Designed ankyrin repeat protein binders for the crystallization of AcrB: Plasticity of the dominant interface

Monroe, Nicole; Sennhauser, Gaby; Seeger, Markus A.; Briand, Christophe; Grütter, Markus G.

doi:10.1016/j.jsb.2011.01.014

Cited by 20 publications

(12 citation statements)

References 43 publications

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“…One prominent example reminiscent to the study presented here is the homotrimeric multidrug transporter AcrB, that has been crystallized alone and in complex with DARPins in an asymmetric conformation in which each conformer represents a consecutive step of the transport cycle (25,27,28). Only two DARPins were found to bind to the trimer, and at the third expected DARPin binding site, a steric clash between a conformationally altered subdomain of AcrB with the back side of the DARPin prevented binding (25,26). The DARPins did not induce the asymmetric state, but rather recognized the intrinsic asymmetry of the targeted protein.…”

Section: Discussionmentioning

confidence: 69%

“…Besides possible improvements of crystal diffraction, DARPins have the potential to trap the targeted protein in alternate conformations. DARPins selected against the multidrug transporter AcrB for example trap this protein in an asymmetric state with two DARPins bound to the homotrimer (25,26). In the absence of DARPins, AcrB was initially crystallized in its symmetric and later in its asymmetric state (27,28).…”

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confidence: 99%

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Asymmetry in the Homodimeric ABC Transporter MsbA Recognized by a DARPin

Mittal

Böhm

Grütter

et al. 2012

Journal of Biological Chemistry

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Background: ABC exporters are suggested to hydrolyze ATP sequentially implying the existence of asymmetries. Results: An in vitro selected binder (DARPin) was identified that binds to homodimeric MsbA at a stoichiometric ratio of 1:1. Conclusion:The DARPin recognizes asymmetries in MsbA. Significance: Selected binding proteins are useful tools to recognize membrane transporters in novel conformational states.

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Section: Discussionmentioning

confidence: 69%

mentioning

confidence: 99%

Asymmetry in the Homodimeric ABC Transporter MsbA Recognized by a DARPin

Mittal

Böhm

Grütter

et al. 2012

Journal of Biological Chemistry

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“…Here, a selected antibody fragment mediated most of the crystal contacts and allowed its structure determination. Further successful examples mainly made use of antibody fragments derived from hybridoma technology [61,62] and recent examples used selected binding proteins based on proteins scaffolds such as DARPins [63] or fibronectin [64]. Camelid nanobodies become increasingly popular due to the recent successes in the field of GPCR structural biology [28,29].…”

Section: Discussionmentioning

confidence: 99%

Nanobody Mediated Crystallization of an Archeal Mechanosensitive Channel

et al. 2013

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Mechanosensitive channels (MS) are integral membrane proteins and allow bacteria to survive sudden changes in external osmolarity due to transient opening of their pores. The efflux of cytoplasmic osmolytes reduces the membrane tension and prevents membrane rupture. Therefore these channels serve as emergency valves when experiencing significant environmental stress. The preparation of high quality crystals of integral membrane proteins is a major bottleneck for structure determination by X-ray crystallography. Crystallization chaperones based on various protein scaffolds have emerged as promising tool to increase the crystallization probability of a selected target protein. So far archeal mechanosensitive channels of small conductance have resisted crystallization in our hands. To structurally analyse these channels, we selected nanobodies against an archeal MS channel after immunization of a llama with recombinant expressed, detergent solubilized and purified protein. Here we present the characterization of 23 different binders regarding their interaction with the channel protein using analytical gel filtration, western blotting and surface plasmon resonance. Selected nanobodies bound the target with affinities in the pico- to nanomolar range and some binders had a profound effect on the crystallization of the MS channel. Together with previous data we show that nanobodies are a versatile and valuable tool in structural biology by widening the crystallization space for highly challenging proteins, protein complexes and integral membrane proteins.

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“…The chaperone recognized a highly charged epitope of the C-terminal domain and was primarily responsible for mediating crystal lattice contacts. Similarly, four Fabs with low nanomolar affinity for the detergent-solubilized citrate carrier K. pneumonia CitS were identified from an optimized phage display library that included highly-expressed and stable members [87], although no structure has yet been reported. Finally, DARPins were selected for high affinity to the E. coli AcrB, leading to different lattices and structure quality [88].…”

Section: Chaperone Technologiesmentioning

confidence: 99%

Crystallization chaperone strategies for membrane proteins

Lieberman

Culver

Entzminger

et al. 2011

Methods

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From G protein-coupled receptors to ion channels, membrane proteins represent over half of known drug targets. Yet, structure-based drug discovery is hampered by the dearth of available three-dimensional models for this large category of proteins. Other than efforts to improve membrane protein expression and stability, current strategies to improve the ability of membrane proteins to crystallize involve examining many orthologs and DNA constructs, testing the effects of different detergents for purification and crystallization, creating a lipidic environment during crystallization, and cocrystallizing with covalent or non-covalent soluble protein chaperones with an intrinsic high propensity to crystallize. In this review, we focus on this last category, highlighting successes of crystallization chaperones in membrane protein structure determination and recent developments in crystal chaperone engineering, including molecular display to enhance chaperone crystallizability, and end with a novel generic approach in development to target any membrane protein of interest.

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Designed ankyrin repeat protein binders for the crystallization of AcrB: Plasticity of the dominant interface

Cited by 20 publications

References 43 publications

Asymmetry in the Homodimeric ABC Transporter MsbA Recognized by a DARPin

Asymmetry in the Homodimeric ABC Transporter MsbA Recognized by a DARPin

Nanobody Mediated Crystallization of an Archeal Mechanosensitive Channel

Crystallization chaperone strategies for membrane proteins

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