Nonsteroidal antiinflammatory drugs (NSAIDs), due to their good efficacy in the treatment of pain, inflammation, and fever, are among the most prescribed class of medicines in the world. The main drawback of NSAIDs is that they induce gastric complications such as peptic ulceration and injury to the intestine. Four NSAIDs, indomethacin, diclofenac, aspirin, and ibuprofen were selected to induce gastropathy in mouse models. It was found that the addition of C-terminal half of bovine lactoferrin (C-lobe) reversed the NSAID-induced injuries to the extent of 47-70% whereas the coadministration of C-lobe prevented it significantly. The C-lobe was prepared proteolytically using serine proteases. The binding studies of C-lobe with NSAIDs showed that these compounds bind to C-lobe with affinities ranging from 2.6 to 4.8 x 10(-4) M. The complexes of C-lobe were prepared with the above four NSAIDs. All four complexes were crystallized and their detailed three-dimensional structures were determined using x-ray crystallographic method. The structures showed that all the four NSAID molecules bound to C-lobe at the newly identified ligand binding site in C-lobe that is formed involving two alpha-helices, alpha10 and alpha11. The ligand binding site is separated from the well known iron binding site by the longest and the most stable beta-strand, betaj, in the structure. Similar results were also obtained with the full length lactoferrin molecule. This novel, to our knowledge, binding site in C-lobe of lactoferrin shows a good complementarity for the acidic and lipophilic compounds such as NSAIDs. We believe this indicates that C-lobe of lactoferrin can be exploited for the prevention of NSAID-induced gastropathy.
During the course of protein synthesis in the cell, the translation process is often terminated due to various reasons. As a result, peptidyl-tRNA molecules are released which are toxic to the cell as well reducing the availability of free amino acid and tRNA molecules for the required protein synthesis in the cell. Such a situation is corrected by an enzyme, Pth (peptidyl-tRNA hydrolase), which catalyses the release of free tRNA and peptide moieties from peptidyl-tRNAs. This means that the active Pth is essential for the survival of bacteria. In order to design inhibitors of PaPth (Pth from Pseudomonas aeruginosa), we determined the structures of PaPth in its native and bound states with compounds amino acylate-tRNA analogue and 5-azacytidine. The structure determination of the native protein revealed that the substrate-binding site was partially occupied by Glu161 from the neigh-bouring molecule. The structure of PaPth indicated that the substrate-binding site can be broadly divided into three distinct subsites. The structures of the two complexes showed that the amino acylate-tRNA analogue filled three subsites, whereas 5-azacytidine filled two subsites. The common sugar and the base moieties of the two compounds occupied identical positions in the cleft. Using surface plasmon resonance, the dissociation constants for the amino acylate-tRNA analogue and 5-azacytidine were found to be 3.53×10-8 M and 5.82×10-8 M respectively.
Bovine lactoferrin, a 76-kDa glycoprotein (Ala1-Arg689) consists of two similar N-and C-terminal molecular halves with the ability to bind two Fe 3+ ions. The N-terminal half, designated as the N-lobe (Ala1-Arg341) and the C-terminal half designated as the C-lobe (Tyr342-Arg689) have similar iron-binding properties, but the resistant C-lobe prolongs the physiological role of bovine lactoferrin in the digestive tract. Here, we report the crystal structure of true C-lobe, which was produced by limited proteolysis of bovine lactoferrin using trypsin. In the first proteolysis step, two fragments of 21 kDa (Glu86-Lys282) and 45 kDa (Ser283-Arg689) were generated because two lysine residues, Lys85 and Lys282, in the structure of iron-saturated bovine lactoferrin were fully exposed. The 45-kDa fragment was further digested at the newly exposed side chain of Arg341, generating a 38-kDa perfect C-lobe (Tyr342-Arg689). By contrast, the apo-lactoferrin was cut by trypsin only at Arg341, which was exposed in the structure of apo-lactoferrin, whereas the other two sites with Lys85 and Lys282 are inaccessible. The purified iron-saturated C-lobe was crystallized at pH 4.0. The structure was determined by the molecular replacement method using coordinates of the C-terminal half (Arg342-Arg689) of intact camel apolactoferrin. The structure determination revealed that the iron atom was absent and the iron-binding cleft was found in a wide-open conformation, whereas in the previously determined structure of iron-saturated C-lobe of bovine lactoferrin, the iron atom was present and the iron-binding site was in the closed confirmation. Structured digital abstract• trypsin cleaves lactoferrin by enzymatic study (View interaction)
A novel plant protein isolated from the underground bulbs of Scadoxus multiflorus, xylanase and α‐amylase inhibitor protein (XAIP), inhibits two structurally and functionally unrelated enzymes: xylanase and α‐amylase. The mature protein contains 272 amino acid residues which show sequence identities of 48% to the plant chitinase hevamine and 36% to xylanase inhibitor protein‐I, a double‐headed inhibitor of GH10 and GH11 xylanases. However, unlike hevamine, it is enzymatically inactive and, unlike xylanase inhibitor protein‐I, it inhibits two functionally different classes of enzyme. The crystal structure of XAIP has been determined at 2.0 Å resolution and refined to Rcryst and Rfree factors of 15.2% and 18.6%, respectively. The polypeptide chain of XAIP adopts a modified triosephosphate isomerase barrel fold with eight β‐strands in the inner circle and nine α‐helices forming the outer ring. The structure contains three cis peptide bonds: Gly33–Phe34, Tyr159–Pro160 and Trp253–Asp254. Although hevamine has a long accessible carbohydrate‐binding channel, in XAIP this channel is almost completely filled with the side‐chains of residues Phe13, Pro77, Lys78 and Trp253. Solution studies indicate that XAIP inhibits GH11 family xylanases and GH13 family α‐amylases through two independent binding sites located on opposite surfaces of the protein. Comparison of the structure of XAIP with that of xylanase inhibitor protein‐I, and docking studies, suggest that loops α3–β4 and α4–β5 may be involved in the binding of GH11 xylanase, and that helix α7 and loop β6–α6 are suitable for the interaction with α‐amylase.
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