Erik Debler - Academia.edu (original) (raw)

Papers by Erik Debler

Journal of Molecular Biology, 2004

Triggering receptor expressed on myeloid cells (TREM) 1 is an activating receptor expressed on my... more Triggering receptor expressed on myeloid cells (TREM) 1 is an activating receptor expressed on myeloid cells whose ligand(s) remain elusive. TREM-1 stimulation activates neutrophils and monocytes and induces the secretion of pro-inflammatory molecules, which amplifies the Toll-like receptor-initiated responses to invading pathogens. In addition, TREM-1 mediates the septic shock pathway, and thus represents a potential therapeutic target. We report the crystal structure of the mouse TREM-1 extracellular domain at 1.76 Å resolution. The mouse extracellular domain is monomeric, consistent with our previous human TREM-1 structure, and strongly supports the contention that the globular TREM-1 head is a monomer contrary to proposals of a symmetric dimer.

Nephron Physiology, 2007

lum and no redistribution was observed after forskolin stimulation. Of note, homology modeling re... more lum and no redistribution was observed after forskolin stimulation. Of note, homology modeling revealed that the two mutations involve two highly conserved residues providing important clues about the role of the wt residues in AQP2 stability and function.

Proceedings of the National Academy of Sciences, 2009

Design of catalysts featuring multiple functional groups is a desirable, yet formidable goal. Ant... more Design of catalysts featuring multiple functional groups is a desirable, yet formidable goal. Antibody 13G5, which accelerates the cleavage of unactivated benzisoxazoles, is one of few artificial enzymes that harness an acid and a base to achieve efficient proton transfer. X-ray structures of the Fab-hapten complexes of wild-type 13G5 and active-site variants now afford detailed insights into its mechanism. The parent antibody preorganizes Asp(H35) and Glu(L34) to abstract a proton from substrate and to orient a water molecule for leaving group stabilization, respectively. Remodeling the environment of the hydrogen bond donor with a compensatory network of ordered waters, as seen in the Glu(L34) to alanine mutant, leads to an impressive 10(9)-fold rate acceleration over the nonenzymatic reaction with acetate, illustrating the utility of buried water molecules in bifunctional catalysis. Generalization of these design principles may aid in creation of catalysts for other important chemical transformations.

Journal of the American Chemical Society, 2007

... Azidoproline Containing Helices: Stabilization of the Polyproline II Structure by a Functiona... more ... Azidoproline Containing Helices: Stabilization of the Polyproline II Structure by a Functionalizable Group. Michael Kümin, Louis-Sebastian Sonntag, and Helma Wennemers. Journal of the American Chemical Society 2007 129 (3), pp 466–467. ...

Journal of Biological Chemistry, 2008

FIGURE 3. Superimposition of the free (blue, four independent copies of the orthorhombic crystal ... more FIGURE 3. Superimposition of the free (blue, four independent copies of the orthorhombic crystal form) and hapten-bound (yellow, four independent copies) 34E4. The complementarity determining regions (CDRs) of the light and heavy chains, which are labeled L1-L3 and H1-H3, respectively, undergo only minor changes upon ligand binding, whereas Trp L91 swings into the binding pocket and serves as surrogate ligand in the apo form. FIGURE 4. Molecular surface representation of free (A) and hapten-bound (B) 34E4. The hapten is displayed as pink stick model. The architecture of the antibody combining site is dramatically different in the two states. The shallow indentation of the predominant closed conformation of the antibody in the unliganded form (A) can convert to an open conformation that can then harbor the hapten in a deep cavity (B).

Angewandte Chemie International Edition, 2006

Journal of Biological Chemistry, 2016

In the family of protein arginine methyltransferases (PRMTs) that predominantly generate either a... more In the family of protein arginine methyltransferases (PRMTs) that predominantly generate either asymmetric or symmetric dimethylarginine (ADMA or SDMA), PRMT7 is unique in producing solely monomethylarginine (MMA) products. The type of methylation on histones and other proteins dictates changes in gene expression, and numerous studies have linked altered profiles of methyl marks with disease phenotypes. Given the importance of specific inhibitor development, it is crucial to understand the mechanisms by which PRMT product specificity is conferred. We have focused our attention on active-site residues of PRMT7 from the protozoan Trypanosoma brucei We have designed 26 single and double mutations in the active site, including residues in the Glu-X8-Glu (double E) loop and the Met-Gln-Trp sequence of the canonical Thr-His-Trp (THW) loop known to interact with the methyl-accepting substrate arginine. Analysis of the reaction products by high-resolution cation exchange chromatography combined with the knowledge of PRMT crystal structures suggests a model where the size of two distinct subregions in the active site determines PRMT7 product specificity. A dual mutation of Glu181 to Asp in the double E loop and Gln329 to Ala in the canonical THW loop enables the enzyme to produce SDMA. Consistent with our model, the mutation of Cys431 to His in the THW loop of human PRMT9 shifts its product specificity from SDMA towards MMA. Together with previous results, these findings provide a structural basis and a general model for product specificity in PRMTs, which will be useful for the rational design of specific PRMT inhibitors.

Proceedings of the National Academy of Sciences of the United States of America, Jan 8, 2016

Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly m... more Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coo...

Nature Structural & Molecular Biology, 2009

Molecular Cell, 2008

We recently proposed a cylindrical coat for the nuclear pore membrane in the nuclear pore complex... more We recently proposed a cylindrical coat for the nuclear pore membrane in the nuclear pore complex (NPC). This scaffold is generated by multiple copies of seven nucleoporins. Here, we report three crystal structures of the nucleoporin pair Seh1 d Nup85, which is part of the coat cylinder. The Seh1 d Nup85 assembly bears resemblance in its shape and dimensions to that of another nucleoporin pair, Sec13 d Nup145C. Furthermore, the Seh1 d Nup85 structures reveal a hinge motion that may facilitate conformational changes in the NPC during import of integral membrane proteins and/or during nucleocytoplasmic transport. We propose that Seh1 d Nup85 and Sec13 d Nup145C form 16 alternating, vertical rods that are horizontally linked by the three remaining nucleoporins of the coat cylinder. Shared architectural and mechanistic principles with the COPII coat indicate a common evolutionary origin and support the notion that the NPC coat represents another class of membrane coats.

Science, 2008

The blue-emissive antibody EP2-19G2 that has been elicited against trans-stilbene has unprecedent... more The blue-emissive antibody EP2-19G2 that has been elicited against trans-stilbene has unprecedented ability to produce bright luminescence and has been used as a biosensor in various applications. We show that the prolonged luminescence is not stilbene fluorescence. Instead, the emissive species is a charge-transfer excited complex of an anionic stilbene and a cationic, parallel pi-stacked tryptophan. Upon charge recombination, this complex generates exceptionally bright blue light. Complex formation is enabled by a deeply penetrating ligand-binding pocket, which in turn results from a noncanonical interface between the two variable domains of the antibody.

Proceedings of the National Academy of Sciences, 2009

The Nup84 complex constitutes a key building block in the nuclear pore complex (NPC). Here we pre... more The Nup84 complex constitutes a key building block in the nuclear pore complex (NPC). Here we present the crystal structure of one of its 7 components, Nup120, which reveals a ␤ propeller and an ␣-helical domain representing a novel fold. We discovered a previously unidentified interaction of Nup120 with Nup133 and confirmed the physiological relevance in vivo. As mapping of the individual components in the Nup84 complex places Nup120 and Nup133 at opposite ends of the heptamer, our findings indicate a head-to-tail arrangement of elongated Nup84 complexes into a ring structure, consistent with a fence-like coat for the nuclear pore membrane. The attachment site for Nup133 lies at the very end of an extended unstructured region, which allows for flexibility in the diameter of the Nup84 complex ring. These results illuminate important roles of terminal unstructured segments in nucleoporins for the architecture, function, and assembly of the NPC.

Proceedings of the National Academy of Sciences, 2005

Antibody 34E4 catalyzes the conversion of benzisoxazoles to salicylonitriles with high rates and ... more Antibody 34E4 catalyzes the conversion of benzisoxazoles to salicylonitriles with high rates and multiple turnovers. The crystal structure of its complex with the benzimidazolium hapten at 2.5-angstroms resolution shows that a combination of hydrogen bonding, pi stacking, and van der Waals interactions is exploited to position both the base, Glu(H50), and the substrate for efficient proton transfer. Suboptimal placement of the catalytic carboxylate, as observed in the 2.8-angstroms structure of the Glu(H50)Asp variant, results in substantially reduced catalytic efficiency. In addition to imposing high positional order on the transition state, the antibody pocket provides a highly structured microenvironment for the reaction in which the carboxylate base is activated through partial desolvation, and the highly polarizable transition state is stabilized by dispersion interactions with the aromatic residue Trp(L91) and solvation of the leaving group oxygen by external water. The enzyme-like efficiency of general base catalysis in this system directly reflects the original hapten design, in which a charged guanidinium moiety was strategically used to elicit an accurately positioned functional group in an appropriate reaction environment and suggests that even larger catalytic effects may be achievable by extending this approach to the induction of acid-base pairs capable of bifunctional catalysis.

Proceedings of the National Academy of Sciences, 2006

IL-2 is a cytokine that functions as a growth factor and central regulator in the immune system a... more IL-2 is a cytokine that functions as a growth factor and central regulator in the immune system and mediates its effects through ligand-induced hetero-trimerization of the receptor subunits IL-2R␣, IL-2R␤, and ␥c. Here, we describe the crystal structure of the trimeric assembly of the human IL-2 receptor ectodomains in complex with IL-2 at 3.0 Å resolution. The quaternary structure is consistent with a stepwise assembly from IL-2͞IL-2R␣ to IL-2͞IL-2R␣͞IL-2R␤ to IL-2͞IL-2R␣͞IL-2R␤͞␥c. The IL-2R␣ subunit forms the largest of the three IL-2͞IL-2R interfaces, which, together with the high abundance of charge-charge interactions, correlates well with the rapid association rate and high-affinity interaction of IL-2R␣ with IL-2 at the cell surface. Surprisingly, IL-2R␣ makes no contacts with IL-2R␤ or ␥c, and only minor changes are observed in the IL-2 structure in response to receptor binding. These findings support the principal role of IL-2R␣ to deliver IL-2 to the signaling complex and act as regulator of signal transduction. Cooperativity in assembly of the final quaternary complex is easily explained by the extraordinarily extensive set of interfaces found within the fully assembled IL-2 signaling complex, which nearly span the entire length of the IL-2R␤ and ␥c subunits. Helix A of IL-2 wedges tightly between IL-2R␤ and ␥c to form a three-way junction that coalesces into a composite binding site for the final ␥c recruitment. The IL-2͞␥c interface itself exhibits the smallest buried surface and the fewest hydrogen bonds in the complex, which is consistent with its promiscuous use in other cytokine receptor complexes.

Proceedings of the National Academy of Sciences, 2009

The heptameric Nup84 complex constitutes an evolutionarily conserved building block of the nuclea... more The heptameric Nup84 complex constitutes an evolutionarily conserved building block of the nuclear pore complex. Here, we present the crystal structure of the heterotrimeric Sec13⅐Nup145C⅐Nup84 complex, the centerpiece of the heptamer, at 3.2-Å resolution. Nup84 forms a U-shaped ␣-helical solenoid domain, topologically similar to two other members of the heptamer, Nup145C and Nup85. The interaction between Nup84 and Nup145C is mediated via a hydrophobic interface located in the kink regions of the two solenoids that is reinforced by additional interactions of two long Nup84 loops. The Nup84 binding site partially overlaps with the homo-dimerization interface of Nup145C, suggesting competing binding events. Fitting of the elongated Z-shaped heterotrimer into electron microscopy (EM) envelopes of the heptamer indicates that structural changes occur at the Nup145C⅐Nup84 interface. Docking the crystal structures of all heptamer components into the EM envelope constitutes a major advance toward the completion of the structural characterization of the Nup84 complex.

Nature Structural & Molecular Biology, 2005

As a counter-defense against antiviral RNA silencing during infection, the insect Flock House vir... more As a counter-defense against antiviral RNA silencing during infection, the insect Flock House virus (FHV) expresses the silencing suppressor protein B2. Biochemical experiments show that B2 binds to double-stranded RNA (dsRNA) without regard to length and inhibits cleavage of dsRNA by Dicer in vitro. A cocrystal structure reveals that a B2 dimer forms a four-helix bundle that binds to one face of an A-form RNA duplex independently of sequence. These results suggest that B2 blocks both cleavage of the FHV genome by Dicer and incorporation of FHV small interfering RNAs into the RNA-induced silencing complex.

Molecular and Cellular Biology, 2005

Journal of Molecular Biology, 2007

A large number of Gram-negative bacteria employ N-acyl homoserine lactones (AHLs) as signaling mo... more A large number of Gram-negative bacteria employ N-acyl homoserine lactones (AHLs) as signaling molecules in quorum sensing, which is a population density-dependent mechanism to coordinate gene expression. Antibody RS2-1G9 was elicited against a lactam mimetic of the N-acyl homoserine lactone and represents the only reported monoclonal antibody that recognizes the naturally-occuring N-acyl homoserine lactone with high affinity. Due to its high cross-reactivity, RS2-1G9 showed remarkable inhibition of quorum sensing signaling in Pseudomonas aeruginosa, a common opportunistic pathogen in humans. The crystal structure of Fab RS2-1G9 in complex with a lactam analog revealed complete encapsulation of the polar lactam moiety in the antibody-combining site. This mode of recognition provides an elegant immunological solution for tight binding to an aliphatic, lipid-like ligand with a small head group lacking typical haptenic features, such as aromaticity or charge, which are often incorporated into hapten design to generate high-affinity antibodies. The ability of RS2-1G9 to discriminate between closely related AHLs is conferred by six hydrogen bonds to the ligand. Conversely, cross-reactivity of RS2-1G9 towards the lactone is likely to originate from conservation of these hydrogen bonds as well as an additional hydrogen bond to the oxygen of the lactone ring. A short, narrow tunnel exiting at the protein surface harbors a portion of the acyl chain and would not allow entry of the head group. The crystal structure of the antibody without its cognate lactam or lactone ligands revealed a considerably altered antibody-combining site with a closed binding pocket. Curiously, a completely buried ethylene glycol molecule mimics the lactam ring and, thus, serves as a surrogate ligand. The detailed structural delineation of this quorum-quenching antibody will aid further development of an antibody-based therapy against bacterial pathogens by interference with quorum sensing.

Cell Death and Differentiation, 2008

Activation of the proapoptotic receptor death receptor5 (DR5) in various cancer cells triggers pr... more Activation of the proapoptotic receptor death receptor5 (DR5) in various cancer cells triggers programmed cell death through the extrinsic pathway. We have generated a fully human monoclonal antibody (Apomab) that induces tumor cell apoptosis through DR5 and investigated the structural features of its interaction with DR5. Biochemical studies showed that Apomab binds DR5 tightly and selectively. X-ray crystallographic analysis of the complex between the Apomab Fab fragment and the DR5 ectodomain revealed an interaction epitope that partially overlaps with both regions of the Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand binding site. Apomab induced DR5 clustering at the cell surface and stimulated a death-inducing signaling complex containing the adaptor molecule Fas-associated death domain and the apoptosis-initiating protease caspase-8. Fc crosslinking further augmented Apomab's proapoptotic activity. In vitro, Apomab triggered apoptosis in cancer cells, while sparing normal hepatocytes even upon anti-Fc crosslinking. In vivo, Apomab exerted potent antitumor activity as a single agent or in combination with chemotherapy in xenograft models, including those based on colorectal, non-small cell lung and pancreatic cancer cell lines. These results provide structural and functional insight into the interaction of Apomab with DR5 and support further investigation of this antibody for cancer therapy.

Annual Review of Biochemistry, 2011

In eukaryotic cells, the spatial segregation of replication and transcription in the nucleus and ... more In eukaryotic cells, the spatial segregation of replication and transcription in the nucleus and translation in the cytoplasm imposes the requirement of transporting thousands of macromolecules between these two compartments. Nuclear pore complexes (NPCs) are the sole gateways that facilitate this macromolecular exchange across the nuclear envelope with the help of soluble transport receptors. Whereas the mobile transport machinery is reasonably well understood at the atomic level, a commensurate structural characterization of the NPC has only begun in the past few years. Here, we describe the recent progress toward the elucidation of the atomic structure of the NPC, highlight emerging concepts of its underlying architecture, and discuss key outstanding questions and challenges. The applied structure determination as well as the described design principles of the NPC may serve as paradigms for other macromolecular assemblies.

Journal of Molecular Biology, 2004

Triggering receptor expressed on myeloid cells (TREM) 1 is an activating receptor expressed on my... more Triggering receptor expressed on myeloid cells (TREM) 1 is an activating receptor expressed on myeloid cells whose ligand(s) remain elusive. TREM-1 stimulation activates neutrophils and monocytes and induces the secretion of pro-inflammatory molecules, which amplifies the Toll-like receptor-initiated responses to invading pathogens. In addition, TREM-1 mediates the septic shock pathway, and thus represents a potential therapeutic target. We report the crystal structure of the mouse TREM-1 extracellular domain at 1.76 Å resolution. The mouse extracellular domain is monomeric, consistent with our previous human TREM-1 structure, and strongly supports the contention that the globular TREM-1 head is a monomer contrary to proposals of a symmetric dimer.

Nephron Physiology, 2007

lum and no redistribution was observed after forskolin stimulation. Of note, homology modeling re... more lum and no redistribution was observed after forskolin stimulation. Of note, homology modeling revealed that the two mutations involve two highly conserved residues providing important clues about the role of the wt residues in AQP2 stability and function.

Proceedings of the National Academy of Sciences, 2009

Design of catalysts featuring multiple functional groups is a desirable, yet formidable goal. Ant... more Design of catalysts featuring multiple functional groups is a desirable, yet formidable goal. Antibody 13G5, which accelerates the cleavage of unactivated benzisoxazoles, is one of few artificial enzymes that harness an acid and a base to achieve efficient proton transfer. X-ray structures of the Fab-hapten complexes of wild-type 13G5 and active-site variants now afford detailed insights into its mechanism. The parent antibody preorganizes Asp(H35) and Glu(L34) to abstract a proton from substrate and to orient a water molecule for leaving group stabilization, respectively. Remodeling the environment of the hydrogen bond donor with a compensatory network of ordered waters, as seen in the Glu(L34) to alanine mutant, leads to an impressive 10(9)-fold rate acceleration over the nonenzymatic reaction with acetate, illustrating the utility of buried water molecules in bifunctional catalysis. Generalization of these design principles may aid in creation of catalysts for other important chemical transformations.

Journal of the American Chemical Society, 2007

... Azidoproline Containing Helices: Stabilization of the Polyproline II Structure by a Functiona... more ... Azidoproline Containing Helices: Stabilization of the Polyproline II Structure by a Functionalizable Group. Michael Kümin, Louis-Sebastian Sonntag, and Helma Wennemers. Journal of the American Chemical Society 2007 129 (3), pp 466–467. ...

Journal of Biological Chemistry, 2008

FIGURE 3. Superimposition of the free (blue, four independent copies of the orthorhombic crystal ... more FIGURE 3. Superimposition of the free (blue, four independent copies of the orthorhombic crystal form) and hapten-bound (yellow, four independent copies) 34E4. The complementarity determining regions (CDRs) of the light and heavy chains, which are labeled L1-L3 and H1-H3, respectively, undergo only minor changes upon ligand binding, whereas Trp L91 swings into the binding pocket and serves as surrogate ligand in the apo form. FIGURE 4. Molecular surface representation of free (A) and hapten-bound (B) 34E4. The hapten is displayed as pink stick model. The architecture of the antibody combining site is dramatically different in the two states. The shallow indentation of the predominant closed conformation of the antibody in the unliganded form (A) can convert to an open conformation that can then harbor the hapten in a deep cavity (B).

Angewandte Chemie International Edition, 2006

Journal of Biological Chemistry, 2016

In the family of protein arginine methyltransferases (PRMTs) that predominantly generate either a... more In the family of protein arginine methyltransferases (PRMTs) that predominantly generate either asymmetric or symmetric dimethylarginine (ADMA or SDMA), PRMT7 is unique in producing solely monomethylarginine (MMA) products. The type of methylation on histones and other proteins dictates changes in gene expression, and numerous studies have linked altered profiles of methyl marks with disease phenotypes. Given the importance of specific inhibitor development, it is crucial to understand the mechanisms by which PRMT product specificity is conferred. We have focused our attention on active-site residues of PRMT7 from the protozoan Trypanosoma brucei We have designed 26 single and double mutations in the active site, including residues in the Glu-X8-Glu (double E) loop and the Met-Gln-Trp sequence of the canonical Thr-His-Trp (THW) loop known to interact with the methyl-accepting substrate arginine. Analysis of the reaction products by high-resolution cation exchange chromatography combined with the knowledge of PRMT crystal structures suggests a model where the size of two distinct subregions in the active site determines PRMT7 product specificity. A dual mutation of Glu181 to Asp in the double E loop and Gln329 to Ala in the canonical THW loop enables the enzyme to produce SDMA. Consistent with our model, the mutation of Cys431 to His in the THW loop of human PRMT9 shifts its product specificity from SDMA towards MMA. Together with previous results, these findings provide a structural basis and a general model for product specificity in PRMTs, which will be useful for the rational design of specific PRMT inhibitors.

Proceedings of the National Academy of Sciences of the United States of America, Jan 8, 2016

Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly m... more Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coo...

Nature Structural & Molecular Biology, 2009

Molecular Cell, 2008

We recently proposed a cylindrical coat for the nuclear pore membrane in the nuclear pore complex... more We recently proposed a cylindrical coat for the nuclear pore membrane in the nuclear pore complex (NPC). This scaffold is generated by multiple copies of seven nucleoporins. Here, we report three crystal structures of the nucleoporin pair Seh1 d Nup85, which is part of the coat cylinder. The Seh1 d Nup85 assembly bears resemblance in its shape and dimensions to that of another nucleoporin pair, Sec13 d Nup145C. Furthermore, the Seh1 d Nup85 structures reveal a hinge motion that may facilitate conformational changes in the NPC during import of integral membrane proteins and/or during nucleocytoplasmic transport. We propose that Seh1 d Nup85 and Sec13 d Nup145C form 16 alternating, vertical rods that are horizontally linked by the three remaining nucleoporins of the coat cylinder. Shared architectural and mechanistic principles with the COPII coat indicate a common evolutionary origin and support the notion that the NPC coat represents another class of membrane coats.

Science, 2008

The blue-emissive antibody EP2-19G2 that has been elicited against trans-stilbene has unprecedent... more The blue-emissive antibody EP2-19G2 that has been elicited against trans-stilbene has unprecedented ability to produce bright luminescence and has been used as a biosensor in various applications. We show that the prolonged luminescence is not stilbene fluorescence. Instead, the emissive species is a charge-transfer excited complex of an anionic stilbene and a cationic, parallel pi-stacked tryptophan. Upon charge recombination, this complex generates exceptionally bright blue light. Complex formation is enabled by a deeply penetrating ligand-binding pocket, which in turn results from a noncanonical interface between the two variable domains of the antibody.

Proceedings of the National Academy of Sciences, 2009

The Nup84 complex constitutes a key building block in the nuclear pore complex (NPC). Here we pre... more The Nup84 complex constitutes a key building block in the nuclear pore complex (NPC). Here we present the crystal structure of one of its 7 components, Nup120, which reveals a ␤ propeller and an ␣-helical domain representing a novel fold. We discovered a previously unidentified interaction of Nup120 with Nup133 and confirmed the physiological relevance in vivo. As mapping of the individual components in the Nup84 complex places Nup120 and Nup133 at opposite ends of the heptamer, our findings indicate a head-to-tail arrangement of elongated Nup84 complexes into a ring structure, consistent with a fence-like coat for the nuclear pore membrane. The attachment site for Nup133 lies at the very end of an extended unstructured region, which allows for flexibility in the diameter of the Nup84 complex ring. These results illuminate important roles of terminal unstructured segments in nucleoporins for the architecture, function, and assembly of the NPC.

Proceedings of the National Academy of Sciences, 2005

Antibody 34E4 catalyzes the conversion of benzisoxazoles to salicylonitriles with high rates and ... more Antibody 34E4 catalyzes the conversion of benzisoxazoles to salicylonitriles with high rates and multiple turnovers. The crystal structure of its complex with the benzimidazolium hapten at 2.5-angstroms resolution shows that a combination of hydrogen bonding, pi stacking, and van der Waals interactions is exploited to position both the base, Glu(H50), and the substrate for efficient proton transfer. Suboptimal placement of the catalytic carboxylate, as observed in the 2.8-angstroms structure of the Glu(H50)Asp variant, results in substantially reduced catalytic efficiency. In addition to imposing high positional order on the transition state, the antibody pocket provides a highly structured microenvironment for the reaction in which the carboxylate base is activated through partial desolvation, and the highly polarizable transition state is stabilized by dispersion interactions with the aromatic residue Trp(L91) and solvation of the leaving group oxygen by external water. The enzyme-like efficiency of general base catalysis in this system directly reflects the original hapten design, in which a charged guanidinium moiety was strategically used to elicit an accurately positioned functional group in an appropriate reaction environment and suggests that even larger catalytic effects may be achievable by extending this approach to the induction of acid-base pairs capable of bifunctional catalysis.

Proceedings of the National Academy of Sciences, 2006

IL-2 is a cytokine that functions as a growth factor and central regulator in the immune system a... more IL-2 is a cytokine that functions as a growth factor and central regulator in the immune system and mediates its effects through ligand-induced hetero-trimerization of the receptor subunits IL-2R␣, IL-2R␤, and ␥c. Here, we describe the crystal structure of the trimeric assembly of the human IL-2 receptor ectodomains in complex with IL-2 at 3.0 Å resolution. The quaternary structure is consistent with a stepwise assembly from IL-2͞IL-2R␣ to IL-2͞IL-2R␣͞IL-2R␤ to IL-2͞IL-2R␣͞IL-2R␤͞␥c. The IL-2R␣ subunit forms the largest of the three IL-2͞IL-2R interfaces, which, together with the high abundance of charge-charge interactions, correlates well with the rapid association rate and high-affinity interaction of IL-2R␣ with IL-2 at the cell surface. Surprisingly, IL-2R␣ makes no contacts with IL-2R␤ or ␥c, and only minor changes are observed in the IL-2 structure in response to receptor binding. These findings support the principal role of IL-2R␣ to deliver IL-2 to the signaling complex and act as regulator of signal transduction. Cooperativity in assembly of the final quaternary complex is easily explained by the extraordinarily extensive set of interfaces found within the fully assembled IL-2 signaling complex, which nearly span the entire length of the IL-2R␤ and ␥c subunits. Helix A of IL-2 wedges tightly between IL-2R␤ and ␥c to form a three-way junction that coalesces into a composite binding site for the final ␥c recruitment. The IL-2͞␥c interface itself exhibits the smallest buried surface and the fewest hydrogen bonds in the complex, which is consistent with its promiscuous use in other cytokine receptor complexes.

Proceedings of the National Academy of Sciences, 2009

The heptameric Nup84 complex constitutes an evolutionarily conserved building block of the nuclea... more The heptameric Nup84 complex constitutes an evolutionarily conserved building block of the nuclear pore complex. Here, we present the crystal structure of the heterotrimeric Sec13⅐Nup145C⅐Nup84 complex, the centerpiece of the heptamer, at 3.2-Å resolution. Nup84 forms a U-shaped ␣-helical solenoid domain, topologically similar to two other members of the heptamer, Nup145C and Nup85. The interaction between Nup84 and Nup145C is mediated via a hydrophobic interface located in the kink regions of the two solenoids that is reinforced by additional interactions of two long Nup84 loops. The Nup84 binding site partially overlaps with the homo-dimerization interface of Nup145C, suggesting competing binding events. Fitting of the elongated Z-shaped heterotrimer into electron microscopy (EM) envelopes of the heptamer indicates that structural changes occur at the Nup145C⅐Nup84 interface. Docking the crystal structures of all heptamer components into the EM envelope constitutes a major advance toward the completion of the structural characterization of the Nup84 complex.

Nature Structural & Molecular Biology, 2005

As a counter-defense against antiviral RNA silencing during infection, the insect Flock House vir... more As a counter-defense against antiviral RNA silencing during infection, the insect Flock House virus (FHV) expresses the silencing suppressor protein B2. Biochemical experiments show that B2 binds to double-stranded RNA (dsRNA) without regard to length and inhibits cleavage of dsRNA by Dicer in vitro. A cocrystal structure reveals that a B2 dimer forms a four-helix bundle that binds to one face of an A-form RNA duplex independently of sequence. These results suggest that B2 blocks both cleavage of the FHV genome by Dicer and incorporation of FHV small interfering RNAs into the RNA-induced silencing complex.

Molecular and Cellular Biology, 2005

Journal of Molecular Biology, 2007

A large number of Gram-negative bacteria employ N-acyl homoserine lactones (AHLs) as signaling mo... more A large number of Gram-negative bacteria employ N-acyl homoserine lactones (AHLs) as signaling molecules in quorum sensing, which is a population density-dependent mechanism to coordinate gene expression. Antibody RS2-1G9 was elicited against a lactam mimetic of the N-acyl homoserine lactone and represents the only reported monoclonal antibody that recognizes the naturally-occuring N-acyl homoserine lactone with high affinity. Due to its high cross-reactivity, RS2-1G9 showed remarkable inhibition of quorum sensing signaling in Pseudomonas aeruginosa, a common opportunistic pathogen in humans. The crystal structure of Fab RS2-1G9 in complex with a lactam analog revealed complete encapsulation of the polar lactam moiety in the antibody-combining site. This mode of recognition provides an elegant immunological solution for tight binding to an aliphatic, lipid-like ligand with a small head group lacking typical haptenic features, such as aromaticity or charge, which are often incorporated into hapten design to generate high-affinity antibodies. The ability of RS2-1G9 to discriminate between closely related AHLs is conferred by six hydrogen bonds to the ligand. Conversely, cross-reactivity of RS2-1G9 towards the lactone is likely to originate from conservation of these hydrogen bonds as well as an additional hydrogen bond to the oxygen of the lactone ring. A short, narrow tunnel exiting at the protein surface harbors a portion of the acyl chain and would not allow entry of the head group. The crystal structure of the antibody without its cognate lactam or lactone ligands revealed a considerably altered antibody-combining site with a closed binding pocket. Curiously, a completely buried ethylene glycol molecule mimics the lactam ring and, thus, serves as a surrogate ligand. The detailed structural delineation of this quorum-quenching antibody will aid further development of an antibody-based therapy against bacterial pathogens by interference with quorum sensing.

Cell Death and Differentiation, 2008

Activation of the proapoptotic receptor death receptor5 (DR5) in various cancer cells triggers pr... more Activation of the proapoptotic receptor death receptor5 (DR5) in various cancer cells triggers programmed cell death through the extrinsic pathway. We have generated a fully human monoclonal antibody (Apomab) that induces tumor cell apoptosis through DR5 and investigated the structural features of its interaction with DR5. Biochemical studies showed that Apomab binds DR5 tightly and selectively. X-ray crystallographic analysis of the complex between the Apomab Fab fragment and the DR5 ectodomain revealed an interaction epitope that partially overlaps with both regions of the Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand binding site. Apomab induced DR5 clustering at the cell surface and stimulated a death-inducing signaling complex containing the adaptor molecule Fas-associated death domain and the apoptosis-initiating protease caspase-8. Fc crosslinking further augmented Apomab's proapoptotic activity. In vitro, Apomab triggered apoptosis in cancer cells, while sparing normal hepatocytes even upon anti-Fc crosslinking. In vivo, Apomab exerted potent antitumor activity as a single agent or in combination with chemotherapy in xenograft models, including those based on colorectal, non-small cell lung and pancreatic cancer cell lines. These results provide structural and functional insight into the interaction of Apomab with DR5 and support further investigation of this antibody for cancer therapy.

Annual Review of Biochemistry, 2011

In eukaryotic cells, the spatial segregation of replication and transcription in the nucleus and ... more In eukaryotic cells, the spatial segregation of replication and transcription in the nucleus and translation in the cytoplasm imposes the requirement of transporting thousands of macromolecules between these two compartments. Nuclear pore complexes (NPCs) are the sole gateways that facilitate this macromolecular exchange across the nuclear envelope with the help of soluble transport receptors. Whereas the mobile transport machinery is reasonably well understood at the atomic level, a commensurate structural characterization of the NPC has only begun in the past few years. Here, we describe the recent progress toward the elucidation of the atomic structure of the NPC, highlight emerging concepts of its underlying architecture, and discuss key outstanding questions and challenges. The applied structure determination as well as the described design principles of the NPC may serve as paradigms for other macromolecular assemblies.