Proline/alanine-rich sequence (PAS) polypeptides as an alternative to PEG precipitants for protein crystallization.

Proline/alanine-rich sequence (PAS) polypeptides represent a novel class of biosynthetic polymers comprising repetitive sequences of the small proteinogenic amino acids L-proline, L-alanine and/or L-serine. PAS polymers are strongly hydrophilic and highly soluble in water, where they exhibit a natively disordered conformation without any detectable secondary or tertiary structure, similar to polyethylene glycol (PEG), which constitutes the most widely applied precipitant for protein crystallization to date. To investigate the potential of PAS polymers for structural studies by X-ray crystallography, two proteins that were successfully crystallized using PEG in the past, hen egg-white lysozyme and the Fragaria × ananassa O-methyltransferase, were subjected to crystallization screens with a 200-residue PAS polypeptide. The PAS polymer was applied as a precipitant using a vapor-diffusion setup that allowed individual optimization of the precipitant concentration in the droplet in the reservoir. As a result, crystals of both proteins showing high diffraction quality were obtained using the PAS precipitant. The genetic definition and precise macromolecular composition of PAS polymers, both in sequence and in length, distinguish them from all natural and synthetic polymers that have been utilized for protein crystallization so far, including PEG, and facilitate their adaptation for future applications. Thus, PAS polymers offer potential as novel precipitants for biomolecular crystallography.

Design of a surrogate Anticalin protein directed against CD98hc for preclinical studies in mice.

The human CD98 heavy chain (CD98hc) offers a promising biomedical target both for tumor therapy and for drug delivery to the brain. We have previously developed a cognate Anticalin protein with picomolar affinity and demonstrated its effectiveness in a xenograft animal model. Due to the lack of cross-reactivity with the murine ortholog, we now report the development and X-ray structural analysis of an Anticalin with high affinity toward CD98hc from mouse. This binding protein recognizes the same protruding epitope loop-despite distinct structure-in the membrane receptor ectodomain as the Anticalin selected against human CD98hc. Thus, this surrogate Anticalin should be useful for the preclinical assessment of CD98hc targeting in vivo and support the translational development for medical application in humans.

Engineering a Highly Active Sucrose Isomerase for Enhanced Product Specificity by Using a "Battleship" Strategy.

The sucrose isomerase SmuA from Serratia plymuthica efficiently catalyses the isomerisation of sucrose into isomaltulose, an artificial sweetener used in the food industry. However, the formation of a hygroscopic by-product, trehalulose, necessitates additional separation to obtain a crystalline product. Therefore, we have improved the product specificity of SmuA by first introducing a few exploratory amino acid exchanges around the active site and investigating their influence. Then, we devised a second set of mutations, either at promising positions from the preceding cycle, but with a different side chain, or at alternative positions in the vicinity. After seven iterative cycles involving just 55 point mutations, we obtained the triple mutant Y219L/D398G/V465E which showed 2.3 times less trehalulose production but still had high catalytic efficiency (kcat /KM =11.8 mM-1 s-1 ). Not only does this mutant SmuA appear attractive as an industrial biocatalyst, but our semirational protein-engineering strategy, which resembles the battleship board game, should be of interest for other challenging enzyme optimization endeavours.

Development of a high affinity Anticalin® directed against human CD98hc for theranostic applications.

Enhanced amino acid supply and dysregulated integrin signaling constitute two hallmarks of cancer and are pivotal for metastatic transformation of cells. In line with its function at the crossroads of both processes, overexpression of CD98hc is clinically observed in various cancer malignancies, thus rendering it a promising tumor target. Methods: We describe the development of Anticalin proteins based on the lipocalin 2 (Lcn2) scaffold against the human CD98hc ectodomain (hCD98hcED) using directed evolution and protein design. X-ray structural analysis was performed to identify the epitope recognized by the lead Anticalin candidate. The Anticalin - with a tuned plasma half-life using PASylation® technology - was labeled with 89Zr and investigated by positron emission tomography (PET) of CD98-positive tumor xenograft mice. Results: The Anticalin P3D11 binds CD98hc with picomolar affinity and recognizes a protruding loop structure surrounded by several glycosylation sites within the solvent exposed membrane-distal part of the hCD98hcED. In vitro studies revealed specific binding activity of the Anticalin towards various CD98hc-expressing human tumor cell lines, suggesting broader applicability in cancer research. PET/CT imaging of mice bearing human prostate carcinoma xenografts using the optimized and 89Zr-labeled Anticalin demonstrated strong and specific tracer accumulation (8.6 ± 1.1 %ID/g) as well as a favorable tumor-to-blood ratio of 11.8. Conclusion: Our findings provide a first proof of concept to exploit CD98hc for non-invasive biomedical imaging. The novel Anticalin-based αhCD98hc radiopharmaceutical constitutes a promising tool for preclinical and, potentially, clinical applications in oncology.

Structural differences between the ectodomains of murine and human CD98hc.

The CD98 heavy chain (CD98hc) constitutes both a promising cell surface target for the treatment of cancers and a transcytosis receptor potentially useful for the brain delivery of therapeutics. However, pharmacokinetic studies and safety assessment of cognate antibodies or nonimmunoglobulin binding proteins in rodents is hampered by cross-species variability of both amino acid sequence and glycosylation pattern. Here, we report the crystal structure of the murine CD98hc extracellular domain and a comprehensive comparison with its human ortholog, revealing only one conserved surface patch that is neither shielded by glycosylation nor by the cell membrane with an accessible surface area typical for an antibody epitope. Our results imply the necessity of a surrogate approach for CD98hc-specific binding proteins with predictive power for clinical investigations.

Structural Basis for the Specific Cotranslational Incorporation of p-Boronophenylalanine into Biosynthetic Proteins.

The site-specific incorporation of the non-natural amino acid p-boronophenylalanine (Bpa) into recombinant proteins enables the development of novel carbohydrate-binding functions as well as bioorthogonal chemical modification. To this end, Bpa is genetically encoded by an amber stop codon and cotranslationally inserted into the recombinant polypeptide chain at the ribosome by means of an artificial aminoacyl-tRNA synthetase (aaRS) in combination with a compatible suppressor tRNA. We describe the crystal structure of an aaRS specific for Bpa, which had been engineered on the basis of the TyrRS from Methanocaldococcus jannaschii, in complex with both Bpa and AMP. The substrates are bound in an orientation resembling the aminoacyl-AMP mixed anhydride intermediate and engaged in a network of four hydrogen bonds that allows specific recognition of the boronate moiety by the aaRS. The key determinant of this interaction is the coplanar alignment of its Glu162 carboxylate group with Bpa, which results in a double hydrogen bond with the boronic acid substituent. Our structural study elucidates how a small set of five side chain exchanges within the TyrRS active site can switch its substrate specificity to the hydrophilic amino acid Bpa, thus stimulating the reprogramming of other aaRS to recruit useful non-natural amino acids for next-generation protein engineering.

Anticalins Reveal High Plasticity in the Mode of Complex Formation with a Common Tumor Antigen.

We describe the comparative X-ray structural analysis of three Anticalin proteins directed against the extra-domain B (ED-B) of oncofetal fibronectin (Fn), a validated marker of tumor neoangiogenesis. The Anticalins were engineered from the human lipocalin 2 (Lcn2) scaffold via targeted randomization of the structurally variable loop region and selection by phage display, resulting in 15-19 exchanged residues. While the four reshaped loops exhibit diverse conformations (with shifts in Cα positions up to 20.4 Å), the ß-barrel core of the lipocalin remains strongly conserved, thus confirming the extraordinary robustness of this scaffold. All three Anticalins bind the cc' hairpin loop of ED-B, the most exposed motif in the context of its neighboring Fn domains, but reveal entirely different binding modes, with orientations differing by up to 180°. Hence, each Anticalin recognizes its molecular target in an individual manner, in line with the distinct epitope specificities previously seen in binding experiments.

Structural basis for cellulose binding by the type A carbohydrate-binding module 64 of Spirochaeta thermophila.

Spirochaeta thermophila secretes seven glycoside hydrolases for plant biomass degradation that carry a carbohydrate-binding module 64 (CBM64) appended at the C-terminus. CBM64 adsorbs to various ß1-4-linked pyranose substrates and shows high affinity for cellulose. We present the first crystal structure of a CBM64 at 1.2 Å resolution, which reveals a jelly-roll-like fold corresponding to a surface-binding type A CBM. Modeling of its interaction with cellulose indicates that CBM64 achieves association with the hydrophobic face of ß-linked pyranose chains via a unique coplanar arrangement of four exposed tryptophan side chains. Proteins 2016; 84:855-858. © 2016 Wiley Periodicals, Inc.

Structural Basis for the Interconversion of Maltodextrins by MalQ, the Amylomaltase of Escherichia coli.

Amylomaltase MalQ is essential for the metabolism of maltose and maltodextrins in Escherichia coli. It catalyzes transglycosylation/disproportionation reactions in which glycosyl or dextrinyl units are transferred among linear maltodextrins of various lengths. To elucidate the molecular basis of transglycosylation by MalQ, we have determined three crystal structures of this enzyme, i.e. the apo-form, its complex with maltose, and an inhibitor complex with the transition state analog acarviosine-glucose-acarbose, at resolutions down to 2.1 Å. MalQ represents the first example of a mesophilic bacterial amylomaltase with known structure and exhibits an N-terminal extension of about 140 residues, in contrast with previously described thermophilic enzymes. This moiety seems unique to amylomaltases from Enterobacteriaceae and folds into two distinct subdomains that associate with different parts of the catalytic core. Intriguingly, the three MalQ crystal structures appear to correspond to distinct states of this enzyme, revealing considerable conformational changes during the catalytic cycle. In particular, the inhibitor complex highlights the requirement of both a 3-OH group and a 4-OH group (or α1-4-glycosidic bond) at the acceptor subsite +1 for the catalytically competent orientation of the acid/base catalyst Glu-496. Using an HPLC-based MalQ enzyme assay, we could demonstrate that the equilibrium concentration of maltodextrin products depends on the length of the initial substrate; with increasing numbers of glycosidic bonds, less glucose is formed. Thus, both structural and enzymatic data are consistent with the extremely low hydrolysis rates observed for amylomaltases and underline the importance of MalQ for the metabolism of maltodextrins in E. coli.

Crystal structure of the human odorant binding protein, OBPIIa.

Human odorant-binding protein, OBPIIa , is expressed by nasal epithelia to facilitate transport of hydrophobic odorant molecules across the aqueous mucus. Here, we report its crystallographic analysis at 2.6 Å resolution. OBPIIa is a monomeric protein that exhibits the classical lipocalin fold with a conserved eight-stranded ß-barrel harboring a remarkably large hydrophobic pocket. Basic residues within the four loops that shape the entrance to this ligand-binding site evoke a positive electrostatic potential. Human OBPIIa shows distinct features compared with other mammalian OBPs, including a potentially reactive Cys side chain within its pocket similar to human tear lipocalin.

The menagerie of human lipocalins: a natural protein scaffold for molecular recognition of physiological compounds.

While immunoglobulins are well-known for their characteristic ability to bind macromolecular antigens (i.e., as antibodies during an immune response), the lipocalins constitute a family of proteins whose role is the complexation of small molecules for various physiological processes. In fact, a number of low-molecular-weight substances in multicellular organisms show poor solubility, are prone to chemical decomposition, or play a pathophysiological role and thus require specific binding proteins for transport through body fluids, storage, or sequestration. In many cases, lipocalins are involved in such tasks. Lipocalins are small, usually monomeric proteins with 150-180 residues and diameters of approximately 40 Å, adopting a compact fold that is dominated by a central eight-stranded up-and-down ß-barrel. At the amino-terminal end, this core is flanked by a coiled polypeptide segment, while its carboxy-terminal end is followed by an α-helix that leans against the ß-barrel as well as an amino acid stretch in a more-or-less extended conformation, which finally is fixed by a disulfide bond. Within the ß-barrel, the antiparallel strands (designated A to H) are arranged in a (+1)7 topology and wind around a central axis in a right-handed manner such that part of strand A is hydrogen-bonded to strand H again. Whereas the lower region of the ß-barrel is closed by short loops and densely packed hydrophobic side chains, including many aromatic residues, the upper end is usually open to solvent. There, four long loops, each connecting one pair of ß-strands, together form the entrance to a cup-shaped cavity. Depending on the individual structure of a lipocalin, and especially on the lengths and amino acid sequences of its four loops, this pocket can accommodate chemical ligands of various sizes and shapes, including lipids, steroids, and other chemical hormones as well as secondary metabolites such as vitamins, cofactors, or odorants. While lipocalins are ubiquitous in all higher organisms, physiologically important members of this family have long been known in the human body, for example with the plasma retinol-binding protein that serves for the transport of vitamin A. This prototypic human lipocalin was the first for which a crystal structure was solved. Notably, several other lipocalins were discovered and assigned to this protein class before the term itself became familiar, which explains their diverse names in the scientific literature. To date, up to 15 distinct members of the lipocalin family have been characterized in humans, and during the last two decades the three-dimensional structures of a dozen major subtypes have been elucidated. This Account presents a comprehensive overview of the human lipocalins, revealing common structural principles but also deviations that explain individual functional features. Taking advantage of modern methods for combinatorial protein design, lipocalins have also been employed as scaffolds for the construction of artifical binding proteins with novel ligand specificities, so-called Anticalins, hence opening perspectives as a new class of biopharmaceuticals for medical therapy.

The dimeric crystal structure of the human fertility lipocalin glycodelin reveals a protein scaffold for the presentation of complex glycans.

Human glycodelin (Gd) is an abundant glycoprotein from the lipocalin family and is involved in crucial biological processes such as reproduction and immune reaction. In females and males, Gd is found in four distinct glycoforms-A, C, F and S-that arise from different N-linked oligosaccharide side chains at amino acid residues Asn28 and Asn63. We have expressed Gd (carrying two amino acid substitutions to improve solubility) as a non-glycosylated protein in Escherichia coli via periplasmic secretion and determined its X-ray structure at 2.45 Å resolution. Gd reveals a classical lipocalin fold including two disulfide bridges, which is however unusually compact and lacks a pronounced central pocket inside the ß-barrel, in line with its low affinity for hydrophobic ligands. Instead, this lipocalin exhibits a unique homodimeric quaternary structure that appears ideally suited as a scaffold for the presentation of specific glycans. In fact, the four oligosaccharides are presented in close proximity on the same side of the dimer surface, which increases avidity for cellular receptors, e.g. during sperm-egg recognition. A bioinformatic analysis indicated that Gd orthologues exclusively occur in certain suborders of primates that have a menstrual cycle, suggesting that this lipocalin with its role in fertility only recently emerged during evolution.

Structural and mutational analyses of Aes, an inhibitor of MalT in Escherichia coli.

The acyl esterase Aes effectively inhibits the transcriptional activity of MalT-the central activator of maltose and maltodextrin utilizing genes in Escherichia coli. To provide better insight into the nature of the interaction between Aes and MalT, we determined two different crystal structures of Aes-in its native form and covalently modified by a phenylmethylsulfonyl moiety at its active site serine. Both structures show distinct space groups and were refined to a resolution of 1.8 Å and 2.3 Å, respectively. The overall structure of Aes resembles a canonical α/ß-hydrolase fold, which is extended by a funnel-like cap structure that forms the substrate-binding site. The catalytic triad of Aes, comprising residues Ser165, His292, and Asp262, is located at the bottom of this funnel. Analysis of the crystal-packing contacts of the two different space groups as well as analytical size-exclusion chromatography revealed a homodimeric arrangement of Aes. The Aes dimer adopts an antiparallel contact involving both the hydrolase core and the cap, with its twofold axis perpendicular to the largest dimension of Aes. To identify the surface area of Aes that is responsible for the interaction with MalT, we performed a structure-based alanine-scanning mutagenesis to pinpoint Aes residues that are significantly impaired in MalT inhibition, but still exhibit wild-type expression and enzymatic activity. These residues map to a shallow slightly concave surface patch of Aes at the opposite site of the dimerization interface and indicate the surface area that interacts with MalT.

A photoswitchable neurotransmitter analogue bound to its receptor.

Incorporation of the azobenzene derivative gluazo, a synthetic photochromic ligand, into a kainate receptor allows for the optical control of neuronal activity. The crystal structure of gluazo bound to a dimeric GluK2 ligand-binding domain reveals one monomer in a closed conformation, occupied by gluazo, and the other in an open conformation, with a bound buffer molecule. The glutamate group of gluazo interacts like the natural glutamate ligand, while its trans-azobenzene moiety protrudes into a tunnel. This elongated cavity presumably cannot accommodate a cis-azobenzene, which explains the reversible activation of the receptor upon photoisomerization.

Cutting Edge: CD1a tetramers and dextramers identify human lipopeptide-specific T cells ex vivo.

Human CD1a mediates foreign Ag recognition by a T cell clone, but the nature of possible TCR interactions with CD1a/lipid are unknown. After incubating CD1a with a mycobacterial lipopeptide Ag, dideoxymycobactin (DDM), we identified and measured binding to a recombinant TCR (TRAV3/ TRBV3-1, KD of ≈100 µM). Detection of ternary CD1a/lipid/TCR interactions enabled development of CD1a tetramers and CD1a multimers with carbohydrate backbones (dextramers), which specifically stained T cells using a mechanism that was dependent on the precise stereochemistry of the peptide backbone and was blocked with a soluble TCR. Furthermore, sorting of human T cells from unrelated tuberculosis patients for bright DDM-dextramer staining allowed recovery of T cells that were activated by CD1a and DDM. These studies demonstrate that the mechanism of T cell activation by lipopeptides occurs via ternary interactions of CD1a/Ag/TCR. Furthermore, these studies demonstrate the existence of lipopeptide-specific T cells in humans ex vivo.

Crystallographic analysis and structure-guided engineering of NADPH-dependent Ralstonia sp. alcohol dehydrogenase toward NADH cosubstrate specificity.

The NADP⁺-dependent alcohol dehydrogenase from Ralstonia sp. (RasADH) belongs to the protein superfamily of short-chain dehydrogenases/reductases (SDRs). As an enzyme that accepts different types of substrates--including bulky-bulky as well as small-bulky secondary alcohols or ketones--with high stereoselectivity, it offers potential as a biocatalyst for industrial biotechnology. To understand substrate and cosubstrate specificities of RasADH we determined the crystal structure of the apo-enzyme as well as its NADP⁺-bound state with resolutions down to 2.8 Å. RasADH displays a homotetrameric quaternary structure that can be described as a dimer of homodimers while in each subunit a seven-stranded parallel ß-sheet, flanked by three α-helices on each side, forms a Rossmann fold-type dinucleotide binding domain. Docking of the well-known substrate (S)-1-phenylethanol clearly revealed the structural determinants of stereospecificity. To favor practical RasADH application in the context of established cofactor recycling systems, for example, those involving an NADH-dependent amino acid dehydrogenase, we attempted to rationally change its cosubstrate specificity from NADP⁺ to NAD⁺ utilizing the structural information that NADP⁺ specificity is largely governed by the residues Asn15, Gly37, Arg38, and Arg39. Furthermore, an extensive sequence alignment with homologous dehydrogenases that have different cosubstrate specificities revealed a modified general SDR motif ASNG (instead of NNAG) at positions 86-89 of RasADH. Consequently, we constructed mutant enzymes with one (G37D), four (N15G/G37D/R38V/R39S), and six (N15G/G37D/R38V/R39S/A86N/S88A) amino acid exchanges. RasADH (N15G/G37D/R38V/R39S) was better able to accept NAD⁺ while showing much reduced catalytic efficiency with NADP⁺, leading to a change in NADH/NADPH specificity by a factor of ∼3.6 million.

Structural basis for the enzymatic formation of the key strawberry flavor compound 4-hydroxy-2,5-dimethyl-3(2H)-furanone.

The last step in the biosynthetic route to the key strawberry flavor compound 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) is catalyzed by Fragaria x ananassa enone oxidoreductase (FaEO), earlier putatively assigned as quinone oxidoreductase (FaQR). The ripening-induced enzyme catalyzes the reduction of the exocyclic double bond of the highly reactive precursor 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone (HMMF) in a NAD(P)H-dependent manner. To elucidate the molecular mechanism of this peculiar reaction, we determined the crystal structure of FaEO in six different states or complexes at resolutions of ≤1.6 Å, including those with HDMF as well as three distinct substrate analogs. Our crystallographic analysis revealed a monomeric enzyme whose active site is largely determined by the bound NAD(P)H cofactor, which is embedded in a Rossmann-fold. Considering that the quasi-symmetric enolic reaction product HDMF is prone to extensive tautomerization, whereas its precursor HMMF is chemically labile in aqueous solution, we used the asymmetric and more stable surrogate product 2-ethyl-4-hydroxy-5-methyl-3(2H)-furanone (EHMF) and the corresponding substrate (2E)-ethylidene-4-hydroxy-5-methyl-3(2H)-furanone (EDHMF) to study their enzyme complexes as well. Together with deuterium-labeling experiments of EDHMF reduction by [4R-(2)H]NADH and chiral-phase analysis of the reaction product EHMF, our data show that the 4R-hydride of NAD(P)H is transferred to the unsaturated exocyclic C6 carbon of HMMF, resulting in a cyclic achiral enolate intermediate that subsequently becomes protonated, eventually leading to HDMF. Apart from elucidating this important reaction of the plant secondary metabolism our study provides a foundation for protein engineering of enone oxidoreductases and their application in biocatalytic processes.

Combinatorial design of an Anticalin directed against the extra-domain b for the specific targeting of oncofetal fibronectin.

The oncofetal isoform of the extracellular matrix protein fibronectin (Fn), which carries the extra-domain B (ED-B) and is exclusively expressed in neovasculature, has gained interest for tumor diagnosis and therapy using engineered antibody fragments. We have employed the human lipocalin 2 (Lcn2) as a small and robust non-immunoglobulin scaffold to select ED-B-specific Anticalins from a new advanced random library using bacterial phage display and ELISA screening against appropriately engineered Fn fragments. As a result, we have isolated and biochemically characterized four different Anticalins that all show low nanomolar affinities for ED-B, right in the range between the monomeric and dimeric forms of the single-chain variable antibody fragment L19 that has been widely applied in this area before. All Anticalins can be readily expressed in Escherichia coli as soluble and strictly monomeric proteins, and they show specific staining of ED-B-positive tumor cells in immunofluorescence microscopy while BIAcore affinity analyses indicate recognition of distinct ED-B epitopes. The crystal structure for one Anticalin, N7A, in complex with the Fn7B8 fragment, was solved at 2.6Å resolution and reveals binding to the gfcc' sheet and cc' loop on ED-B. This is the second example of a protein-specific Lcn2-based Anticalin, which illustrates the remarkable plasticity of the calyx-like ligand pocket of lipocalins with their four structurally hypervariable loops supported by a highly conserved ß-barrel. The ED-B-specific Anticalins resulting from this study should provide useful reagents in research and biomedical drug development, both for in vivo imaging and for directed cancer therapy.

Crystal structure of the ω-aminotransferase from Paracoccus denitrificans and its phylogenetic relationship with other class III aminotransferases that have biotechnological potential.

Apart from their crucial role in metabolism, pyridoxal 5'-phosphate (PLP)-dependent aminotransferases (ATs) constitute a class of enzymes with increasing application in industrial biotechnology. To provide better insight into the structure-function relationships of ATs with biotechnological potential we performed a fundamental bioinformatics analysis of 330 representative sequences of pro- and eukaryotic Class III ATs using a structure-guided approach. The calculated phylogenetic maximum likelihood tree revealed six distinct clades of which the first segregates with a very high bootstrap value of 92%. Most enzymes in this first clade have been functionally well characterized, whereas knowledge about the natural functions and substrates of enzymes in the other branches is sparse. Notably, in those clades 2-6 members of the peculiar class of ω-ATs prevail, many of which have proven useful for the preparation of chiral amines or artificial amino acids. One representative is the ω-AT from Paracoccus denitrificans (PD ω-AT) which catalyzes, for example, the transamination in a novel biocatalytic process for the production of L-homoalanine from L-threonine. To gain structural insight into this important enzyme, its X-ray analysis was carried out at a resolution of 2.6 Å, including the covalently bound PLP as well as 5-aminopentanoate as a putative amino donor substrate. On the basis of this crystal structure in conjunction with our phylogenetic analysis, we have identified a generic set of active site residues of ω-ATs that are associated with a strong preference for aromatic substrates, thus guiding the discovery of novel promising enzymes for the biotechnological production of corresponding chiral amines.

Extra-domain B in oncofetal fibronectin structurally promotes fibrillar head-to-tail dimerization of extracellular matrix protein.

The type III extra-domain B (ED-B) is specifically spliced into fibronectin (Fn) during embryogenesis and neoangiogenesis, including many cancers. The x-ray structure of the recombinant four-domain fragment Fn(III)7B89 reveals a tightly associated, extended head-to-tail dimer, which is stabilized via pair-wise shape and charge complementarity. A tendency toward ED-B-dependent dimer formation in solution was supported by size exclusion chromatography and analytical ultracentrifugation. When amending the model with the known three-dimensional structure of the Fn(III)10 domain, its RGD loop as well as the adhesion synergy region in Fn(III)9-10 become displayed on the same face of the dimer; this should allow simultaneous binding of at least two integrins and, thus, receptor clustering on the cell surface and intracellular signaling. Insertion of ED-B appears to stabilize overall head-to-tail dimerization of two separate Fn chains, which, together with alternating homodimer formation via disulfide bridges at the C-terminal Fn tail, should lead to the known macromolecular fibril formation.