4mvk
Crystal structure of an engineered lipocalin (Anticalin US7) in complex with the Alzheimer amyloid peptide fragment VFFAED
Structural highlights
FunctionNGAL_HUMAN Iron-trafficking protein involved in multiple processes such as apoptosis, innate immunity and renal development. Binds iron through association with 2,5-dihydroxybenzoic acid (2,5-DHBA), a siderophore that shares structural similarities with bacterial enterobactin, and delivers or removes iron from the cell, depending on the context. Iron-bound form (holo-24p3) is internalized following binding to the SLC22A17 (24p3R) receptor, leading to release of iron and subsequent increase of intracellular iron concentration. In contrast, association of the iron-free form (apo-24p3) with the SLC22A17 (24p3R) receptor is followed by association with an intracellular siderophore, iron chelation and iron transfer to the extracellular medium, thereby reducing intracellular iron concentration. Involved in apoptosis due to interleukin-3 (IL3) deprivation: iron-loaded form increases intracellular iron concentration without promoting apoptosis, while iron-free form decreases intracellular iron levels, inducing expression of the proapoptotic protein BCL2L11/BIM, resulting in apoptosis. Involved in innate immunity, possibly by sequestrating iron, leading to limit bacterial growth.[1] Publication Abstract from PubMedAbeta peptides, in particular Abeta42 and Abeta40, exert neurotoxic effects and their overproduction leads to amyloid deposits in the brain, thus constituting an important biomolecular target for treatments of Alzheimer's disease. We describe the engineering of cognate Anticalins as a novel type of neutralising protein reagent based on the human lipocalin scaffold. Phage display selection from a genetic random library comprising variants of the human lipocalin 2 with mutations targeted at 20 exposed amino acid positions in the four loops that form the natural binding site was performed using both recombinant and synthetic target peptides and resulting in three different Anticalins. Biochemical characterisation of the purified proteins produced by periplasmic secretion in <em>E. coli</em> revealed high folding stability in a monomeric state, with Tmvalues ranging from 53.4 degrees C to 74.5 degrees C, as well as high affinities for Abeta40, between 95 pM and 563 pM, as measured by real-time surface plasmon resonance analysis. The central linear VFFAED epitope within the Abeta sequence was mapped using a synthetic peptide array on membranes and was shared by all three Anticalins, despite up to 13 mutual amino acid differences in their binding sites. All Anticalins had the ability - with varying extent - to inhibit Abeta aggregation <em>in vitro</em> according to the thioflavin-T fluorescence assay and, furthermore, they abolished Abeta42-mediated toxicity in neuronal cell culture. Thus, these Anticalins provide not only useful protein reagents to study the molecular pathology of Alzheimer's disease but they also show potential as alternative drug candidates <em>versus</em> antibodies. High-affinity Anticalins with aggregation-blocking activity directed against the Alzheimer beta-amyloid peptide.,Rauth S, Hinz D, Borger M, Uhrig M, Mayhaus M, Riemenschneider M, Skerra A Biochem J. 2016 Mar 30. pii: BCJ20160114. PMID:27029347[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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