[28] Molecular design and modeling of protein—heparin interactions

Cardin, Alan D.; Demeter, David A.; Weintraub, Herschel J. R.; Jackson, Richard L.

doi:10.1016/0076-6879(91)03030-k

Cited by 64 publications

(50 citation statements)

References 23 publications

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“…The autochaperone domain contains a highly basic region located on the C-terminal ␣-helix, which is well conserved within K5 lyases and K1 endosialidases (41) and which closely resembles a Cardin-Weintraub motif (43,56,57). This motif is implicated in binding of glycosaminoglycans and was previously proposed to contribute to substrate recognition and binding by KflA (43).…”

Section: Discussionmentioning

confidence: 99%

The K5 Lyase KflA Combines a Viral Tail Spike Structure with a Bacterial Polysaccharide Lyase Mechanism

Thompson

Pourhossein

Waterhouse

et al. 2010

Journal of Biological Chemistry

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K5 lyase A (KflA) is a tail spike protein (TSP) encoded by a K5A coliphage, which cleaves K5 capsular polysaccharide, a glycosaminoglycan with the repeat unit [-4)-␤GlcA-(1,4)-␣GlcNAc(1-], displayed on the surface of Escherichia coli K5 strains. The crystal structure of KflA reveals a trimeric arrangement, with each monomer containing a right-handed, singlestranded parallel ␤-helix domain. Stable trimer formation by the intertwining of strands in the C-terminal domain, followed by proteolytic maturation, is likely to be catalyzed by an autochaperone as described for K1F endosialidase. The structure of KflA represents the first bacteriophage tail spike protein combining polysaccharide lyase activity with a single-stranded parallel ␤-helix fold. We propose a catalytic site and mechanism representing convergence with the syn-␤-elimination site of heparinase II from Pedobacter heparinus.The KflA protein is a K5 lyase, an enzyme found as a tail spike protein of coliphages K5A (1, 2) and K1-5 (3), where it catalyzes depolymerization of K5 capsular polysaccharide. K5 lyase enables the phage to recognize and remove the protective K5 capsule around host bacteria, thereby exposing phage receptors in the outer membrane. The K5 capsular polysaccharide is a virulence factor of Escherichia coli K5 isolates, which are responsible for extraintestinal infections (4, 5). The polysaccharide is identical to N-acetyl-heparosan (heparan), a structure present in nonmodified regions of heparan sulfate, and KflA has been utilized previously to study the domain structure of heparan sulfate (6). A bacterial K5 lyase, ElmA, from E. coli K5 strain SEBR 3282 also has been described (7).The structures of several bacterial glycosaminoglycan-degrading lyases have been published: hyaluronate lyases (EC 4.2.2.1) and chondroitin AC lyases (EC 4.2.2.5) have catalytic N-terminal domains with (␣/␣) 5 toroid topology and C-terminal ␤-sheet/sandwich domains (8 -10), they act at GlcA residues within their substrates. Chondroitin ABC lyases (11,12) and heparinase II from Pedobacter heparinus (13) have overall structural similarity with these enzymes and can act at GlcA or IdoA (C5-epimerised GlcA) substrate residues, with each enzyme using two overlapping and epimer-specific catalytic groupings (12, 13). A heparin lyase (EC 4.2.2.7, heparinase I) from Bacteroides thetaiotaomicron recently has been shown to have a ␤-jellyroll domain containing an IdoA-specific active site similar to the IdoA-specific site of heparinase II (14). Heparinsulfate lyases (heparitinase or heparinase III, EC 4.2.2.8) act at GlcA residues of suitable substrates (15); no representative structure has yet been published, but these enzymes have sequence homology within the C-terminal ␤-sheet/sandwich domain of heparinase II. Chondroitinase B (EC 4.2.2.4) from Pedobacter heparinus (16, 17) is the only glycosaminoglycan lyase known to contain the right-handed parallel ␤-helix topology that we report here for KflA. It acts at IdoA residues within the substrate dermatan sulfate.The first...

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Section: Discussionmentioning

confidence: 99%

The K5 Lyase KflA Combines a Viral Tail Spike Structure with a Bacterial Polysaccharide Lyase Mechanism

Thompson

Pourhossein

Waterhouse

et al. 2010

Journal of Biological Chemistry

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“…Based on the structures of human secretory class I1 PLA, 163, 641 and A.h. blomhofii PLA,[44] and the proposed consensus sequence of heparin-binding site [31,32], the most likely candidate for the heparin-binding site in PLA, is the amino-terminal region (Fig. 6).…”

Section: Discussionmentioning

confidence: 99%

Inhibition of human secretory class II phospholipase A₂ by heparin

Dua

Cho

1994

European Journal of Biochemistry

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By means of kinetic analyses using Triton X-100/deoxycholic acid/dilauroylglycerophosphoethanolamine (4 : 2 : 1, molar ratio) mixed micelles we examined the effects of heparin on the activity of several phospholipases A, (PLA,). Heparin avidly bound cationic PLA,s including human secretory class I1 PLA, and thereby inhibited their hydrolysis of phospholipids in the mixed micelles. Initial velocity measurements indicated that heparin behaved as a competitive inhibitor for human secretory class I1 PLA, and closely related A.h. blomhoffii PLA, and A.p. piscivorus PLA,. In particular, heparin showed the highest specificity for human secretory class I1 PLA,. In the absence of deoxycholic acid in mixed micelles, A.h. blomhoffii PLA, was also strongly inhibited by heparin. The observed inhibition was not due to the interaction of heparin with the active site of PLA, because heparin did not inhibit the hydrolysis of monomeric substrates by PLA,s. Both kinetic measurements and fluorescence measurements of PLA,-bound 8-anilino-1-naphthalene sulfonate in the presence of varying amounts of heparin showed that a heparin molecule bound about seven molecules of PLA,. When positive charges of four lysines in the amino-terminal region of A.h. blomhoffii PLA, were neutralized by limited carbamoylation, heparin neither bound the carbamoylated A.h. blomhofii PLA, nor inhibited the hydrolysis of Triton X-l OO/dilauroylglycerophosphocholine mixed micelles by the carbamoylated A.h. blornhoffii PLA, that retained 50% activity of native A.h. blornhofii PLA,. Also, heparin did not inhibit the hydrolysis of mixed micelles by 7,lObis(octanoy1)ated A.p. piscivorus PLA, in which two lysines in the amino-terminal a-helix are acylated. These results indicate that the inhibition of human secretory class I1 PLA, and related cationic PLA,s by heparin originates from the interaction of heparin with cationic residues in the aminoterminal region that forms a part of interfacial binding site. In addition, unique structural features of human secretory class I1 PLA,, together with its unique mode of interaction with heparin, suggest that this PLA, might have an additional heparin-binding site. Although the heparin-PLA, binding diminished as the ionic strength of reaction medium increased, the inhibition of human secretory class T I PLA, by heparin remained significant at the physiological ionic strength. An estimated value of inhibition constant (K,) was 0.1 pM under physiological conditions, which suggests that a normal pharmaceutical dose of heparin might inhibit human secretory class I1 PLA, and regulate its biological effects.Phospholipase A, (PLA,) catalyzes the hydrolysis of the fatty acid ester in the 2-position of 3-sn-phospholipids [ 1, 21. This reaction is generally believed to produce arachidonic acid which is a precursor of eicosanoids [3, 41. In certain inflammatory cells, the activation of PLA, leads to the formation of platelet activating factor [51. Because these lipids are potent mediators of inflammatory reactions, PLA, has been impl...

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“…Fragments of VCP were expressed in Pichia pastoris cells by using the secretory expression vector pPIC9. The fragments corresponded to amino acids 18 to 146 [rVCP SCR (1,2)], 82 to 204 [rVCP SCR (2,3)], 145 to 263 [rVCP SCR (3,4)], and 18 to 262 [rVCP SCR (1)(2)(3)(4)]. Genomic DNA from vaccinia virus was used as template for the amplification of the DNA fragments encoding the above protein fragments by PCR.…”

Section: Methodsmentioning

confidence: 99%

Conserved Surface-Exposed K/R-X-K/R Motifs and Net Positive Charge on Poxvirus Complement Control Proteins Serve as Putative Heparin Binding Sites and Contribute to Inhibition of Molecular Interactions with Human Endothelial Cells: a Novel Mechanism for Evasion of Host Defense

Smith

Mullin

Parkinson

et al. 2000

J Virol

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Vaccinia virus complement control protein (VCP) has been shown to possess the ability to inhibit both classical and alternative complement pathway activation. The newly found ability of this protein to bind to heparin has been shown in previous studies to result in uptake by mast cells, possibly promoting tissue persistence. It has also been shown to reduce chemotactic migration of leukocytes by blocking chemokine binding. In addition, this study shows that VCP-through its ability to bind to glycosaminoglycans (heparin-like molecules) on the surface of human endothelial cells-is able to block antibody binding to surface major histocompatibility complex class I molecules. Since heparin binding is critical for many functions of this protein, we have attempted to characterize the molecular basis for this interaction. Segments of this protein, generated by genetic engineering of the DNA encoding VCP into the Pichia pastoris expression system, were used to localize the regions with heparin binding activity. These regions were then analyzed to more specifically define their properties for binding. It was found that the number of putative binding sites (K/R-X-K/R), the overall positive charge, and the percentage of positively charged amino acids within the protein were responsible for this interaction.

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[28] Molecular design and modeling of protein—heparin interactions

Cited by 64 publications

References 23 publications

The K5 Lyase KflA Combines a Viral Tail Spike Structure with a Bacterial Polysaccharide Lyase Mechanism

The K5 Lyase KflA Combines a Viral Tail Spike Structure with a Bacterial Polysaccharide Lyase Mechanism

Inhibition of human secretory class II phospholipase A₂ by heparin

Conserved Surface-Exposed K/R-X-K/R Motifs and Net Positive Charge on Poxvirus Complement Control Proteins Serve as Putative Heparin Binding Sites and Contribute to Inhibition of Molecular Interactions with Human Endothelial Cells: a Novel Mechanism for Evasion of Host Defense

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[28] Molecular design and modeling of protein—heparin interactions

Cited by 64 publications

References 23 publications

The K5 Lyase KflA Combines a Viral Tail Spike Structure with a Bacterial Polysaccharide Lyase Mechanism

The K5 Lyase KflA Combines a Viral Tail Spike Structure with a Bacterial Polysaccharide Lyase Mechanism

Inhibition of human secretory class II phospholipase A2 by heparin

Conserved Surface-Exposed K/R-X-K/R Motifs and Net Positive Charge on Poxvirus Complement Control Proteins Serve as Putative Heparin Binding Sites and Contribute to Inhibition of Molecular Interactions with Human Endothelial Cells: a Novel Mechanism for Evasion of Host Defense

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Inhibition of human secretory class II phospholipase A₂ by heparin