Akiko Koide - Academia.edu (original) (raw)

Papers by Akiko Koide

Proceedings of the National Academy of Sciences of the United States of America, Jan 10, 2011

Discriminating closely related molecules remains a major challenge in the engineering of binding ... more Discriminating closely related molecules remains a major challenge in the engineering of binding proteins and inhibitors. Here we report the development of highly selective inhibitors of small ubiquitin-related modifier (SUMO) family proteins. SUMOylation is involved in the regulation of diverse cellular processes. Functional differences between two major SUMO isoforms in humans, SUMO1 and SUMO2/3, are thought to arise from distinct interactions mediated by each isoform with other proteins containing SUMO-interacting motifs (SIMs). However, the roles of such isoform-specific interactions are largely uncharacterized due in part to the difficulty in generating high-affinity, isoform-specific inhibitors of SUMO/SIM interactions. We first determined the crystal structure of a "monobody," a designed binding protein based on the fibronectin type III scaffold, bound to the yeast homolog of SUMO. This structure illustrated a mechanism by which monobodies bind to the highly conserv...

Nature Communications, 2014

Fluc-type F(-) channels--used by microorganisms for resisting fluoride toxicity--are unusual in t... more Fluc-type F(-) channels--used by microorganisms for resisting fluoride toxicity--are unusual in their quaternary architecture: they are thought to associate as dimers with the two subunits in antiparallel transmembrane orientation. Here, we subject this unusual structural feature to a direct test. Single purified Fluc channels recorded in planar lipid bilayers are constitutively open, with rare, short-lived closings. Using combinatorial libraries, we generated synthetic binding proteins, 'monobodies,' that specifically bind to Fluc homologues with nanomolar affinity. Reversible binding of monobodies to two different Fluc channel homologues is seen in single-channel recordings as long-lived nonconducting events that follow bimolecular kinetics. By applying monobodies sequentially to the two sides of the bilayer in a double-sided perfusion manoeuvre, we show that Fluc channels present monobody-binding epitopes to both sides of the membrane. The result establishes that Fluc subunits are arranged in dimeric antiparallel orientation.

Biophysical Journal, 2015

Protein Expression and Purification, 2006

Affinity chromatography coupled with an &... more Affinity chromatography coupled with an "affinity tag" has become a powerful and routine technology for the purification of recombinant proteins. However, such tag-based affinity chromatography usually cannot separate different conformational states (e.g., folded and misfolded) of a protein to be purified. Here, we describe a strategy to separate different conformations of a protein by using "tailor-made" affinity chromatography based on engineered binding proteins. Our method involves: (i) engineering of a binding protein specific to a particular conformation of the protein of interest, and (ii) production and immobilization of the binding protein to prepare conformation-specific affinity chromatography media. Using "monobodies," small antibody mimics based on the fibronectin type III domain, as the target-binding proteins, we demonstrated the effectiveness of our method by separating the active form of the estrogen receptor alpha ligand-binding domain (ERalpha-LBD) from a mixture of active and misfolded species and by discriminating two different conformations of ERalpha-LBD bound to different ligands. Our strategy should be generally applicable to the preparation of conformationally homogeneous protein samples.

Protein Engineering Design and Selection, 2009

Proceedings of the National Academy of Sciences, 2007

Proceedings of the National Academy of Sciences, 2009

Proceedings of the National Academy of Sciences, 2008

Proceedings of the National Academy of Sciences, 2008

Proceedings of the National Academy of Sciences, 2013

Proceedings of the National Academy of Sciences, 2013

Multidrug transporters belonging to the multidrug and toxic compound extrusion (MATE) family expe... more Multidrug transporters belonging to the multidrug and toxic compound extrusion (MATE) family expel dissimilar lipophilic and cationic drugs across cell membranes by dissipating a preexisting Na(+) or H(+) gradient. Despite its clinical relevance, the transport mechanism of MATE proteins remains poorly understood, largely owing to a lack of structural information on the substrate-bound transporter. Here we report crystal structures of a Na(+)-coupled MATE transporter NorM from Neisseria gonorrheae in complexes with three distinct translocation substrates (ethidium, rhodamine 6G, and tetraphenylphosphonium), as well as Cs(+) (a Na(+) congener), all captured in extracellular-facing and drug-bound states. The structures revealed a multidrug-binding cavity festooned with four negatively charged amino acids and surprisingly limited hydrophobic moieties, in stark contrast to the general belief that aromatic amino acids play a prominent role in multidrug recognition. Furthermore, we discovered an uncommon cation-π interaction in the Na(+)-binding site located outside the drug-binding cavity and validated the biological relevance of both the substrate- and cation-binding sites by conducting drug resistance and transport assays. Additionally, we uncovered potential rearrangement of at least two transmembrane helices upon Na(+)-induced drug export. Based on our structural and functional analyses, we suggest that Na(+) triggers multidrug extrusion by inducing protein conformational changes rather than by directly competing for the substrate-binding amino acids. This scenario is distinct from the canonical antiport mechanism, in which both substrate and counterion compete for a shared binding site in the transporter. Collectively, our findings provide an important step toward a detailed and mechanistic understanding of multidrug transport.

Nature Structural & Molecular Biology, 2010

Nature, 2013

The functions of G-protein-coupled receptors (GPCRs) are primarily mediated and modulated by thre... more The functions of G-protein-coupled receptors (GPCRs) are primarily mediated and modulated by three families of proteins: the heterotrimeric G proteins, the G-protein-coupled receptor kinases (GRKs) and the arrestins. G proteins mediate activation of second-messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors, and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization. Arrestins activated by interaction with phosphorylated receptors can also mediate G-protein-independent signalling by serving as adaptors to link receptors to numerous signalling pathways. Despite their central role in regulation and signalling of GPCRs, a structural understanding of β-arrestin activation and interaction with GPCRs is still lacking. Here we report the crystal structure of β-arrestin-1 (also called arrestin-2) in complex with a fully phosphorylated 29-amino-acid carboxy-terminal peptide derived from the human V2 vasopressin receptor (V2Rpp). This peptide has previously been shown to functionally and conformationally activate β-arrestin-1 (ref. 5). To capture this active conformation, we used a conformationally selective synthetic antibody fragment (Fab30) that recognizes the phosphopeptide-activated state of β-arrestin-1. The structure of the β-arrestin-1-V2Rpp-Fab30 complex shows marked conformational differences in β-arrestin-1 compared to its inactive conformation. These include rotation of the amino- and carboxy-terminal domains relative to each other, and a major reorientation of the 'lariat loop' implicated in maintaining the inactive state of β-arrestin-1. These results reveal, at high resolution, a receptor-interacting interface on β-arrestin, and they indicate a potentially general molecular mechanism for activation of these multifunctional signalling and regulatory proteins.

Methods, 2013

A set of phage display sorting strategies and validation methodologies are presented that are cap... more A set of phage display sorting strategies and validation methodologies are presented that are capable of producing high performance synthetic antibodies (sABs) with customized properties. Exquisite control of antigen and conditions during the phage display selection process can yield sABs that: (1) recognize conformational states, (2) target specific regions of the surface of a protein, (3) induce conformational changes, and (4) capture and stabilize multi-protein complexes. These unique capabilities open myriad opportunities to study complex macromolecular processes inaccessible to traditional affinity reagent technology. We present detailed protocols for de novo isolation of binders, as well as examples of downstream biophysical characterization. The methods described are generalizable and can be adapted to other in vitro direct evolution approaches based on yeast or mRNA display.

Journal of Molecular Biology, 2007

Proceedings of the National Academy of Sciences of the United States of America, Jan 10, 2011

Discriminating closely related molecules remains a major challenge in the engineering of binding ... more Discriminating closely related molecules remains a major challenge in the engineering of binding proteins and inhibitors. Here we report the development of highly selective inhibitors of small ubiquitin-related modifier (SUMO) family proteins. SUMOylation is involved in the regulation of diverse cellular processes. Functional differences between two major SUMO isoforms in humans, SUMO1 and SUMO2/3, are thought to arise from distinct interactions mediated by each isoform with other proteins containing SUMO-interacting motifs (SIMs). However, the roles of such isoform-specific interactions are largely uncharacterized due in part to the difficulty in generating high-affinity, isoform-specific inhibitors of SUMO/SIM interactions. We first determined the crystal structure of a "monobody," a designed binding protein based on the fibronectin type III scaffold, bound to the yeast homolog of SUMO. This structure illustrated a mechanism by which monobodies bind to the highly conserv...

Nature Communications, 2014

Fluc-type F(-) channels--used by microorganisms for resisting fluoride toxicity--are unusual in t... more Fluc-type F(-) channels--used by microorganisms for resisting fluoride toxicity--are unusual in their quaternary architecture: they are thought to associate as dimers with the two subunits in antiparallel transmembrane orientation. Here, we subject this unusual structural feature to a direct test. Single purified Fluc channels recorded in planar lipid bilayers are constitutively open, with rare, short-lived closings. Using combinatorial libraries, we generated synthetic binding proteins, 'monobodies,' that specifically bind to Fluc homologues with nanomolar affinity. Reversible binding of monobodies to two different Fluc channel homologues is seen in single-channel recordings as long-lived nonconducting events that follow bimolecular kinetics. By applying monobodies sequentially to the two sides of the bilayer in a double-sided perfusion manoeuvre, we show that Fluc channels present monobody-binding epitopes to both sides of the membrane. The result establishes that Fluc subunits are arranged in dimeric antiparallel orientation.

Biophysical Journal, 2015

Protein Expression and Purification, 2006

Affinity chromatography coupled with an &... more Affinity chromatography coupled with an "affinity tag" has become a powerful and routine technology for the purification of recombinant proteins. However, such tag-based affinity chromatography usually cannot separate different conformational states (e.g., folded and misfolded) of a protein to be purified. Here, we describe a strategy to separate different conformations of a protein by using "tailor-made" affinity chromatography based on engineered binding proteins. Our method involves: (i) engineering of a binding protein specific to a particular conformation of the protein of interest, and (ii) production and immobilization of the binding protein to prepare conformation-specific affinity chromatography media. Using "monobodies," small antibody mimics based on the fibronectin type III domain, as the target-binding proteins, we demonstrated the effectiveness of our method by separating the active form of the estrogen receptor alpha ligand-binding domain (ERalpha-LBD) from a mixture of active and misfolded species and by discriminating two different conformations of ERalpha-LBD bound to different ligands. Our strategy should be generally applicable to the preparation of conformationally homogeneous protein samples.

Protein Engineering Design and Selection, 2009

Proceedings of the National Academy of Sciences, 2007

Proceedings of the National Academy of Sciences, 2009

Proceedings of the National Academy of Sciences, 2008

Proceedings of the National Academy of Sciences, 2008

Proceedings of the National Academy of Sciences, 2013

Proceedings of the National Academy of Sciences, 2013

Multidrug transporters belonging to the multidrug and toxic compound extrusion (MATE) family expe... more Multidrug transporters belonging to the multidrug and toxic compound extrusion (MATE) family expel dissimilar lipophilic and cationic drugs across cell membranes by dissipating a preexisting Na(+) or H(+) gradient. Despite its clinical relevance, the transport mechanism of MATE proteins remains poorly understood, largely owing to a lack of structural information on the substrate-bound transporter. Here we report crystal structures of a Na(+)-coupled MATE transporter NorM from Neisseria gonorrheae in complexes with three distinct translocation substrates (ethidium, rhodamine 6G, and tetraphenylphosphonium), as well as Cs(+) (a Na(+) congener), all captured in extracellular-facing and drug-bound states. The structures revealed a multidrug-binding cavity festooned with four negatively charged amino acids and surprisingly limited hydrophobic moieties, in stark contrast to the general belief that aromatic amino acids play a prominent role in multidrug recognition. Furthermore, we discovered an uncommon cation-π interaction in the Na(+)-binding site located outside the drug-binding cavity and validated the biological relevance of both the substrate- and cation-binding sites by conducting drug resistance and transport assays. Additionally, we uncovered potential rearrangement of at least two transmembrane helices upon Na(+)-induced drug export. Based on our structural and functional analyses, we suggest that Na(+) triggers multidrug extrusion by inducing protein conformational changes rather than by directly competing for the substrate-binding amino acids. This scenario is distinct from the canonical antiport mechanism, in which both substrate and counterion compete for a shared binding site in the transporter. Collectively, our findings provide an important step toward a detailed and mechanistic understanding of multidrug transport.

Nature Structural & Molecular Biology, 2010

Nature, 2013

The functions of G-protein-coupled receptors (GPCRs) are primarily mediated and modulated by thre... more The functions of G-protein-coupled receptors (GPCRs) are primarily mediated and modulated by three families of proteins: the heterotrimeric G proteins, the G-protein-coupled receptor kinases (GRKs) and the arrestins. G proteins mediate activation of second-messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors, and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization. Arrestins activated by interaction with phosphorylated receptors can also mediate G-protein-independent signalling by serving as adaptors to link receptors to numerous signalling pathways. Despite their central role in regulation and signalling of GPCRs, a structural understanding of β-arrestin activation and interaction with GPCRs is still lacking. Here we report the crystal structure of β-arrestin-1 (also called arrestin-2) in complex with a fully phosphorylated 29-amino-acid carboxy-terminal peptide derived from the human V2 vasopressin receptor (V2Rpp). This peptide has previously been shown to functionally and conformationally activate β-arrestin-1 (ref. 5). To capture this active conformation, we used a conformationally selective synthetic antibody fragment (Fab30) that recognizes the phosphopeptide-activated state of β-arrestin-1. The structure of the β-arrestin-1-V2Rpp-Fab30 complex shows marked conformational differences in β-arrestin-1 compared to its inactive conformation. These include rotation of the amino- and carboxy-terminal domains relative to each other, and a major reorientation of the 'lariat loop' implicated in maintaining the inactive state of β-arrestin-1. These results reveal, at high resolution, a receptor-interacting interface on β-arrestin, and they indicate a potentially general molecular mechanism for activation of these multifunctional signalling and regulatory proteins.

Methods, 2013

A set of phage display sorting strategies and validation methodologies are presented that are cap... more A set of phage display sorting strategies and validation methodologies are presented that are capable of producing high performance synthetic antibodies (sABs) with customized properties. Exquisite control of antigen and conditions during the phage display selection process can yield sABs that: (1) recognize conformational states, (2) target specific regions of the surface of a protein, (3) induce conformational changes, and (4) capture and stabilize multi-protein complexes. These unique capabilities open myriad opportunities to study complex macromolecular processes inaccessible to traditional affinity reagent technology. We present detailed protocols for de novo isolation of binders, as well as examples of downstream biophysical characterization. The methods described are generalizable and can be adapted to other in vitro direct evolution approaches based on yeast or mRNA display.

Journal of Molecular Biology, 2007