The primary structure of the core protein of the small, leucine-rich proteoglycan (PG I) from bovine articular cartilage

Two forms of small, interstitial proteoglycans have been isolated from bovine articular cartilage and have different core proteins, based on NH2-terminal analysis and peptide mapping (Choi, H. U., Johnson, T. L., Pal, S., Tang, L-H., Rosenberg, L. C., and Neame, P. J. (1989) J. Biol. Chem. 264, 2876-2884). These proteoglycans have been called PG I and PG II. Since they were first described, they have also been called "biglycan" (PG I), "decorin," and "DS-PG" (PG II). This report describes the primary structure of PG I from bovine articular cartilage. The protein core consists of 331 amino acids with a molecular mass of 37,280 Da. The amino acid sequence shows 55% identity to the cDNA-derived sequence of PG II from bovine bone. There are four discrete domains in the amino acid sequence. Domain 1, at the NH2 terminus (approximately 23 amino acids), contains two sites of attachment of dermatan sulfate, both of which match the consensus sequence of Asp/Glu-X-X-Ser-Gly-hydrophobic. Neither of these sites is substituted to 100% with glycosaminoglycan in native PG I. Domain 2, near the NH2 terminus and containing approximately 28 amino acids, has a cysteine pattern similar to a domain near the COOH terminus of mouse metallothionein and contains at least one disulfide bond (between the first and fourth cysteine residues). The majority of the core protein of PG I (domain 3) is a leucine-rich domain containing ten repeating units (approximately 231 amino acids). Patthy [1987) J. Mol. Biol. 198, 567-577) has shown that for PG II, the majority of domain 3 shows considerable similarity to leucine-rich alpha 2-glycoprotein (LRG) from serum. Domain 2 of PG I or PG II also has an analog in LRG, in that it has two cysteines in a similar place. The major motif in the PG I described here, in PG II and in LRG, is a series of leucine-rich repeats. PG I and PG II both contain 10 leucine-rich repeats which are 14 amino acids long and which are somewhat irregularly spaced, while LRG contains 9 leucine-rich repeats spaced 10 amino acids apart. Other proteins which contain leucine repeats are the platelet glycoprotein Ib, which is involved in platelet adherence to subendothelium (eight repeats in the alpha chain and two in the beta chain), the protein encoded by the Toll gene (involved in lateral and ventral spatial organization in Drosophila) and chaoptin (a protein involved in Drosophila photoreceptor morphogenesis).(ABSTRACT TRUNCATED AT 400 WORDS)     

cDNA sequence for rat dermatan sulfate proteoglycan-II (decorin).

A cDNA clone for dermatan sulfate proteoglycan-II, or decorin, has been isolated from a rat uterus library and sequenced. The cDNA and deduced amino acid sequences are 79 and 77% identical to the previously reported human and bovine sequences, respectively. The rat protein contains potential attachment sites for two glycosaminoglycan chains and four N-linked oligosaccharides, six conserved cysteine residues and multiple repeats of a leucine-rich sequence, LXXLXLXXNXL/I. Overlapping the C-end of one of these repeats is an NKISK sequence, which has been implicated in binding to fibronectin.     

Designing repeat proteins: modular leucine-rich repeat protein libraries based on the mammalian ribonuclease inhibitor family

We present a novel approach to design repeat proteins of the leucine-rich repeat (LRR) family for the generation of libraries of intracellular binding molecules. From an analysis of naturally occurring LRR proteins, we derived the concept to assemble repeat proteins with randomized surface positions from libraries of consensus repeat modules. As a guiding principle, we used the mammalian ribonuclease inhibitor (RI) family, which comprises cytosolic LRR proteins known for their extraordinary affinities to many RNases. By aligning the amino acid sequences of the internal repeats of human, pig, rat, and mouse RI, we derived a first consensus sequence for the characteristic alternating 28 and 29 amino acid residue A-type and B-type repeats. Structural considerations were used to replace all conserved cysteine residues, to define less conserved positions, and to decide where to introduce randomized amino acid residues. The so devised consensus RI repeat library was generated at the DNA level and assembled by stepwise ligation to give libraries of 2-12 repeats. Terminal capping repeats, known to shield the continuous hydrophobic core of the LRR domain from the surrounding solvent, were adapted from human RI. In this way, designed LRR protein libraries of 4-14 LRRs (equivalent to 130-415 amino acid residues) were obtained. The biophysical analysis of randomly chosen library members showed high levels of soluble expression in the Escherichia coli cytosol, monomeric behavior as characterized by gel-filtration, and alpha-helical CD spectra, confirming the success of our design approach.     

The trap-like relaxin-binding site of the leucine-rich G-protein-coupled receptor 7

The pleated sheet region of the leucine-rich G-protein-coupled receptor 7 supports a relaxin-binding group of amino acids that perfectly matches the binding cassette of relaxin. Arginines B13 and B17 are each chelated by an aspartic acid/glutamic acid pair and by isoleucine B20, which, offset by a one-quarter helix turn from a straight line connecting the arginines, interacts with a cluster of hydrophobic amino acids. The binding cassette of relaxin cuts at an angle of approximately 45 degrees across five parallel leucine-rich repeats. The arginine residues 13 and 17, which evolve parallel from the B-chain alpha-helix of relaxin, neutralize the charge repulsion of the juxta-posed acidic groups on the receptor and thereby trigger closure of a hydrogen bonding network around the guanidinium groups. Thus, relaxin is bound by synchronized chelation of two arginines and stabilized by hydrophobic interaction of isoleucine B20 with tryptophan, isoleucine, and leucine in neighboring leucine-rich repeats of the receptor. Deletion of any one of the three features diminishes interaction to the level of nonspecific binding. This model explains the exquisite sensitivity of relaxin binding avidity to minute changes in the disposition of the guanidinium and the size dependence of the hydrophobic binding residue in position B20.     

Amphipathic beta structure of a leucine-rich repeat peptide.

Long tandem arrays of a characteristic leucine-rich repeat motif on the order of 24 amino acids in length have been found in the primary structure of an increasing number of proteins. The most striking feature of these repeats is an amphipathic sequence, with leucine as the predominant hydrophobic residue. Based on this amphipathic sequence and the function of the proteins in which they have been found, the repeats have been proposed to be involved in protein-protein and protein-lipid interactions. As a step toward elucidating the structure and biochemical properties of the leucine-rich repeat motif, we have studied a synthetic leucine-rich repeat peptide (LRP32) representing one of the repeats found in Drosophila chaoptin. We have shown that: (i) LRP32 is soluble in aqueous solution but will bind quantitatively to phospholipid vesicles; (ii) LRP32 has a partial beta structure in aqueous solution and is predominantly a beta structure in the presence of phospholipid; (iii) LRP32 integrates into lipid bilayers to form 60-A intramembrane particles as seen using freeze-fracture electron microscopy (these putative oligomeric structures appear to contain a central aqueous core as indicated by their ability to generate conductances in planar lipid bilayers); and (iv) LRP32-lipid complexes generate 2H NMR spectra characteristic of integral membrane proteins. This study is consistent with LRP32 forming an amphipathic beta sheet. We propose that protein segments containing tandem arrays of leucine-rich repeats also may form amphipathic beta sheets.     

The decorin sequence SYIRIADTNIT binds collagen type I

Decorin belongs to the small leucine-rich repeat proteoglycan family, interacts with fibrillar collagens, and regulates the assembly, structure, and biomechanical properties of connective tissues. The decorin-collagen type I-binding region is located in leucine-rich repeats 5-6. Site-directed mutagenesis of this 54-residue-long collagen-binding sequence identifies Arg-207 and Asp-210 in leucine-rich repeat 6 as crucial for the binding to collagen. The synthetic peptide SYIRIADTNIT, which includes Arg-207 and Asp-210, inhibits the binding of full-length recombinant decorin to collagen in vitro. These collagen-binding amino acids are exposed on the exterior of the beta-sheet-loop structure of the leucine-rich repeat. This resembles the location of interacting residues in other leucine-rich repeat proteins.     

Necessity of conserved asparagine residues in the leucine-rich repeats of platelet glycoprotein Ib alpha for the proper conformation and function of the ligand-binding region

The polypeptides of the platelet von Willebrand factor (vWf) receptor, the GP Ib-IX-V complex, each contain tandem repeats of a sequence that assigns them to the leucine-rich repeat protein family. Here, we studied the role of conserved Asn residues in the leucine-rich repeats of GP Ib alpha, the ligand-binding subunit of the complex. We replaced the Asn residue in the sixth position of the first or sixth leucine-rich repeat (of seven) either with a bulky, charged Lys residue or with a Ser residue (sometimes found in the same position of other leucine-rich repeats) and studied the effect of the mutations on complex expression, modulator-dependent vWf binding, and interactions with immobilized vWf under fluid shear stress. As predicted, the Lys substitutions yielded more severe phenotypes, producing proteins that either were rapidly degraded within the cell (mutant N158K) or failed to bind vWf in the presence of ristocetin or roll on immobilized vWf under fluid shear stress (mutant N41K). The binding of function-blocking GP Ib alpha antibodies to the N41K mutant was either significantly reduced (AK2 and SZ2) or abolished (AN51 and CLB-MB45). Ser mutations were tolerated much better, although both mutants demonstrated subtle defects in vWf binding. These results suggest a vital role for the conserved asparagine residues in the leucine-rich repeats of GP Ib alpha for the structure and functions of this polypeptide. The finding that mutations in the first leucine-rich repeat had a much more profound effect on vWf binding indicates that the more N-terminal repeats may be directly involved in this interaction.     

A collagen-binding 59-kd protein (fibromodulin) is structurally related to the small interstitial proteoglycans PG-S1 and PG-S2 (decorin).

We have determined the primary structure of a 59 kd collagen binding protein which is present in many types of connective tissues, e.g. cartilage, tendon, skin, sclera and cornea. The amino acid sequence, deducted from a 2662 bp cDNA clone, predicts a 42 kd protein with a high content of leucine residues. Most of the protein consists of homologous 23 amino acid residues repeats with predominantly leucine residues in conserved positions. Similar leucine rich repeats have been identified in a number of proteins including the small interstitial proteoglycans decorin and PG-S1. The 59 kd protein and the two proteoglycans are homologous in their entire sequences suggesting that they have evolved from a common ancestral gene. The 59 kd protein and decorin are also functionally related in that both bind to collagen type I and II, and affect their fibrillogenesis. The substitution with glycosaminoglycan chains appears to be a feature shared by all three members of this family of leucine rich motif extracellular proteins, since the 59 kd protein isolated from cartilage is substituted with at least one keratan sulfate chain.     

Leucine periodicity of U2 small nuclear ribonucleoprotein particle (snRNP) A'' protein is implicated in snRNP assembly via protein-protein interactions.

Recombinant A' protein could be reconstituted into U2 small nuclear ribonucleoprotein particles (snRNPs) upon addition to HeLa cell extracts as determined by coimmunoprecipitation and particle density; however, direct binding to U2 RNA could not be demonstrated except in the presence of the U2 snRNP B" protein. Mutational analysis indicated that a central core region of A' was required for particle reconstitution. This region consists of five tandem repeats of approximately 24 amino acids each that exhibit a periodicity of leucine and asparagine residues that is distinct from the leucine zipper. Similar leucine-rich (Leu-Leu motif) repeats are characteristic of a diverse array of soluble and membrane-associated proteins from yeasts to humans but have not been reported previously to reside in nuclear proteins. Several of these proteins, including Toll, chaoptin, RNase/angiogenin inhibitors, lutropin-choriogonadotropin receptor, carboxypeptidase N, adenylyl cyclase, CD14, and human immunodeficiency virus type 1 Rev, may be involved in protein-protein interactions. Our findings suggest that in cell extracts the Leu-Leu motif of A' is required for reconstitution with U2 snRNPs and perhaps with other components involved in splicing through protein-protein interactions.     

OMgp LRR281～228：OMgp神经突起生长抑制功能的主要结构域

OMgp(oligodendrocyte-myelin glycoprotein)可以通过与MAG、nogo-66等神经再生抑制因子竞争结合同一受体NgR而诱使生长锥溃变和抑制神经突起的生长。以前的研究表明,在OMgp与NgR结合抑制神经突起生长的过程中,OMgp的亮氨酸富含重复序列(LRR)是必需的。为进一步了解OMgp LRR在神经突起生长中的作用及其结构与功能之间的关系,采用PCR-定点突变法对OMgp LRR结构域分段删除,表达了删除不同基因片段后的OMgp LRR蛋白,通过对表达有NgR的CHO细胞(NgR-CHO)的黏附实验和对原代培养神经细胞的抑制实验对其功能进行了研究。结果显示,分别删除了OMgp 25～56、57～133、134～180位氨基酸的OMgp LRR蛋白仍具有结合NgR-CHO和抑制原代培养的神经元突起生长的作用;而删除了第181～228位氨基酸的OMgp LRR蛋白则失去了对原代培养神经元的生长抑制作用,但仍然具有结合NgR的能力。表明OMgp181～228在OMgp的功能中具有重要的意义。删除了第181～228位氨基酸的OMgp LRR蛋白可望作为OMgp的竞争性抑制剂,用于中枢神经系统损伤后神经再生的治疗。     

Primary structure of human placental ribonuclease inhibitor

The primary structure of the human placental ribonuclease inhibitor (PRI), a tight-binding inhibitor of angiogenin and pancreatic ribonucleases, has been determined from the cDNA. The sequence of the mature protein is composed of 460 amino acids, yielding a molecular mass of 49,847 g/mol. Peptides comprising 92% of the predicted sequence were isolated from a tryptic digest of PRI, and direct sequence information obtained for 65% of the molecule agreed at all positions with the sequence predicted from the cDNA. The amino acid sequence of PRI contains seven direct internal repeat units, each 57 amino acids in length. These repeat units comprise 87% of the molecule. The average degree of identity between any two is 39%. A region within each repeat unit displays similarity to tandem, leucine-rich repeats found in six other proteins. Modification of PRI with iodoacetic acid, p-(hydroxymercuri)benzoate, and 5,5'-dithiobis(2-nitrobenzoic acid) reveals that at least 30 of the 32 cysteine residues of PRI are in the reduced form.     

The Pib gene for rice blast resistance belongs to the nucleotide binding and leucine-rich repeat class of plant disease resistance genes

Rice blast, caused by the fungal pathogen Magnaporthe grisea, is one of the most serious diseases of rice. Here we describe the isolation and characterization of Pib, one of the rice blast resistance genes. The Pib gene was isolated by a map-based cloning strategy. The deduced amino acid sequence of the Pib gene product contains a nucleotide binding site (NBS) and leucine-rich repeats (LRRs); thus, Pib is a member of the NBS-LRR class of plant disease resistance genes. Interestingly, a duplication of the kinase 1a, 2 and 3a motifs of the NBS region was found in the N-terminal half of the Pib protein. In addition, eight cysteine residues are clustered in the middle of the LRRs, a feature which has not been reported for other R genes. Pib gene expression was induced upon altered environmental conditions, such as altered temperatures and darkness.     

The novel fold of scytovirin reveals a new twist for antiviral entry inhibitors

The solution structure of the potent 95 residue anti-HIV protein scytovirin has been determined and two carbohydrate-binding sites have been identified. This unique protein, containing five structurally important disulfide bonds, demonstrates a novel fold with no elements of extended regular secondary structure. Scytovirin contains two 39 residue sequence repeats, differing in only three amino acid residues, and each repeat has primary sequence similarity to chitin binding proteins. Both sequence repeats form similarly structured domains, with the exception of one region. The result is two carbohydrate-binding sites with substantially different affinities. The unusual fold clusters aromatic residues in both sites, suggesting a binding mechanism similar to other known hevein-like carbohydrate-binding proteins but differing in carbohydrate specificity. Scytovirin, originally isolated from the cyanobacterium Scytonema varium, holds potential as an HIV entry inhibitor for both therapeutic and prophylactic anti-HIV applications. The high-resolution structural studies reported are an important initial step in unlocking the therapeutic potential of scytovirin.     

Crystallographic analysis of a complex between human immunodeficiency virus type 1 protease and acetyl-pepstatin at 2.0-A resolution

The mode of binding of acetyl-pepstatin to the protease from the human immunodeficiency virus type 1 (HIV-1) has been determined by x-ray diffraction analysis. Crystals of an acetyl-pepstatin-HIV-1 protease complex were obtained in space group P2(1)2(1)2 (unit cell dimensions a = 58.39 A, b = 86.70 A, c = 46.27 A) by precipitation with sodium chloride. The structure was phased by molecular replacement methods, and a model for the structure was refined using diffraction data to 2.0 A resolution (R = 0.176 for 12901 reflections with I greater than sigma (I); deviation of bond distances from ideal values = 0.018 A; 172 solvent molecules included). The structure of the protein in the complex has been compared with the structure of the enzyme without the ligand. A core of 44 amino acids in each monomer, including residues in the active site and residues at the dimer interface, remains unchanged on binding of the inhibitor (root mean square deviation of alpha carbon positions = 0.39 A). The remaining 55 residues in each monomer undergo substantial rearrangement, with the most dramatic changes occurring at residues 44-57 (these residues comprise the so-called flaps of the enzyme). The flaps interact with one another and with the inhibitor so as to largely preserve the 2-fold symmetry of the protein. The inhibitor is bound in two approximately symmetric orientations. In both orientations the peptidyl backbone of the inhibitor is extended; a network of hydrogen bonds is formed between the inhibitor and the main body of the protein as well as between the inhibitor and the flaps. Hydrophobic side chains of residues in the body of the protein form partial binding sites for the side chains of the inhibitor; hydrophobic side chains of residues in the flaps complete these binding sites.     

Identification of inhibitor binding sites of the cAMP-specific phosphodiesterase 4

Using the technique of site-directed mutagenesis, point mutants of human PDE4A have been developed in order to identify amino acids involved in inhibitor binding. Relevant amino acids were selected according to a peptidic binding site model for PDE4 inhibitors, which suggests interaction with two tryptophan residues, one histidine and one tyrosine residue, as well as one Zn(2+) ion. Mutations were directed at those tryptophan, histidine, and tyrosine residues, which are conserved among the PDE4 subtypes (PDE4A-D) and lie within the high-affinity 4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone (rolipram) binding domain of human PDE4A (amino acids 276-681 according to the PDE4A sequence L20965). Truncations to this region do not alter enzyme activity or inhibitor sensitivity. The mutants were expressed in COS1 cells, and the recombinant cyclic nucleotide phosphodiesterase (PDE) forms have been characterized in terms of their catalytic activity and inhibitor sensitivities. Tyrosine residues 432 and 602, as well as histidine 588, were found to be involved in inhibitor binding, but no interaction was detected between tryptophan and PDE inhibitors tested. To test the possibility that other amino acids are of importance for hydrophobic interactions, selected phenylalanine residues were also mutated. We found phenylalanine 613 and 645 to influence inhibitor binding to PDE4. The significant differences in the inhibitor sensitivities of the mutants show that the various inhibitors have different enzyme binding sites. Based on the assumption that the known side effects of PDE4 inhibitors (like emesis and nausea) are caused directly by selective inhibition of different conformation states of PDE4, our results may be a hint to differ between PDE4 inhibitors, which have emetic side effects (like rolipram), and those that do not have side effects (like N-(3,5-dichlorpyrid-4-yl)-[1-(4-fluorbenzyl)-5-hydroxy-indol-3-yl]-glyoxylateamide [AWD12-281]) by the differences of their binding sites and in that context contribute to the development of novel drugs. Furthermore, the identification of amino acid interactions proposed by the peptidic binding site model, which was used for the mutant selection, verifies the PrGen modeling as a useful method for the prediction of inhibitor binding sites in cases where detailed knowledge of the protein structure is not available.     

Factor XI interacts with the leucine-rich repeats of glycoprotein Ibalpha on the activated platelet

Factor XI (FXI) binds specifically and reversibly to high affinity sites on the surface of stimulated platelets (Kd app of approximately 10 nm; Bmax of approximately 1,500 sites/platelet) utilizing residues exposed on the Apple 3 domain in the presence of high molecular weight kininogen and Zn2+ or prothrombin and Ca2+. Because the FXI receptor in the platelet membrane is contained within the glycoprotein Ibalpha subunit of the glycoprotein Ib-IX-V complex (Baglia, F. A., Badellino, K. O., Li, C. Q., Lopez, J. A., and Walsh, P. N. (2002) J. Biol. Chem. 277, 1662-1668), we utilized mocarhagin, a cobra venom metalloproteinase, to generate a fragment (His1-Glu282) of glycoprotein Ibalpha that contains the leucine-rich repeats of the NH2-terminal globular domain and excludes the macroglycopeptide portion of glycocalicin, the soluble extracytoplasmic portion of glycoprotein Ibalpha. This fragment was able to compete with FXI for binding to activated platelets (Ki of 3.125 +/- 0.25 nm) with a potency similar to that of intact glycocalicin (Ki of 3.72 +/- 0.30 nm). However, a synthetic glycoprotein Ibalpha peptide, Asp269-Asp287, containing a thrombin binding site had no effect on the binding of FXI to activated platelets. Moreover, the binding of 125I-labeled thrombin to glycocalicin was unaffected by the presence of FXI at concentrations up to 10(-5) m. The von Willebrand factor A1 domain, which binds the leucine-rich repeats, inhibited the binding of FXI to activated platelets. Thus, we examined the effect of synthetic peptides of each of the seven leucine-rich repeats on the binding of 125I-FXI to activated platelets. All leucine-rich repeat (LRR) peptides derived from glycoprotein Ibalpha were able to inhibit FXI binding to activated platelets in the following order of decreasing potency: LRR7, LRR1, LRR4, LRR5, LRR6, LRR3, and LRR2. However, the leucine-rich repeat synthetic peptides derived from glycoprotein Ibbeta and Toll protein had no effect. We conclude that FXI binds to glycoprotein Ibalpha at sites comprising the leucine-rich repeat sequences within the NH2-terminal globular domain that are separate and distinct from the thrombin-binding site.     

Deduced protein sequence of bone small proteoglycan I (biglycan) shows homology with proteoglycan II (decorin) and several nonconnective tissue proteins in a variety of species

The small proteoglycans (PG) of bone consist of two different molecular species: one containing one chondroitin sulfate chain (PG II) and the other, two chains (PG I). These two proteoglycans are found in many connective tissues and have Mr = 45,000 core proteins with clear differences in their NH2-terminal sequences. Using antisera produced against synthetic peptides derived from the human PG I and PG II NH2 termini, we have isolated several cDNA clones from a lambda gt11 expression library made against mRNA isolated from human bone-derived cells. The clones, which reacted with antisera to the PG II peptide, were sequenced and found to be identical with the PG II class of proteoglycan from human fibroblasts known as PG-40 or decorin. The clones reacting to the PG I antisera, however, had a unique sequence. The derived protein sequence of PG I showed sufficient homology with the PG II sequence (55% of the amino acids are identical, with most others involving chemically similar amino acid substitutions) to strongly suggest that the two proteins were the result of a gene duplication. PG II (decorin) contains one attached glycosaminoglycan chain, while PG I probably contains two chains. For this reason, we suggest that PG I be called biglycan. The biglycan protein sequence contains 368 residues (Mr = 42,510 for the complete sequence and Mr = 37,983 for the secreted form) that appears to consist predominantly of a series of 12 tandem repeats of 24 residues. The repeats are recognized by their conserved leucines (and leucine-like amino acids) in positions previously reported for a diverse collection of proteins (none of which is thought to be proteoglycans) including: two morphogenic proteins (toll and chaoptin) in the fruit fly; a yeast adenylate cyclase; and two human proteins, the von Willebrand Factor-binding platelet membrane protein, GPIb, and a rare serum protein, leucine-rich glycoprotein.     

Localization and characterization of the calsequestrin-binding domain of triadin 1. Evidence for a charged beta-strand in mediating the protein-protein interaction

Triadin is an integral membrane protein of the junctional sarcoplasmic reticulum that binds to the high capacity Ca(2+)-binding protein calsequestrin and anchors it to the ryanodine receptor. The lumenal domain of triadin contains multiple repeats of alternating lysine and glutamic acid residues, which have been defined as KEKE motifs and have been proposed to promote protein associations. Here we identified the specific residues of triadin responsible for binding to calsequestrin by mutational analysis of triadin 1, the major cardiac isoform. A series of deletional fusion proteins of triadin 1 was generated, and by using metabolically labeled calsequestrin in filter-overlay assays, the calsequestrin-binding domain of triadin 1 was localized to a single KEKE motif comprised of 25 amino acids. Alanine mutagenesis within this motif demonstrated that the critical amino acids of triadin binding to calsequestrin are the even-numbered residues Lys(210), Lys(212), Glu(214), Lys(216), Gly(218), Gln(220), Lys(222), and Lys(224). Replacement of the odd-numbered residues within this motif by alanine had no effect on calsequestrin binding to triadin. The results suggest a model in which residues 210-224 of triadin form a beta-strand, with the even-numbered residues in the strand interacting with charged residues of calsequestrin, stabilizing a "polar zipper" that links the two proteins together. This small, highly charged beta-strand of triadin may tether calsequestrin to the junctional face membrane, allowing calsequestrin to sequester Ca(2+) in the vicinity of the ryanodine receptor during Ca(2+) uptake and Ca(2+) release.     

Fibronectin binding to the Salmonella enterica serotype Typhimurium ShdA autotransporter protein is inhibited by a monoclonal antibody recognizing the A3 repeat

ShdA is a large outer membrane protein of the autotransporter family whose passenger domain binds the extracellular matrix proteins fibronectin and collagen I, possibly by mimicking the host ligand heparin. The ShdA passenger domain consists of approximately 1,500 amino acid residues that can be divided into two regions based on features of the primary amino acid sequence: an N-terminal nonrepeat region followed by a repeat region composed of two types of imperfect direct amino acid repeats, called type A and type B. The repeat region bound bovine fibronectin with an affinity similar to that for the complete ShdA passenger domain, while the nonrepeat region exhibited comparatively low fibronectin-binding activity. A number of fusion proteins containing truncated fragments of the repeat region did not bind bovine fibronectin. However, binding of the passenger domain to fibronectin was inhibited in the presence of immune serum raised to one truncated fragment of the repeat region that contained repeats A2, B8, A3, and B9. Furthermore, a monoclonal antibody that specifically recognized an epitope in a recombinant protein containing the A3 repeat inhibited binding of ShdA to fibronectin.     

Structure and expression of an inhibitor of fungal polygalacturonases from tomato

A polygalacturonase inhibitor protein (PGIP) was characterized from tomato fruit. Differential glycosylation of a single polypeptide accounted for heterogeneity in concanavalin A binding and in molecular mass. Tomato PGIP had a native molecular mass of 35 to 41 kDa, a native isoelectric point of 9.0, and a chemically deglycosylated molecular mass of 34 kDa, suggesting shared structural similarities with pear fruit PGIP. When purified PGIPs from pear and tomato were compared, tomato PGIP was approximately twenty-fold less effective an inhibitor of polygalacturonase activity isolated from cultures of Botrytis cinerea. Based on partial amino acid sequence, polymerase chain reaction products and genomic clones were isolated and used to demonstrate the presence of PGIP mRNA in both immature and ripening fruit as well as cell suspension cultures. Nucleotide sequence analysis indicates that the gene, uninterrupted by introns, encodes a predicted 36.5 kDa polypeptide containing amino acid sequences determined from the purified protein and sharing 68% and 50% amino acid sequence identity with pear and bean PGIPs, respectively. Analysis of the PGIP sequences also revealed that they belong to a class of proteins which contain leucine-rich tandem repeats. Because these sequence domains have been associated with protein-protein interactions, it is possible that they contribute to the interaction between PGIP and fungal polygalacturonases.