Journal Article Efficient deletion mutagenesis by PCR Get access Zumrut B. Ogel, Zumrut B. Ogel Department of Biochemistry and Molecular Biology, University of LeedsLeeds LS2 9JT, UK Search for other works by this author on: Oxford Academic PubMed Google Scholar Michael J. McPherson Michael J. McPherson 1 Department of Biochemistry and Molecular Biology, University of LeedsLeeds LS2 9JT, UK 1To whom correspondence should be addressed Search for other works by this author on: Oxford Academic PubMed Google Scholar Protein Engineering, Design and Selection, Volume 5, Issue 5, July 1992, Pages 467–468, https://doi.org/10.1093/protein/5.5.467 Published: 01 July 1992 Article history Received: 02 December 1991 Revision received: 13 May 1992 Accepted: 13 May 1992 Published: 01 July 1992
Significance Autoimmune disease pathogenesis is driven by inflammation, induced partly by IgG autoantibody-containing immune complexes binding to Fc gamma receptors (FcγRs). These receptors are valid therapeutic targets in the treatment of autoimmunity. FcγRIIIa is one of a family of highly homologous receptors for IgG antibodies; previous attempts at therapeutic blockade have resulted in off-target effects involving cells that express the almost identical protein FcγRIIIb. Here we report the identification of functionally specific protein-based inhibitors (Affimer proteins) of FcγRIIIa and the structural/functional basis of their selectivity. As molecular research tools FcγRIIIa-specific Affimer proteins provide the ability to block IgG interaction with a single receptor. Our findings suggest that highly selective protein-based blocking agents that may have therapeutic applications can be readily produced.
The crystal structure of the complex between the copper amine oxidase from Escherichia coli (ECAO) and a covalently bound inhibitor, 2-hydrazinopyridine, has been determined to a resolution of 2.0 A. The inhibitor covalently binds at the 5 position of the quinone ring of the cofactor, 2,4,5-trihydroxyphenylalaninequinone (TPQ). The inhibitor complex is analogous to the substrate Schiff base formed during the reaction with natural monoamine substrate. A proton is abstracted from a methylene group adjacent to the amine group by a catalytic base during the reaction. The inhibitor, however, has a nitrogen at this position, preventing proton abstraction and trapping the enzyme in a covalent complex. The electron density shows this nitrogen is hydrogen bonded to the side chain of Asp383, a totally conserved residue, identifying it as the probable catalytic base. The positioning of Asp383 is such that the pro-S proton of a substrate would be abstracted, consistent with the stereospecificity of the enzyme determined by 1H NMR spectroscopy. Site-directed mutagenesis and in vivo suppression have been used to substitute Asp383 for 12 other residues. The resulting proteins either lack or, in the case of glutamic acid, have very low enzyme activity consistent with an essential catalytic role for Asp383. The O4 position on the quinone ring is involved in a short hydrogen bond with the hydroxyl of conserved residue Tyr369. The distance between the oxygens is less than 2.5 A, consistent with a shared proton, and suggesting ionization at the O4 position of the quinone ring. The Tyr369 residue appears to play an important role in stabilizing the position of the quinone/inhibitor complex. The O2 position on the quinone ring is hydrogen bonded to the apical water ligand of the copper. The basal water ligand, which lies 2.0 A from the copper in the native structure, is at a distance of 3.0 A in the complex. In the native structure, the active site is completely buried, with no obvious route for entry of substrate. In the complex, the tip of the pyridine ring of the bound inhibitor is on the surface of the protein at the edge of the interface between domains 3 and 4, suggesting this as the entry point for the amine substrate.
Abstract Ras proteins are small GTPases that are mutationally activated in around 30% of all human cancers. Oncogenic mutations in Ras trigger uncontrolled cellular differentiation and division through uninhibited Ras-GTP signaling. Despite major efforts in developing inhibitors, lack of treatments directly targeting Ras in cancer led to the current assumption that Ras is undruggable. A new approach, which involves use of biologics, has shown great potential for development of Ras inhibitors, as demonstrated by recent increase in the number of antibody mimetic reagents targeting Ras, including single domain antibodies, monobodies and DARPins. We have developed modulators of Ras activity using novel artificial binding proteins, termed Affimers. Affimers are small 91-amino-acid scaffold proteins that constrain one or two randomized nine amino acid loop regions for molecular recognition. Affimers isolated against KRas, the most commonly mutated Ras family member, displayed low nanomolar binding affinities, were shown to be effective at inhibiting nucleotide exchange and blocked interaction between Ras and its effector Raf. When expressed in mammalian cells, Affimers bound with endogenous Ras and inhibited Ras-mediated signaling. Site-directed mutagenesis of Affimer variable regions revealed three residues critical for binding and inhibition. X-ray crystal structure of Affimer in complex with KRas demonstrated that these residues bind into a hydrophobic pocket on Ras, previously described with small molecules. Currently, we are determining whether this interaction gives insight into new modes of therapeutic development using molecular docking in attempt to mimic the Affimer residues with small molecules. This project provides a unique opportunity to further our understanding of Ras biology through the development of reagents that modulate Ras activity. In addition, structural insights into mode of inhibition allow the modelling and design of small-molecule compounds, providing novel therapeutic strategies to slow Ras-addicted tumor growth. Citation Format: Katarzyna Haza, Heather Martin, Christian Tiede, Kevin Tipping, Chi Trinh, Holly Foster, Rachel Trowbridge, Richard Foster, Thomas Edwards, Alexander Breeze, Michael McPherson, Darren Tomlinson. Development of small-molecule RAS inhibitors using Affimer reagents [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B24.
Copper amine oxidases are homodimeric enzymes containing one Cu(2+) ion and one 2,4,5-trihydroxyphenylalanine quinone (TPQ) per monomer. Previous studies with the copper amine oxidase from Escherichia coli (ECAO) have elucidated the structure of the active site and established the importance in catalysis of an active-site base, Asp-383. To explore the early interactions of substrate with enzyme, we have used tranylcypromine (TCP), a fully reversible competitive inhibitor, with wild-type ECAO and with the active-site base variants D383E and D383N. The formation of an adduct, analogous to the substrate Schiff base, between TCP and the TPQ cofactor in the active site of wild-type ECAO and in the D383E and D383N variants has been investigated over the pH range 5.5-9.4. For the wild-type enzyme, the plot of the binding constant for adduct formation (K(b)) against pH is bell-shaped, indicating two pK(a)s of 5.8 and approximately 8, consistent with the preferred reaction partners being the unprotonated active-site base and the protonated TCP. For the D383N variant, the reaction pathway involving unprotonated base and protonated TCP cannot occur, and binding must follow a less favoured pathway with unprotonated TCP as reactant. Surprisingly, for the D383E variant, the K(b) versus pH behaviour is qualitatively similar to that of D383N, supporting a reaction pathway involving unprotonated TCP. The TCP binding data are consistent with substrate binding data for the wild type and the D383E variant using steady-state kinetics. The results provide strong support for a protonated amine being the preferred substrate for the wild-type enzyme, and emphasize the importance of the active-site base, Asp-383, in the primary binding event.
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