36 mediates binding of soluble thrombospondin-1 but not cell adhesion and haptotaxis on immobilized thrombospondin-1

In this study, we examined the binding of soluble TSP1 (and ox-LDL) to CD36-transfected cells and the mechanisms by which immobilized TSP1 mediated attachment and haptotaxis (cell migration towards a substratum-bound ligand) of these transfected cells. CD36 cDNA transfection of NIH 3T3 cells clearly induced a dramatic increase in binding of both soluble [¹²⁵I]-TSP1 and [¹²⁵I]-ox-LDL to the surface of CD36-transfected cells, indicating that there was a gain of function with CD36 transfection in NIH 3T3 cells. Despite this gain of function, mock- and CD36-transfected NIH 3T3 cells attached and migrated to a similar extent on immobilized TSP1. An anti-TSP1 oligoclonal antibody inhibited CD36-transfected cell attachment to TSP1 while function blocking anti-CD36 antibodies, alone or in combination with heparin, did not. A series of fusion proteins encompassing cell-recognition domains of TSP1 was then used to delineate mechanisms by which NIH 3T3 cells adhere to TSP1. Although CD36 binds soluble TSP1 through a CSVTCG sequence located within type 1 repeats,^18,19 CD36-transfected NIH 3T3 cells did not attach to immobilized type 1 repeats while they did adhere to the N-terminal, type 3 repeats (in an RGD-dependent manner) and the C-terminal domain of TSP1. Conversely, Bowes melanoma cells attached to type 1 repeats and the N- and C-terminal domains of TSP1. However, CD36 cDNA transfection of Bowes cells did not increase cell attachment to type 1 repeats compared to that observed with mock-transfected Bowes cells. Moreover, a function blocking anti-CSVTCG peptide antibody did not inhibit the attachment of mock- and CD36-transfected Bowes cells to type 1 repeats. It is suggested that CD36/TSP1 interaction does not occur upon cell–matrix adhesion and haptotaxis because TSP1 undergoes conformational changes that do not allow the exposure of the CD36 binding site. © 1998 John Wiley & Sons, Ltd.     

Modification of native collagen with cell‐adhesive peptide to promote RPE cell attachment on Bruch's membrane

Current efforts to reverse loss of visual function due to Age‐related Macular Degeneration point to the restoration of the Retinal Pigment Epithelial (RPE) layer. Restoration of the RPE layer involves replacing lost RPE cells as well as addressing the degeneration of the underlying Bruch's membrane (BM). To advance the potential of using donor BM, we present a strategy to achieve specific and controllable modification of the inner collagenous layer (ICL) of the Bruch's membrane. In particular, interaction between a collagen binding peptide (CBP) sequence with exposed collagen fibers on the ICL surface is utilized to anchor bioactive molecules. Here, a cell‐adhesion sequence is added to the collagen binding sequence to promote attachment and survival of ARPE‐19. First, the binding specificity of the CBP sequence is verified with a fluorescent binding assay. Subsequently, the effect of modification using the peptide is studied qualitatively using confocal fluorescent imaging and quantitatively through a cell proliferation assay. Results of these experiments indicate that the peptide sequence binds specifically to collagen fibers. Additionally, modification using the peptide enhanced cell adhesion, allowing large uniform cell networks to be formed on the surface. Furthermore, modification with the peptide also delayed the onset of apoptosis on adherent cells. Biotechnol. Bioeng. 2009;102: 1723–1729. © 2008 Wiley Periodicals, Inc.     

Three-dimensional structure of the rSly1 N-terminal domain reveals a conformational change induced by binding to syntaxin 5

Sec1/Mun18-like (SM) proteins and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play central roles in intracellular membrane fusion. Diverse modes of interaction between SM proteins and SNAREs from the syntaxin family have been described. However, the observation that the N-terminal domains of Sly1 and Vps45, the SM proteins involved in traffic at the endoplasmic reticulum, the Golgi, the trans-Golgi network and the endosomes, bind to similar N-terminal sequences of their cognate syntaxins suggested a unifying theme for SM protein/SNARE interactions in most internal membrane compartments. To further understand this mechanism of SM protein/SNARE coupling, we have elucidated the structure in solution of the isolated N-terminal domain of rat Sly1 (rSly1N) and analyzed its complex with an N-terminal peptide of rat syntaxin 5 by NMR spectroscopy. Comparison with the crystal structure of a complex between Sly1p and Sed5p, their yeast homologues, shows that syntaxin 5 binding requires a striking conformational change involving a two-residue shift in the register of the C-terminal beta-strand of rSly1N. This conformational change is likely to induce a significant alteration in the overall shape of full-length rSly1 and may be critical for its function. Sequence analyses indicate that this conformational change is conserved in the Sly1 family but not in other SM proteins, and that the four families represented by the four SM proteins found in yeast (Sec1p, Sly1p, Vps45p and Vps33p) diverged early in evolution. These results suggest that there are marked distinctions between the mechanisms of action of each of the four families of SM proteins, which may have arisen from different regulatory requirements of traffic in their corresponding membrane compartments.     

Identification by photoaffinity labeling of the extracellular N-terminal domain of PAC1 receptor as the major binding site for PACAP

Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts many crucial biological functions through the interaction with its specific PAC1 receptor (PAC1-R), a class B G protein-coupled receptor (GPCR). To identify the binding sites of PACAP in the PAC1-R, three peptide derivatives containing a photoreactive p-benzoyl-phenylalanine (Bpa) residue were developed. These photosensitive PACAP analogs were fully biologically active and competent to displace radiolabeled Ac-PACAP27 from the PAC1-R. Subsequently, the ¹²⁵I-labeled photoprobes were used to anchor the PAC1-R expressed in Chinese hamster ovary cells. Photolabeling led to the formation of two protein complexes of 76 and 67 kDa, representing different glycosylated forms of the receptor. Proteinase and chemical cleavages of the peptide-receptor complexes revealed that ¹²⁵I[Bpa⁰, Nle¹⁷]PACAP27, ¹²⁵I[Bpa⁶, Nle¹⁷]PACAP27 and ¹²⁵I[Nle¹⁷, Bpa²²]PACAP27 covalently labeled the Ser⁹⁸ - Met¹¹¹ segment, the Ser¹²⁴ - Glu¹²⁵ dipeptide and the Ser¹⁴¹ - Met¹⁷² fragment, respectively. Taking into account the topology of the PAC1-R, these segments are mainly located within the extracellular N-terminal domain, indicating that this PAC1-R domain is the major binding site of PACAP27. The present study constitutes the first characterization of the binding domains of PACAP to its specific receptor and suggests heterogeneity within the binding mode of peptide ligands to class B GPCRs.     

Kinetic analysis of the attachment of a biological particle to a surface by macromolecular binding

Motivated by the problem of microbial deposition, a dynamic model is developed for the attachment of a Brownian particle to a surface mediated by colloidal forces as well as macromolecular bridging. The model predicts the attachment probability of the particle to the surface based upon the free energy as a function of fluctuating bond number and separation distance from the surface. From this model, the mean first-passage time approach is used to predict the mean time required for the particle moving from the unattached state to the attached state based on the properties of the binding macromolecules. This approach provides an analytical approximation for mean transition time from the secondary energy minimum as well as the attachment rate constant for the general case where neither binding nor particle diffusion are necessarily rate-limiting.     

Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site.

Ezrin is a membrane-cytoskeletal linking protein that is concentrated in actin-rich surface structures. It is closely related to the microvillar proteins radixin and moesin and to the tumor suppressor merlin/schwannomin. Cell extracts contain ezrin dimers and ezrin-moesin heterodimers in addition to monomers. Truncated ezrin fusion proteins were assayed by blot overlay to determine which regions mediate self-association. Here we report that ezrin self-association occurs by head-to-tail joining of distinct N-terminal and C-terminal domains. It is likely that these domains, termed N- and C-ERMADs (ezrin-radixin-moesin association domain), are responsible for homotypic and heterotypic associations among ERM family members. The N-ERMAD of ezrin resided within amino acids 1-296; deletion of 10 additional residues resulted in loss of activity. The C-ERMAD was mapped to the last 107 amino acids of ezrin, residues 479-585. The two residues at the C-terminus were required for activity, and the region from 530-585 was insufficient. The C-ERMAD was masked in the native monomer. Exposure of this domain required unfolding ezrin with sodium dodecyl sulfate or expressing the domain as part of a truncated protein. Intermolecular association could not occur unless the C-ERMAD had been made accessible to its N-terminal partner. It can be inferred that dimerization in vivo requires an activation step that exposes this masked domain. The conformationally inaccessible C-terminal region included the F-actin binding site, suggesting that this activity is likewise regulated by masking.     

Lactobacillus plantarum AS1 binds to cultured human intestinal cell line HT-29 and inhibits cell attachment by enterovirulent bacterium Vibrio parahaemolyticus

Aim: Lactobacillus plantarum AS1 was incubated with HT‐29 adenocarcinoma cell line to assess its adhesion potency and examined for its inhibitory effect on the cell attachment by an enterovirulent bacterium Vibrio parahaemolyticus. Methods and Results: Lactobacillus plantarum AS1 attached efficiently to HT‐29 cells as revealed by scanning electron microscopy and bacterial adhesion assay. Lactobacillus plantarum AS1 significantly reduced V. parahaemolyticus attached to HT‐29 cells by competition, exclusion and displacement mode. Lactobacillus plantarum AS1 seems to adhere to human intestinal cells via mechanisms that involve different combinations of carbohydrate and protein factors on the bacteria and eukaryotic cell surface. Conclusion: Strain Lact. plantarum AS1 inhibits the cell attachment of a pathogen V. parahaemolyticus by steric hindrance mechanism. Also, antibacterial factors such as bacteriocins, lactic acid and exopolysaccharides could be involved. Significance and Impact of the Study: The ability to inhibit the adhesion of V. parahaemolyticus to intestinal cell line warrants further investigation to explore the use of probiotic strain Lact. plantarum AS1 in the management of gastroenteritis caused by V. parahaemolyticus.     

Cloning and characterization of angiocidin, a tumor cell binding protein for thrombospondin-1

Thrombospondin-1 (TSP-1) is a matrix protein that has been implicated in mechanisms of tumor progression. Our laboratory previously showed that the CSVTCG (cys-ser-val-thr-cys-gly) sequence of TSP-1 functioned as a tumor cell adhesion domain and CSVTCG peptides as well as an anti-peptide antibody possessed anti-metastatic activity in a murine model of lung metastasis. In a subsequent study, a putative TSP-1 binding protein from lung carcinoma was isolated by CSVTCG-peptide affinity chromatography. In this study, we present the full-length cDNA of this binding protein isolated from a prostate cancer cell (PC3-NI) cDNA library. The purified recombinant protein, termed angiocidin, is a potent inhibitor of tumor growth of Lewis Lung carcinoma in vivo and tumor invasion and angiogenesis in vitro. In addition, the recombinant protein inhibits tumor and endothelial cell proliferation and induces apoptosis. The activity of angiocidin both in vivo and in vitro is partially dependent on its TSP-1 binding activity, since an angiocidin deletion mutant missing a high affinity-binding site for TSP-1 failed to inhibit tumor growth in vivo and was less active in its anti-tumor and anti-angiogenic activities in vitro. These results suggest that the anti-tumor activity of TSP-1 reported in many studies may be mediated in part by binding proteins such as angiocidin. Such proteins may function as tumor-suppressor proteins, which limit the growth of tumors by inhibiting angiogenesis and cell matrix interaction.     

The structures of the thrombospondin-1 N-terminal domain and its complex with a synthetic pentameric heparin

The N-terminal domain of thrombospondin-1 (TSPN-1) mediates the protein's interaction with (1) glycosaminoglycans, calreticulin, and integrins during cellular adhesion, (2) low-density lipoprotein receptor-related protein during uptake and clearance, and (3) fibrinogen during platelet aggregation. The crystal structure of TSPN-1 to 1.8 A resolution is a beta sandwich with 13 antiparallel beta strands and 1 irregular strand-like segment. Unique structural features of the N- and C-terminal regions, and the disulfide bond location, distinguish TSPN-1 from the laminin G domain and other concanavalin A-like lectins/glucanases superfamily members. The crystal structure of the complex of TSPN-1 with heparin indicates that residues R29, R42, and R77 in an extensive positively charged patch at the bottom of the domain specifically associate with the sulfate groups of heparin. The TSPN-1 structure and identified adjacent linker region provide a structural framework for the analysis of the TSPN domain of various molecules, including TSPs, NELLs, many collagens, TSPEAR, and kielin.     

Crosslinking of an iodo-uridine-RNA hairpin to a single site on the human U1A N-terminal RNA binding domain.

The N-terminal RNA binding domain (RBD) of the human U1A snRNP protein binds tightly and specifically to an RNA hairpin that contains a 10-nucleotide loop. The protein is one of a class of RNA binding proteins that adopts a beta alpha beta beta alpha beta global fold, which in turn forms a four-stranded antiparallel beta-sheet. This sheet forms the primary binding surface for the RNA, as shown by the crosslinking results described here, and in more detail by a recently described co-crystal of this RBD with an RNA hairpin (Oubridge C, et al., 1994, Nature 372:432-438). The RNA hairpin sequence used in the crosslinking experiments, containing 5-iodo-uridine, is a variant of the normal U1 snRNA sequence which is able to form a crosslink with the protein, in contrast to the wild-type sequence, which does not. This single uridine substitution in the 10-nucleotide loop is the site of cross-linking to one tyrosine (Tyr 13) in the beta 1 strand of the U1A N-terminal RBD. This same uridine is also crosslinked to a mutant Tyr 13 Phe RBD, at this Phe 13 substitution.     

Identification of active peptide sequences in the carboxyl-terminal cell binding domain of human thrombospondin-1.

Thrombospondin (TS) mediates attachment, spreading, and motility of several cell types through at least four cell binding domains: the amino-terminal heparin binding domain, the type I repeats containing the CSVTCG sequence, the RGDA sequence in the last of the type III calcium binding repeats and the carboxyl-terminal cell or platelet binding domain (CBD). The attachment of human melanoma cells (G361) to the COOH-terminal domain is independent of the RGDA sequence and is inhibited by the monoclonal antibody C6.7. To define the cell binding site(s) within this 212-residue COOH-terminal domain, we have synthesized eight overlapping peptides (seven 30-mers and a final 37-mer) representing the entire sequence of the CBD. Several of these peptides are insoluble in aqueous buffers at high concentration. Cell adhesion assays have been devised which employ covalent coupling of peptides in chaotropic solvents to chemically derivatized plastic 96-well plates. Three synthetic peptides, two of which are nonadjacent in the linear sequence, are potent attachment factors for G361 cells. C6.7 blocks adhesion to one of these peptides, whereas sulfated glycoconjugates inhibit adhesion of cells to all three. Polyclonal antibodies raised against the peptides inhibit cell adhesion to the peptides, the recombinant CBD, and to intact TS. The peptides GRGDSP and VTCG are not inhibitory. These sites are thus independent from the type I repeats and the RGDA sequence of TS. Each of the active peptides inhibits cell attachment to the other active peptides as well as to the CBD and to intact TS. This mutual inhibition suggests that the peptides share a common cellular receptor which may contain an associated glycoconjugate chain. These data indicate that the COOH-terminal cell binding domain of TS contains at least two peptide sequences which contribute to the attachment of a wide variety of cells.     

The tetrapeptide analogue of the cell attachment site of fibronectin inhibits platelet aggregation and fibrinogen binding to activated platelets

Fibrinogen binding to receptors on activated platelets is a prerequisite for platelet aggregation. However, the regions of fibrinogen interacting with these receptors have not been completely characterized. Fibronectin also binds to platelet fibrinogen receptors. Moreover, the amino acid sequence Arg-Gly-Asp-Ser, corresponding to the cell attachment site of fibronectin, is located near the carboxyl-terminal region of the alpha-chain of fibrinogen. We have examined the ability of this tetrapeptide to inhibit platelet aggregation and fibrinogen binding to activated platelets. Arg-Gly-Asp-Ser, but not the peptide Arg-Gly-Tyr-Ser-Leu-Gly, inhibited platelet aggregation stimulated by ADP, collagen, and gamma-thrombin without inhibiting platelet shape change or secretion. At a concentration of 60-80 microM, Arg-Gly-Asp-Ser inhibited the aggregation of ADP-stimulated gel-filtered platelets approximately equal to 50%. Arg-Gly-Asp-Ser, but not Arg-Gly-Tyr-Ser-Leu-Gly, also inhibited fibrinogen binding to ADP-stimulated platelets. This inhibition was competitive with a Ki of approximately equal to 25 microM but was incomplete even at higher tetrapeptide concentrations, indicating that Arg-Gly-Asp-Ser is a partial competitive inhibitor of fibrinogen binding. These data suggest that a region near the carboxyl-terminus of the alpha-chain of fibrinogen interacts with the fibrinogen receptor on activated platelets. The data also support the concept that the sequence Arg-Gly-Asp-Ser has been conserved for use in a variety of cellular adhesive processes.     

1-Oleoyl-2-acetylglycerol stimulates 5-lipoxygenase activity via a putative (phospho)lipid binding site within the N-terminal C2-like domain

5-Lipoxygenase (5-LO) catalysis is positively regulated by Ca2+ ions and phospholipids that both act via the N-terminal C2-like domain of 5-LO. Previously, we have shown that 1-oleoyl-2-acetylglycerol (OAG) functions as an agonist for human polymorphonuclear leukocytes (PMNL) in stimulating 5-LO product formation. Here we have demonstrated that OAG directly stimulates 5-LO catalysis in vitro. In the absence of Ca2+ (chelated using EDTA), OAG strongly and concentration-dependently stimulated crude 5-LO in 100,000 x g supernatants as well as purified 5-LO enzyme from PMNL. Also, the monoglyceride 1-O-oleyl-rac-glycerol and 1,2-dioctanoyl-sn-glycerol were effective, whereas various phospholipids did not stimulate 5-LO. However, in the presence of Ca2+, OAG caused no stimulation of 5-LO. Also, phospholipids or cellular membranes abolished the effects of OAG. As found previously for Ca2+, OAG renders 5-LO activity resistant against inhibition by glutathione peroxidase activity, and this effect of OAG is reversed by phospholipids. Intriguingly, a 5-LO mutant lacking tryptophan residues (Trp-13, -75, and -102) important for the binding of the 5-LO C2-like domain to phospholipids was not stimulated by OAG. We conclude that OAG directly stimulates 5-LO by acting at a phospholipid binding site located within the C2-like domain.     

Limited cell attachment time as a method to synchronize cells grown in monolayer culture

Summary A novel method of synchronizing monolayer tissue culture cells is described. By limiting the period of attachment of trypsinized cells and the subsequent removal of unattached cells a G₁ population of cells is isolated. Evaluation of the degree of synchrony has been carried out by measuring the labeling index and incorporation of [³H]thymidine into DNA. Further conformation of synchrony, as well as a comparison with synchrony by isoleucine deprivation, was obtained by flow cytometry. The expected peak in DNA synthesis rate following limited attachment was observed. This peak becomes more prominent and shifts to earlier times with shorter attachment intervals. The synchronization method described is simple, rapid, yields a substantial number of cells, and is applicable to many cell lines. This research was supported by a Basic Science Research Grant and a grant from the American Heart Foundation to K. Janakidevi. Paul Held and Christian Sell were also supported by predoctoral training grant HL07194 from the National Institutes of Health, Bethesda, MD.     

Fibroblast growth factor-2 binding to the thrombospondin-1 type III repeats, a novel antiangiogenic domain

Thrombospondin-1, an antiangiogenic matricellular protein, binds with high affinity to the angiogenic fibroblast growth factor-2, affecting its bioavailability and activity. The present work aimed at further locating the fibroblast growth factor-2 binding site of thrombospondin-1 and investigating its activity, using recombinant thrombospondin-1 proteins. Only recombinant constructs containing the thrombospondin-1 type III repeats bound fibroblast growth factor-2, whereas other domains, including the known anti-angiogenic type I repeats, were inactive. Binding was specific and inhibited by the anti thrombospondin-1 monoclonal antibody B5.2. Surface plasmon resonance analysis on BIAcore revealed a binding affinity (K(d)) of 310nM for the type III repeats and 11nM for intact thrombospondin-1. Since the type III repeats bind calcium, the effect of calcium on thrombospondin-1 binding to fibroblast growth factor-2 was investigated. Binding was modulated by calcium, as thrombospondin-1 or the type III repeats bound to fibroblast growth factor-2 only in calcium concentrations <0.3mM. The type III repeats inhibited binding of fibroblast growth factor-2 to endothelial cells, fibroblast growth factor-2-induced endothelial cell proliferation in vitro and angiogenesis in the chorioallantoic membrane assay in vivo, thus indicating the antiangiogenic activity of the domain. In conclusion, this study demonstrates that the fibroblast growth factor-2 binding site of thrombospondin-1 is located in the type III repeats. The finding that this domain is active in inhibiting angiogenesis indicates that the type III repeats represent a novel antiangiogenic domain of thrombospondin-1.     

Identification of a Bcl-XL binding region within the ATPase domain of Apaf-1

CED-4, a pro-apoptotic factor in Caenorhabditis elegans, activates the cell death protease CED-3. CED-9 directly binds to CED-4 and represses this. However, it has remained unclear whether a mammalian CED-9 homologue, Bcl-XL, inhibits the function of the mammalian CED-4 homologue, Apaf-1, by direct binding. To analyze the interaction, we adopted a yeast two-hybrid system. Since Bcl-XL and the CED-4-like portion of Apaf-1 failed to exhibit a positive result in the assay, we prepared "fragment libraries" of bcl-XL or apaf-1 cDNA. By screening of the apaf-1 "fragment library," we obtained nine clones interacting with Bcl-XL, all containing the same region within the ATPase domain, designated BBR: the Bcl-XL binding region. Binding of BBR to Bcl-XL was also confirmed by immunoprecipitation assays. Bcl-2, Bcl-w, A1/Bfl-1, and Boo/Diva failed to show the same capacity for binding to BBR as Bcl-XL. These results indicate that Bcl-XL directly binds to a specific region in Apaf-1.     

Muskelin, a novel intracellular mediator of cell adhesive and cytoskeletal responses to thrombospondin-1.

We have used an expression cloning strategy based on a cell-attachment assay screen to seek identification of molecules required in cellular responses to thrombospondin-1, a regulated macromolecular component of extracellular matrix. We report the identification and functional characterization of a novel, widely expressed, intracellular protein, named muskelin, which contains dispersed motifs with homology to the tandem repeats first identified in the Drosophila kelch ORF1 protein. In adherent C2C12 cells, muskelin localizes in the cytoplasm and at cell margins. Over-expression of muskelin in C2C12 cells promotes cell attachment to the thrombospondin-1 C-terminal domain, alters the mechanisms of attachment to intact thrombospondin-1 and correlates with decreased formation of fascin microspikes and increased assembly of focal contacts by cells adherent on thrombospondin-1. Reciprocally, cell attachment, spreading and cytoskeletal organization are specifically reduced in TSP-1-adherent cells after antisense depletion of muskelin. These results establish a requirement for muskelin in cell responses to thrombospondin-1 and demonstrate that such responses involve a novel process which is integrated into the regulation of cell-adhesive behaviour and cytoskeletal organization.     

Lumazine proteins from photobacteria: localization of the single ligand binding site to the N-terminal domain

Lumazine protein is believed to serve as an optical transponder in bioluminescence emission by certain marine bacteria. Sequence arguments suggest that the protein comprises two similarly folded riboflavin synthase-type domains, but earlier work also suggested that only one domain binds 6,7-dimethyl-8-ribityllumazine (DMRL). We show that the replacement of serine-48 or threonine-50 in the N-terminal domain of lumazine protein of Photobacterium leiognathi modulates the absorbance and fluorescence properties of bound DMRL or riboflavin. Moreover, the replacement of these amino acids is accompanied by reduced ligand affinity. Replacement of serine-48 by tryptophan shifts the (13)C NMR signal of the 6-methyl group in bound DMRL upfield by 2.9 ppm as compared to the wild-type protein complex. Replacement of threonine-50 causes a downfield shift of approximately 20 ppm for the (15)N NMR signal of N-5, as well as an upfield shift of 3 ppm for the (13)C NMR signal of C-7 in bound DMRL, respectively. The replacement of the topologically equivalent serine-144 and proline-146 in the C-terminal domain had no significant impact on optical properties, chemical shifts and apparent binding constants of bound DMRL. These data show that the N-terminal domain is the unique site for ligand binding in lumazine protein.