The Anticalin CL31d, an engineered lipocalin protein previously designed to specifically bind rare-earth and related metal ions as chelate complexes with p-NH2-Bn-CHX-A″-DTPA (DTPA-NH2), was subjected to structural and binding studies with a series of 11 different MIII·DTPA-NH2 complexes. These complexes include various lanthanide and main-group metal(III) ions whose radioisotopes are useful in nuclear medicine, with ionic radii ranging from 0.62 to 1.03 Å. Binding activities of the Anticalin for the MIII·DTPA-NH2 complexes were quantified by fluorescence titration (probing the intrinsic Tyr/Trp emission), revealing Kd values of 0.8-2.4 nM for most of the lanthanide ions investigated (rion = 0.75-0.96 Å), but showing markedly reduced affinities towards the small and large main-group metal ions Ga3+ (Kd = 15.3 nM) and Bi3+ (Kd = 47.8 nM), respectively. The crystal structures of six representative MIII·DTPA-NH2 complexes bound to the Anticalin were solved at high resolution (1.5-1.8 Å) using synchrotron X-ray diffraction. Superposition onto the previously described Anticalin CL31 with bound Y3+·DTPA-SCN indicated an essentially invariant conformation both for the binding protein and its metal-chelate ligands, including conserved hydrogen bonds, and a surprisingly uniform ninefold metal coordination via five carboxylate groups, three N atoms and one water molecule. However, there were two exceptions: the small Sc3+ ion appeared to be coordinated only eightfold with the DTPA-NH2 chelator but lacking the water ligand, whereas no metal electron density was observed for the Ga3+ ion, in line with its known noncanonical DTPA complex geometry. Interestingly, in this case a water molecule was detected at the expected position of the central metal ion within the protein-bound DTPA-NH2 chelator. Our investigation of the influence of chelate geometry on complex stability establishes the Anticalin CL31d as a small and robust universal binding protein for medically relevant MIII·DTPA complexes with surprisingly broad tolerance towards varying ionic radii, thus enabling flexible radionuclide-targeting strategies in nuclear medicine.

Azobenzene derivatives, which show light‐induced reversible trans ↔ cis isomerization, have gained increasing attention in the area of protein science. p ‐(Phenylazo)‐L‐phenylalanine (Pap) was recently employed to enable the light‐controlled affinity purification of biosynthetic proteins as part of the Azo‐tag. Specific supramolecular complex formation with immobilized α ‐cyclodextrin ( α ‐CD) groups is mediated by the Pap side chain in its low‐energy trans ‐configuration, whereas photoisomerization to the cis ‐state leads to immediate dissociation. Here, we describe the X‐ray crystallographic analysis of super‐folder green fluorescent protein (sfGFP) displaying Pap at amino acid position 39 on its surface in complex with α ‐CD. While this experimental structure generally confirms the mode of host–guest interaction predicted by molecular modeling, there are two unexpected observations: (i) the conically shaped α ‐CD binds with its narrow end toward the aminoacyl moiety of Pap, despite appearing sterically more demanding, and (ii) the azobenzene side chain shows a considerably twisted conformation of its two phenyl rings, which contrasts with the fully coplanar arrangement usually anticipated for unmodified azobenzene and its chemical derivatives. Thus, this crystal structure of the photoswitchable noncanonical amino acid Pap (also known as AzoF or AzoPhe) provides valuable insight for future molecular engineering endeavors to endow proteins with light‐controllable functions.

While protein A affinity chromatography is widely established for antibody purification, the acidic elution conditions often lead to protein aggregation and deamidation. Here, an alternative approach is described for the purification of antibodies utilizing an engineered binding protein based on the archaebacterial Sac7d scaffold in combination with light-controlled α-CD affinity chromatography (Excitography). Starting from a published affitin molecule, a monomeric protein version (C3A24) was engineered by substituting the unpaired thiol side chain Cys24 within the binding site by Ala, and, unexpectedly, its binding activity towards the human IgG1 Fc region was even improved (KD = 76 nM). X-ray analysis of the cocrystallized C3A24 with a recombinant human Fc fragment revealed a 2:1 stoichiometry, with a binding site at the junction between the CH2 and CH3 domains. Interestingly, this binding site coincides with the ones of protein A, protein G, and the neonatal Fc receptor (FcRn). The affitin/Fc interaction is dominated by a network of hydrogen bonds, whereas, unpredicted by the initial affitin design, the two C-terminal Lys residues are also involved via a salt bridge and another hydrogen bond. Using the Azo-tagged C3A24, we purified clinically relevant antibodies from cell culture medium in a single step under physiological buffer conditions.

Neurotoxic organophosphorus compounds (OPs) pose a severe threat if misused in military conflicts or by terrorists. Administration of a hydrolytic enzyme that can decompose the circulating nerve agent into non-toxic metabolites in vivo offers a potential treatment. A promising candidate is the homo-dimeric phosphotriesterase originating from the bacterium Brevundimonas diminuta (BdPTE), which has been subject to several rational and combinatorial protein design studies. A series of engineered versions with much improved catalytic efficiencies toward medically relevant nerve agents was described, carrying up to 22 mutations per enzyme subunit. To provide a basis for further rational design, we have determined the crystal structure of the highly active variant 10-2-C3(C59V/C227V)─stabilized against oxidation by substitution of two unpaired Cys residues─in complex with a substrate analogue at 1.5 Å resolution. Unexpectedly, the long loop segment (residues 253-276) that covers the active site shows a totally new conformation, with drastic structural deviations up to 19 Å, which was neither predicted in any of the preceding protein design studies nor seen in previous crystallographic analyses of less far evolved enzyme versions. Inspired by this structural insight, additional amino acid exchanges were introduced and their effects on protein stability as well as on the catalytic efficiency toward several neurotoxic OPs were investigated. Somewhat surprisingly, our results suggest that the presently available engineered version of BdPTE, in spite of its design on the basis of partly false structural assumptions, constitutes a fairly optimized enzyme for the detoxification of relevant OP nerve agents.

Using Anticalin technology, a lipocalin protein dubbed Colchicalin, with the ability to bind the toxic plant alkaloid colchicine with picomolar affinity, has previously been engineered, thus offering a potential antidote in vivo and also allowing its sensitive detection in biological samples. To further analyze the mode of ligand recognition, the crystal structure of Colchicalin is now reported in its unliganded form and is compared with the colchicine complex. A superposition of the protein structures revealed major rearrangements in the four structurally variable loops of the engineered lipocalin. Notably, the binding pocket in the unbound protein is largely occupied by the inward-bent loop #3, in particular Ile97, as well as by the phenylalanine side chain at position 71 in loop #2. Upon binding of colchicine, a dramatic shift of loop #3 by up to 11.1 Å occurs, in combination with a side-chain flip of Phe71, thus liberating the necessary space within the ligand pocket. Interestingly, the proline residue at the neighboring position 72, which arose during the combinatorial engineering of Colchicalin, remained in a cis configuration in both structures. These findings provide a striking example of a conformational adaptation mechanism, which is a long-known phenomenon for antibodies in immunochemistry, during the recognition of a small ligand by an engineered lipocalin, thus illustrating the general similarity between the mode of antigen/ligand binding by immunoglobulins and lipocalins.

We describe the structural analysis of two Anticalin® proteins that tightly bind Aβ40, a peptide involved in the pathophysiology of Alzheimer's disease. These anticalins, US7 and H1GA, were engineered on the basis of the human lipocalin 2, thus yielding compact single-domain binding proteins as an alternative to antibodies. Albeit selected under different conditions and mutually deviating in 13 amino acid positions within the binding pocket (of 17 mutated residues in total), both crystallised anticalins recognize the same epitope in the middle of the β-amyloid peptide. In the two complexes with the Aβ40 peptide, its central part comprising residues LysP16 to LysP28 shows well defined electron density whereas the flanking regions appear structurally disordered. The compact zigzag-bend conformation which is seen in both structures may indicate a role during conversion of the soluble monomeric form into pathogenic Aβ state(s) and, thus, explain the aggregation-inhibiting effect of the anticalins. In contrast to solanezumab, which targets the same Aβ region in a different conformation, the anticalin H1GA does not show cross-reactivity with sequence-related human plasma proteins. Consequently, anticalins offer promising reagents to prevent oligomerization of Aβ peptides to neurotoxic species in vivo and their small size may enable new routes for brain delivery.

The lack of a non-invasive test for malignant thyroid nodules makes the diagnosis of thyroid cancer (TC) challenging. Human galectin-3 (hGal3) has emerged as a promising target for medical TC imaging and diagnosis because of its exclusive overexpression in malignant thyroid tissues. We previously developed a human-chimeric αhGal3 Fab fragment derived from the rat monoclonal antibody (mAb) M3/38 with optimized clearance characteristics using PASylation technology. Here, we describe the elucidation of the hGal3 epitope recognized by mAb M3/38, X-ray crystallographic analysis of its complex with the chimeric Fab and, based on the three-dimensional structure, the rational humanization of the Fab by CDR grafting. Four CDR-grafted versions were designed using structurally most closely related fully human immunoglobulin V H /V L regions of which one—employing the acceptor framework regions of the HIV-1 neutralizing human antibody m66—showed the highest antigen affinity. By introducing two additional back-mutations to the rodent donor sequence, an affinity toward hGal3 indistinguishable from the chimeric Fab was achieved (K D = 0.34 ± 0.02 nM in SPR). The PASylated humanized Fab was site-specifically labelled with the fluorescent dye Cy7 and applied for the immuno-histochemical staining of human tissue sections representative for different TCs. The same protein was conjugated with the metal chelator Dfo, followed by radiolabelling with 89 Zr(IV). The resulting protein tracer allowed the highly sensitive and specific PET/CT imaging of orthotopic tumors in mice, which was confirmed by quantitative analysis of radiotracer accumulation. Thus, the PASylated humanized αhGal3 Fab offers clinical potential for the diagnostic imaging of TC.

Bovine butyrophilin (BTN1A1) is an abundant type I transmembrane glycoprotein exposed on the surface of milk fat globules. We have solved the crystal structure of its extracellular region via multiple wavelength anomalous dispersion after incorporation of selenomethionine into the bacterially produced protein. The butyrophilin ectodomain exhibits two subdomains with immunoglobulin fold, each comprising a β-sandwich with a central disulfide bridge as well as one N-linked glycosylation. The fifth Cys residue at position 193 is unpaired and prone to forming disulfide crosslinks. The apparent lack of a ligand-binding site or receptor activity suggests a function predominantly as hydrophilic coat protein to prevent coagulation of the milk fat droplets. While there is less structural resemblance to members of the human butyrophilin family such as BTN3A, which play a role as immune receptors, the N-terminal bovine butyrophilin subdomain shows surprising similarity to the human myelin oligodendrocyte glycoprotein, a protein exposed on the surface of myelin sheaths. Thus, our study lends structural support to earlier hypotheses of a correlation between the consumption of cow milk and prevalence of neurological autoimmune diseases and may offer guidance for the breeding of cattle strains that express modified butyrophilin showing less immunological cross-reactivity.

The affinity system based on the artificial peptide ligand Strep-tag (R) II and engineered tetrameric strep-tavidin, known as Strep-Tactin (R), offers attractive applications for the study of recombinant proteins, from detection and purification to functional immobilization. To further improve binding of the Strep-tag II to streptavidin we have subjected two protruding loops that shape its ligand pocket for the peptide - instead of D-biotin recognized by the natural protein - to iterative random mutagenesis. Sequence analyses of hits from functional screening assays revealed several unexpected structural motifs, such as a disulfide bridge at the base of one loop, replacement of the crucial residue Trp120 by Gly and a two-residue deletion in the second loop. The mutant m1-9 (dubbed Strep-Tactin XT) showed strongly enhanced affinity towards the Strep-tag II, which was further boosted in case of the bivalent Twin-Strep-tag (R). Four representative strep-tavidin mutants were crystallized in complex with the Strep-tag II peptide and their X-ray structures were solved at high resolutions. In addition, the crystal structure of the complex between Strep-Tactin XT and the Twin-Strep-tag was elucidated, indicating a bivalent mode of binding and explaining the experimentally observed avidity effect. Our study illustrates the structural plasticity of streptavidin as a scaffold for ligand binding and reveals interaction modes that would have been difficult to predict. As result, Strep-Tactin XT offers a convenient reagent for the kinetically stable immobilization of recombinant proteins fused with the Twin-Strep-tag. The possibility of reversibly dissociating such complexes simply with D-biotin as a competing ligand enables functional studies in protein science as well as cell biology. (C) 2021 The Author(s). Published by Elsevier Ltd.

Boronic acids have long been known to form cyclic diesters with cis-diol compounds, including many carbohydrates. This phenomenon was previously exploited to create an artificial lectin by incorporating p-borono-l-phenylalanine (Bpa) into the ligand pocket of an engineered lipocalin, resulting in a so-called Borocalin. Here we describe the X-ray analysis of its covalent complex with 4-nitrocatechol as a high-affinity model ligand. As expected, the crystal structure reveals the formation of a cyclic diester between the biosynthetic boronate side chain and the two ortho-hydroxy substituents of the benzene ring. Interestingly, the boron also has a hydroxide ion associated, despite an only moderately basic pH 8.5 in the crystallization buffer. The complex is stabilized by a polar contact to the side chain of Asn134 within the ligand pocket, thus validating the functional design of the Borocalin as an artificial sugar-binding protein. Our structural analysis demonstrates how a boronate can form a thermodynamically stable diester with a vicinal diol in a tetrahedral configuration in aqueous solution near physiological pH. Moreover, our data provide a basis for the further engineering of the Borocalin with the goal of specific recognition of biologically relevant glycans.

The first crystal structure of a specific cyclic diester formed between a solvated boronic acid, located in the ligand pocket of an engineered lipocalin protein, and a cis-diol compound is reported. Even though the sugar/diol binding activity of boric acid and its derivatives has been known for almost a century, structural information on corresponding complexes has remained scarce up to now. More information can be found in the communication by A. Skerra et al. on page 469 in Issue 4, 2020 (DOI: 10.1002/cbic.201900405).

AbstractBacillus anthracis verdankt seine ausgeprägte Virulenz – neben spezifischen Toxinen – einem doppelten Importmechanismus für FeIII‐Ionen. Das pathogene Bakterium sekretiert die Siderophore Bacillibactin (BB) und Petrobactin (PB), von denen nur BB von menschlichem Siderocalin, einem im Plasma vorherrschenden Lipocalin, neutralisiert wird. Wir beschreiben dessen Umgestaltung durch kombinatorisches Protein‐Design, um PB⋅FeIII anstelle von BB⋅FeIII zu binden, und dies sogar mit höherer Affinität (KD≈20 pm). Die Röntgen‐kristallographische Analyse des erhaltenen “Petrocalins” in Komplex mit PB⋅GaIII offenbart eine positiv geladene Ligandentasche, wobei die ausgedehnte schmetterlingsartige Konformation des gebundenen PB die fehlende Erkennung durch das natürliche Siderocalin erklärt. In mikrobiologischen Studien unterdrückte eine Kombination aus Petrocalin und Siderocalin das Wachstum eines BB+/PB+‐Stamms von Bacillus cereus unter eisenlimitierenden Kulturbedingungen. Das neuprogrammierte Lipocalin vermag daher neuartige Behandlungsmöglichkeiten für verheerende Infektionen durch B. anthracis zu eröffnen.