Allosteric Inhibition of Aminoglycoside Phosphotransferase by a Designed Ankyrin Repeat Protein

Abstract

Aminoglycoside phosphotransferase (3')-IIIa (APH) is a bacterial kinase that confers antibiotic resistance to many pathogenic bacteria and shares structural homology with eukaryotic protein kinases. We report here the crystal structure of APH, trapped in an inactive conformation by a tailor-made inhibitory ankyrin repeat (AR) protein, at 2.15 A resolution. The inhibitor was selected from a combinatorial library of designed AR proteins. The AR protein binds the C-terminal lobe of APH and thereby stabilizes three alpha helices, which are necessary for substrate binding, in a significantly displaced conformation. BIAcore analysis and kinetic enzyme inhibition experiments are consistent with the proposed allosteric inhibition mechanism. In contrast to most small-molecule kinase inhibitors, the AR proteins are not restricted to active site binding, allowing for higher specificity. Inactive conformations of pharmaceutically relevant enzymes, as can be elucidated with the approach presented here, represent powerful starting points for rational drug design.

Structural and functional analysis of phosphorylation-specific binders of the kinase ERK from designed ankyrin repeat protein libraries

“…S5) as the ones whose structures were determined, underlining that DARPin libraries in combination with ribosome display offer multiple solutions for a certain binding epitope. Interestingly, DARPins targeting two other protein kinases with structural homology to MAPK (35,36) bind to an epitope located near the observed structural changes in ERK2 upon phosphorylation. Thus, this region appears to be amenable for the comparatively rigid-body interaction surface of DARPins and provides specificity through structural changes in the activation loop.…”

Amyloid-β Peptide-specific DARPins as a Novel Class of Potential Therapeutics for Alzheimer Disease

“…DARPin D23 at 50 nM was preincubated with a 50-fold molar excess of the peptide fragments A␤(Ϫ4 -6), A␤(Ϫ3-7), A␤(Ϫ2-8), A␤(Ϫ1-9), A␤(1-8), A␤(2-11), A␤ (3)(4)(5)(6)(7)(8)(9)(10)(11)(12), A␤(4 -13), A␤ (5)(6)(7)(8)(9)(10)(11)(12)(13)(14), A␤(6 -15) (peptides&elephants, Potsdam, Germany), A␤(1-11), A␤ (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16), A␤ (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)…”

“…DARPins are built from several ankyrin repeat modules that are tightly packed and capped by terminating repeats that shield the hydrophobic core, resulting in high stability and solubility with a low aggregation tendency (11,12). Additionally, the absence of redox-sensitive disulfide bonds has enabled DARPins to be used for both intracellular and extracellular applications (13,14) and, combined with the lack of endogenous receptors, allows an improved fine-tuning of tissue distribution and clearance (15) as compared with conventional immunoglobulinbased (IgG) scaffolds. At one-tenth the molecular weight of IgGs, DARPins might cross the blood-brain barrier more efficiently than antibodies upon peripheral administration (16,17).…”

Exploring the Relationship between Sequences, Structures, Dynamical Behaviors and Functions of New Type Protein Drugs: DARPins

“…The SASA values of residues N45, T46, T48, L53, Y56, S57, V78, F79, Y81, L86, Y89, W90, N102, N107, M109, D110, S111, W123 and K144 were decreased, which were mainly on the regions of linking the -helixes of adjacent repeat units and near the binding sites to the target protein. The hydrophobic core of each repeat unit contained residues 8,11,12,19,20,23,24, 28 and 30 [18]. The SASA value of hydrophobic core of the AR1 for DARPin off7 wasn't changed very much after binding to target protein, but the SASA values of hydrophobic core of AR2 and AR3 were decreased, and the value of AR3 was not decreased obviously.…”

“…Based on designed ankyrin repeat proteins, developing a proteinaceous APH inhibitor which could bind to the C-terminal lobe of APH outside the substrate-binding pocket, and the combination between the two proteins was allosteric inhibition mechanism. The crystal structure (PDB code: 2bkk) of APH in complex with AR protein inhibitor (AR_3a) was resolved [20]. Using the selected DARPins to bind polo-like kinase-1(PIK-1) as an effective drug target for cancer therapy, the selected DARPins could partially inhibit the activity of PIK-1 with competitive inhibition or as allosteric inhibitors and assist in the crystallization of PIK-1.…”