The von Willebrand factor D'D3 assembly and structural principles for factor VIII binding and concatemer biogenesis.

D assemblies make up half of the von Willebrand factor (VWF), yet are of unknown structure. D1 and D2 in the prodomain and D'D3 in mature VWF at Golgi pH form helical VWF tubules in Weibel Palade bodies and template dimerization of D3 through disulfides to form ultralong VWF concatemers. D'D3 forms the binding site for factor VIII. The crystal structure of monomeric D'D3 with cysteine residues required for dimerization mutated to alanine was determined at an endoplasmic reticulum (ER)-like pH. The smaller C8-3, TIL3 (trypsin inhibitor-like 3), and E3 modules pack through specific interfaces as they wind around the larger, N-terminal, Ca2+-binding von Willebrand D domain (VWD) 3 module to form a wedge shape. D' with its TIL' and E' modules projects away from D3. The 2 mutated cysteines implicated in D3 dimerization are buried, providing a mechanism for protecting them against premature disulfide linkage in the ER, where intrachain disulfide linkages are formed. D3 dimerization requires co-association with D1 and D2, Ca2+, and Golgi-like acidic pH. Associated structural rearrangements in the C8-3 and TIL3 modules are required to expose cysteine residues for disulfide linkage. Our structure provides insight into many von Willebrand disease mutations, including those that diminish factor VIII binding, which suggest that factor VIII binds not only to the N-terminal TIL' domain of D' distal from D3 but also extends across 1 side of D3. The organizing principle for the D3 assembly has implications for other D assemblies and the construction of higher-order, disulfide-linked assemblies in the Golgi in both VWF and mucins.

Development of an aggregate-selective, human-derived α-synuclein antibody BIIB054 that ameliorates disease phenotypes in Parkinson's disease models.

Aggregation of α-synuclein (α-syn) is neuropathologically and genetically linked to Parkinson's disease (PD). Since stereotypic cell-to-cell spreading of α-syn pathology is believed to contribute to disease progression, immunotherapy with antibodies directed against α-syn is considered a promising therapeutic approach for slowing disease progression. Here we report the identification, binding characteristics, and efficacy in PD mouse models of the human-derived α-syn antibody BIIB054, which is currently under investigation in a Phase 2 clinical trial for PD. BIIB054 was generated by screening human memory B-cell libraries from healthy elderly individuals. Epitope mapping studies conducted using peptide scanning, X-ray crystallography, and mutagenesis show that BIIB054 binds to α-syn residues 1-10. BIIB054 is highly selective for aggregated forms of α-syn with at least an 800-fold higher apparent affinity for fibrillar versus monomeric recombinant α-syn and a strong preference for human PD brain tissue. BIIB054 discriminates between monomers and oligomeric/fibrillar forms of α-syn based on high avidity for aggregates, driven by weak monovalent affinity and fast binding kinetics. In efficacy studies in three different mouse models with intracerebrally inoculated preformed α-syn fibrils, BIIB054 treatment attenuated the spreading of α-syn pathology, rescued motor impairments, and reduced the loss of dopamine transporter density in dopaminergic terminals in striatum. The preclinical data reported here provide a compelling rationale for clinical development of BIIB054 for the treatment and prevention of PD.

Antibody humanization by redesign of complementarity-determining region residues proximate to the acceptor framework.

Antibodies are key components of the adaptive immune system and are well-established protein therapeutic agents. Typically high-affinity antibodies are obtained by immunization of rodent species that need to be humanized to reduce their immunogenicity. The complementarity-determining regions (CDRs) contain the residues in a defined loop structure that confer antigen binding, which must be retained in the humanized antibody. To design a humanized antibody, we graft the mature murine CDRs onto a germline human acceptor framework. Structural defects due to mismatches at the graft interface can be fixed by mutating some framework residues to murine, or by mutating some residues on the CDRs' backside to human or to a de novo designed sequence. The first approach, framework redesign, can yield an antibody with binding better than the CDR graft and one equivalent to the mature murine, and reduced immunogenicity. The second approach, CDR redesign, is presented here as a new approach, yielding an antibody with binding better than the CDR graft, and immunogenicity potentially less than that from framework redesign. Application of both approaches to the humanization of anti-α4 integrin antibody HP1/2 is presented and the concept of the hybrid humanization approach that retains "difficult to match" murine framework amino acids and uses de novo CDR design to minimize murine amino acid content and reduce cell-mediated cytotoxicity liabilities is discussed.

Structure of the LINGO-1-anti-LINGO-1 Li81 antibody complex provides insights into the biology of LINGO-1 and the mechanism of action of the antibody therapy.

Multiple sclerosis (MS) is an autoimmune-inflammatory disease of the central nervous system (CNS) with prominent demyelination and axonal injury. While most MS therapies target the immunologic response, there is a large unmet need for treatments that can promote CNS repair. LINGO-1 (leucine-rich repeat and Ig-containing Nogo receptor interacting protein-1) is a membrane protein selectively expressed in the CNS that suppresses myelination, preventing the repair of damaged axons. We are investigating LINGO-1 antagonist antibodies that lead to remyelination as a new paradigm for treatment of individuals with MS. The anti-LINGO-1 Li81 antibody,BIIB033, is currently in clinical trials and is the first MS treatment targeting CNS repair. Here, to elucidate the mechanism of action of the antibody, we solved the crystal structure of the LINGO-1-Li81 Fab complex and used biochemical and functional studies to investigate structure-function relationships. Li81 binds to the convex surface of the leucine-rich repeat domain of LINGO-1 within repeats 4-8. Fab binding blocks contact points used in the oligomerization of LINGO-1 and produces a stable complex containing two copies each of LINGO-1 and Fab that results from a rearrangement of contacts stabilizing the quaternary structure of LINGO-1. The formation of the LINGO-1-Li81 Fab complex masks functional epitopes within the Ig domain of LINGO-1 that are important for its biologic activity in oligodendrocyte differentiation. These studies provide new insights into the structure and biology of LINGO-1 and how Li81 monoclonal antibody can block its function.

EC144, a synthetic inhibitor of heat shock protein 90, blocks innate and adaptive immune responses in models of inflammation and autoimmunity.

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in folding and stabilizing multiple intracellular proteins that have roles in cell activation and proliferation. Many Hsp90 client proteins in tumor cells are mutated or overexpressed oncogenic proteins driving cancer cell growth, leading to the acceptance of Hsp90 as a potential therapeutic target for cancer. Because several signal transduction molecules that are dependent on Hsp90 function are also involved in activation of innate and adaptive cells of the immune system, we investigated the mechanism by which inhibiting Hsp90 leads to therapeutic efficacy in rodent models of inflammation and autoimmunity. EC144, a synthetic Hsp90 inhibitor, blocked LPS-induced TLR4 signaling in RAW 264.7 cells by inhibiting activation of ERK1/2, MEK1/2, JNK, and p38 MAPK but not NF-κB. Ex vivo LPS-stimulated CD11b(+) peritoneal exudate cells from EC144-treated mice were blocked from phosphorylating tumor progression locus 2, MEK1/2, and ERK1/2. Consequently, EC144-treated mice were resistant to LPS administration and had suppressed systemic TNF-α release. Inhibiting Hsp90 also blocked in vitro CD4(+) T cell proliferation in mouse and human MLRs. In vivo, semitherapeutic administration of EC144 blocked disease development in rat collagen-induced arthritis by suppressing the inflammatory response. In a mouse collagen-induced arthritis model, EC144 also suppressed disease development, which correlated with a suppressed Ag-specific Ab response and a block in activation of Ag-specific CD4(+) T cells. Our results describe mechanisms by which blocking Hsp90 function may be applicable to treatment of autoimmune diseases involving inflammation and activation of the adaptive immune response.

Structural basis of cell surface receptor recognition by botulinum neurotoxin B.

Botulinum neurotoxins (BoNTs) are potent bacterial toxins that cause paralysis at femtomolar concentrations by blocking neurotransmitter release. A 'double receptor' model has been proposed in which BoNTs recognize nerve terminals via interactions with both gangliosides and protein receptors that mediate their entry. Of seven BoNTs (subtypes A-G), the putative receptors for BoNT/A, BoNT/B and BoNT/G have been identified, but the molecular details that govern recognition remain undefined. Here we report the crystal structure of full-length BoNT/B in complex with the synaptotagmin II (Syt-II) recognition domain at 2.6 A resolution. The structure of the complex reveals that Syt-II forms a short helix that binds to a hydrophobic groove within the binding domain of BoNT/B. In addition, mutagenesis of amino acid residues within this interface on Syt-II affects binding of BoNT/B. Structural and sequence analysis reveals that this hydrophobic groove is conserved in the BoNT/G and BoNT/B subtypes, but varies in other clostridial neurotoxins. Furthermore, molecular docking studies using the ganglioside G(T1b) indicate that its binding site is more extensive than previously proposed and might form contacts with both BoNT/B and synaptotagmin. The results provide structural insights into how BoNTs recognize protein receptors and reveal a promising target for blocking toxin-receptor recognition.

Discovery of a potent and highly selective PDK1 inhibitor via fragment-based drug discovery.

We report the use of a fragment-based lead discovery method, Tethering with extenders, to discover a pyridinone fragment that binds in an adaptive site of the protein PDK1. With subsequent medicinal chemistry, this led to the discovery of a potent and highly selective inhibitor of PDK1, which binds in the 'DFG-out' conformation.

Molecular evolution of antibody cross-reactivity for two subtypes of type A botulinum neurotoxin.

Broadening antibody specificity without compromising affinity should facilitate detection and neutralization of toxin and viral subtypes. We used yeast display and a co-selection strategy to increase cross-reactivity of a single chain (sc) Fv antibody to botulinum neurotoxin type A (BoNT/A). Starting with a scFv that binds the BoNT/A1 subtype with high affinity (136 pM) and the BoNT/A2 subtype with low affinity (109 nM), we increased its affinity for BoNT/A2 1,250-fold, to 87 pM, while maintaining high-affinity binding to BoNT/A1 (115 pM). To find the molecular basis for improved cross-reactivity, we determined the X-ray co-crystal structures of wild-type and cross-reactive antibodies complexed to BoNT/A1 at resolutions up to 2.6 A, and measured the thermodynamic contribution of BoNT/A1 and A2 amino acids to wild-type and cross-reactive antibody binding. The results show how an antibody can be engineered to bind two different antigens despite structural differences in the antigen-antibody interface and may provide a general strategy for tuning antibody specificity and cross-reactivity.

Functional activity of anti-LINGO-1 antibody opicinumab requires target engagement at a secondary binding site.

LINGO-1 is a membrane protein of the central nervous system (CNS) that suppresses myelination of axons. Preclinical studies have revealed that blockade of LINGO-1 function leads to CNS repair in demyelinating animal models. The anti-LINGO-1 antibody Li81 (opicinumab), which blocks LINGO-1 function and shows robust remyelinating activity in animal models, is currently being investigated in a Phase 2 clinical trial as a potential treatment for individuals with relapsing forms of multiple sclerosis (AFFINITY: clinical trial.gov number NCT03222973). Li81 has the unusual feature that it contains two LINGO-1 binding sites: a classical site utilizing its complementarity-determining regions and a cryptic secondary site involving Li81 light chain framework residues that recruits a second LINGO-1 molecule only after engagement of the primary binding site. Concurrent binding at both sites leads to formation of a 2:2 complex of LINGO-1 with the Li81 antigen-binding fragment, and higher order complexes with intact Li81 antibody. To elucidate the role of the secondary binding site, we designed a series of Li81 variant constructs that eliminate it while retaining the classic site contacts. These Li81 mutants retained the high affinity binding to LINGO-1, but lost the antibody-induced oligodendrocyte progenitor cell (OPC) differentiation activity and myelination activity in OPC- dorsal root ganglion neuron cocultures seen with Li81. The mutations also attenuate antibody-induced internalization of LINGO-1 on cultured cortical neurons, OPCs, and cells over-expressing LINGO-1. Together these studies reveal that engagement at both LINGO-1 binding sites of Li81 is critical for robust functional activity of the antibody.

Structural understanding of stabilization patterns in engineered bispecific Ig-like antibody molecules.

Bispecific immunoglobulin-like antibodies capable of engaging multiple antigens represent a promising new class of therapeutic agents. Engineering of these molecules requires optimization of the molecular properties of one of the domain components. Here, we present a detailed crystallographic and computational characterization of the stabilization patterns in the lymphotoxin-beta receptor (LTbetaR) binding Fv domain of an anti-LTbetaR/anti-TNF-related apoptosis inducing ligand receptor-2 (TRAIL-R2) bispecific immunoglobulin-like antibody. We further describe a new hierarchical structure-guided approach toward engineering of antibody-like molecules to enhance their thermal and chemical stability.

Structural and kinetic basis for the selectivity of aducanumab for aggregated forms of amyloid-ß.

Aducanumab, a human-derived antibody targeting amyloid-ß (Aß), is in Phase 3 clinical trials for the treatment of Alzheimer's disease. Biochemical and structural analyses show that aducanumab binds a linear epitope formed by amino acids 3-7 of the Aß peptide. Aducanumab discriminates between monomers and oligomeric or fibrillar aggregates based on weak monovalent affinity, fast binding kinetics and strong avidity for epitope-rich aggregates. Direct comparative studies with analogs of gantenerumab, bapineuzumab and solanezumab demonstrate clear differentiation in the binding properties of these antibodies. The crystal structure of the Fab fragment of aducanumab bound to its epitope peptide reveals that aducanumab binds to the N terminus of Aß in an extended conformation, distinct from those seen in structures with other antibodies that target this immunodominant epitope. Aducanumab recognizes a compact epitope that sits in a shallow pocket on the antibody surface. In silico analyses suggest that aducanumab interacts weakly with the Aß monomer and may accommodate a variety of peptide conformations, further supporting its selectivity for Aß aggregates. Our studies provide a structural rationale for the low affinity of aducanumab for non-pathogenic monomers and its greater selectivity for aggregated forms than is seen for other Aß-targeting antibodies.

A structural perspective of the sequence variability within botulinum neurotoxin subtypes A1-A4.

Botulinum neurotoxin (BoNT) is a category A toxin that has been classified within seven serotypes, designated A-G. Recently, it has been discovered that sequence variability occurs in BoNTs produced by serotype A (BoNT/A) variant strains, designated as subtypes A1 and A2, which have significantly different antibody-binding properties. We have therefore made efforts to understand at the molecular level the diversity and its effects on the biological actions of the toxin, including receptor binding, substrate recognition, and catalysis. We provide the results of these studies, including the analysis of two newly sequenced BoNT/A variants, Loch Maree (A3) and 657Ba (A4), and their comparison to A1 and A2. Using sequence analysis, available functional data, molecular modeling, and comparison of models with the crystal structures of BoNT/A1 and the light chain of BoNT/A2, we conclude that these sequence differences within subtypes will impact development of broad-spectrum antibody and small ligand therapeutics, and suggest dissimilarities in binding affinity and cleavage efficiency of the SNAP-25 substrate. In particular, sequence variation in subtypes BoNT/A3 and BoNT/A4 will likely effect alpha-exosite and S1' subsite recognition, respectively.

Engineering potent long-acting variants of the Wnt inhibitor DKK2.

Wnt signaling pathways are required for a wide variety of biological processes ranging from embryonic development to tissue repair and regeneration. Dickkopf-2 (DKK2) is classically defined as a canonical Wnt inhibitor, though it may play a role in activating non-canonical Wnt pathways in the context of endothelial network formation after acute injury. Here we report the discovery of a fusion partner for a DKK2 polypeptide that significantly improves the expression, biochemical properties and pharmacokinetics (PK) of the DKK2 polypeptide. Specifically, human serum albumin (HSA) was identified as a highly effective fusion partner. Substitution of selected amino acid residues in DKK2 designed to decrease heparan sulfate binding by HSA-DKK2 variants, further improved the PK properties of the molecule in rodents. The HSA-DKK2 variants were monomeric, as thermally stable as wild type, and active as measured by their ability to bind to and prevent phosphorylation of the Wnt coreceptor LRP6. Our engineering efforts resulted in potent long-lived variants of the canonical Wnt inhibitor DKK2, applicable for Wnt pathway manipulation either by systematic delivery or focused administration at sites of tissue injury.

The structure of the neurotoxin-associated protein HA33/A from Clostridium botulinum suggests a reoccurring beta-trefoil fold in the progenitor toxin complex.

The hemagglutinating protein HA33 from Clostridium botulinum is associated with the large botulinum neurotoxin secreted complexes and is critical in toxin protection, internalization, and possibly activation. We report the crystal structure of serotype A HA33 (HA33/A) at 1.5 A resolution that contains a unique domain organization and a carbohydrate recognition site. In addition, sequence alignments of the other toxin complex components, including the neurotoxin BoNT/A, hemagglutinating protein HA17/A, and non-toxic non-hemagglutinating protein NTNHA/A, suggests that most of the toxin complex consists of a reoccurring beta-trefoil fold.

Crystal structure of a novel carboxypeptidase from the hyperthermophilic archaeon Pyrococcus furiosus.

The structure of Pyrococcus furiosus carboxypeptidase (PfuCP) has been determined to 2.2 A resolution using multiwavelength anomalous diffraction (MAD) methods. PfuCP represents the first structure of the new M32 family of carboxypeptidases. The overall structure is comprised of a homodimer. Each subunit is mostly helical with its most pronounced feature being a deep substrate binding groove. The active site lies at the bottom of this groove and contains an HEXXH motif that coordinates the metal ion required for catalysis. Surprisingly, the structure is similar to the recently reported rat neurolysin. Comparison of these structures as well as sequence analyses with other homologous proteins reveal several conserved residues. The roles for these conserved residues in the catalytic mechanism are inferred based on modeling and their location.

Structure of botulinum neurotoxin type D light chain at 1.65 A resolution: repercussions for VAMP-2 substrate specificity.

The seven serotypes (A-G) of botulinum neurotoxins (BoNTs) function through their proteolytic cleavage of one of three proteins (SNAP-25, Syntaxin, and VAMP) that form the SNARE complex required for synaptic vesicle fusion. The different BoNTs have very specific protease recognition requirements, between 15 and 50 amino acids in length depending on the serotype. However, the structural details involved in substrate recognition remain largely unknown. Here is reported the 1.65 A resolution crystal structure of the catalytic domain of BoNT serotype D (BoNT/D-LC), providing insight into the protein-protein binding interaction and final proteolysis of VAMP-2. Structural analysis has identified a hydrophobic pocket potentially involved in substrate recognition of the P1' VAMP residue (Leu 60) and a second remote site for recognition of the V1 SNARE motif that is critical for activity. A structural comparison of BoNT/D-LC with BoNT/F-LC that also recognizes VAMP-2 one residue away from the BoNT/D-LC site provides additional molecular details about the unique serotype specific activities. In particular, BoNT/D prefers a hydrophobic interaction for the V1 motif of VAMP-2, while BoNT/F adopts a more hydrophilic strategy for recognition of the same V1 motif.

Crystal structure of botulinum neurotoxin type G light chain: serotype divergence in substrate recognition.

The seven serotypes (A-G) of botulinum neurotoxins (BoNTs) block neurotransmitter release through their specific proteolysis of one of the three proteins of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) complex. BoNTs have stringent substrate specificities that are unique for metalloprotease in that they require exceptionally long substrates (1). To understand the molecular reasons for the unique specificities of the BoNTs, we determined the crystal structure of the catalytic light chain (LC) of Clostridium botulinum neurotoxin type G (BoNT/G-LC) at 2.35 A resolution. The structure of BoNT/G-LC reveals a C-terminal beta-sheet that is critical for LC oligomerization and is unlike that seen in the other LC structures. Its structural comparison with thermolysin and the available pool of LC structures reveals important serotype differences that are likely to be involved in substrate recognition of the P1' residue. In addition, structural and sequence analyses have identified a potential exosite of BoNT/G-LC that recognizes a SNARE recognition motif of VAMP.