A surface plasmon resonance assay for characterisation and epitope mapping of anti‐GLP‐1 antibodies

The incretin hormone glucagon‐like peptide‐1 (GLP‐1) has been subject to substantial pharmaceutical research regarding the treatment of type 2 diabetes mellitus. However, quantification of GLP‐1 levels remains complicated due to the low circulation concentration and concurrent existence of numerous metabolites, homologous peptides, and potentially introduced GLP‐1 receptor agonists. Surface plasmon resonance (SPR) facilitates real‐time monitoring allowing a more detailed characterisation of the interaction compared with conventional enzyme‐linked immunosorbent assays (ELISA). In this paper, we describe the development of the first SPR assays for characterisation of anti‐GLP‐1 antibodies for ELISA purposes. Binding responses were obtained on covalently immobilised anti‐GLP‐1 antibodies at 12°C, 25°C, and 40°C and fitted to a biomolecular (1:1) interaction model showing association rates of 1.01 × 10³ to 4.54 × 10³ M^?1 s^?1 and dissociation rates of 3.56 × 10^?5 to 1.56 × 10^?3 s^?1 leading to affinities of 35.2 to 344 nM, depending on the temperature. Determination of thermodynamic properties revealed an enthalpy driven interaction (ΔH < ΔS < 0) with higher affinities at lower temperatures due to the formation and stabilisation of hydrogen bonds within the binding site primarily composed of polar amino acids (ΔC_p < 0). Pair‐wise epitope mapping was performed on captured anti‐GLP‐1 antibodies followed by subsequent interaction with GLP‐1 (7‐36) and other anti‐GLP‐1 antibodies. A global evaluation of every binding response led to an epitope map elucidating the potential of various anti‐GLP‐1 antibody pairs for sandwich ELISA and hence pinpointing the optimal antibody combinations. The SPR assays proved capable of providing vital information for ELISA development endorsing it as a useful optimisation tool.     

RACK1 (receptor for activated C‐kinase 1) interactions with spectrin repeat elements

Receptor for activated C‐kinase 1 (RACK1) is an intracellular scaffolding protein involved in a multitude of signalling pathways. The cytoskeleton is fundamental for intracellular cell signalling as it forms an interconnected network of regulatory proteins. Here, spectrin is a central component as it forms the actin–spectrin network that serves as docking surfaces for cellular components. The interaction between RACK1 and components of spectrin, the single spectrin repeats R16, R17 and the double spectrin repeat R1617 from the α‐spectrin chain were investigated by biosensor technology and docking analysis. RACK1 associated only weakly to R16 (K_D = 1.0 ± 0.5 × 10^?6 M), about 20 times stronger to R1617 (K_D = 5.3 ± 0.7 × 10^?8 M) and 100 times stronger to R17 (K_D = 0.9 ± 0.3 × 10^?8 M). Docking analysis showed that while R16 alone preferentially docked with its B‐helix, R17 docked through its A‐helix and BC loop. The double repeat and RACK1 mainly formed two different complex conformations. R1617 docked tangentially to the N/C‐terminal of RACK1 or radially along a groove on the outer surface of RACK1. These configurations could account for the slight increase in entropic and the decrease in enthalpic interactions for the R1617–RACK1 interaction, compared with the interactions of RACK1 to the two single repeats. Our results suggest a mode of interaction that allows spectrin to attach to the N/C part of RACK through the inter‐helical AB and BC loops and adopt a multitude of configurations in between the two limiting configurations. Copyright © 2014 John Wiley & Sons, Ltd.     

Identification of Cys255 in HIF‐1α as a novel site for development of covalent inhibitors of HIF‐1α/ARNT PasB domain protein–protein interaction

The heterodimer HIF‐1α (hypoxia inducible factor)/HIF‐β (also known as ARNT‐aryl hydrocarbon nuclear translocator) is a key mediator of cellular response to hypoxia. The interaction between these monomer units can be modified by the action of small molecules in the binding interface between their C‐terminal heterodimerization (PasB) domains. Taking advantage of the presence of several cysteine residues located in the allosteric cavity of HIF‐1α PasB domain, we applied a cysteine‐based reactomics “hotspot identification” strategy to locate regions of HIF‐1α PasB domain critical for its interaction with ARNT. COMPOUND 5 was identified using a mass spectrometry‐based primary screening strategy and was shown to react specifically with Cys255 of the HIF‐1α PasB domain. Biophysical characterization of the interaction between PasB domains of HIF‐1α and ARNT revealed that covalent binding of COMPOUND 5 to Cys255 reduced binding affinity between HIF‐1α and ARNT PasB domains approximately 10‐fold. Detailed NMR structural analysis of HIF‐1α‐PasB‐COMPOUND 5 conjugate showed significant local conformation changes in the HIF‐1α associated with key residues involved in the HIF‐1α/ARNT PasB domain interaction as revealed by the crystal structure of the HIF‐1α/ARNT PasB heterodimer. Our screening strategy could be applied to other targets to identify pockets surrounding reactive cysteines suitable for development of small molecule modulators of protein function.     

Binding kinetics and activity of human poly(ADP‐ribose) polymerase‐1 on oligo‐deoxyribonucleotide substrates

Poly(ADP‐ribose) polymerase‐1 (PARP‐1) is a mammalian enzyme that attaches long branching chains of ADP‐ribose to specific nuclear proteins, including itself. Because its activity in vitro is dependent upon interaction with broken DNA, it has been postulated that PARP‐1 plays an important role in DNA strand‐break repair in vivo. The exact mechanism of binding to DNA and the structural determinants of binding remain to be defined, but regions of transition from single‐stranded to double‐strandedness may be important recognition sites. Here we employ surface plasmon resonance (SPR) to investigate this hypothesis. Oligodeoxynucleotide (ODN) substrates that mimic DNA with different degrees of single‐strandedness were used for measurements of both PARP‐1/DNA binding kinetics and PARP‐1's enzyme activities. We found that binding correlated with activity, but was unrelated to single‐strandedness of the ODN. Instead, PARP‐1 binding and activity were highest on ODNs that modeled a DNA double‐strand break (DSB). These results provide support for PARP‐1 recognizing and binding DSBs in a manner that is independent of single‐stranded features, and demonstrate the usefulness of SPR for simultaneously investigating both PARP‐1 binding and PARP‐1 auto‐poly(ADP‐ribosyl)ation activities within the same in vitro system. Copyright © 2009 John Wiley & Sons, Ltd.     

The investigation of recognition interaction between phenylboronate monolayer and glycated hemoglobin using surface plasmon resonance

Glycated hemoglobin (HbA1c) is formed by a nonenzymatic reaction of glucose with the N-terminal valine of adult hemoglobin's beta-chain. The amount of HbA1c reflects the average concentration of glucose variation level over the preceding 2 to 3 months. Because the boronate has antibody mimicking for HbA1c, often it is used to detect HbA1c. However, factors such as the ratio of the phenylboronic acid derivatives and diol composition, the pH of the solution, and the stereostructure of phenylboronic acid derivatives could influence the interactions between phenylboronic acid derivatives and diol composition. In this study, the factors were evaluated using surface plasmon resonance (SPR). The results show that pH value is an important factor affecting HbA1c and phenylboronic acid to form the complex and Lewis bases. This could change the stereostructure of phenylboronic acid to form B(OH)(3) for binding with saccharine easily. In addition, linear response appeared in HbA1c in the range of 0.43 to 3.49 mug/ml, and the detection limit was 0.01 microg/ml. The results also demonstrated that an SPR biosensor can be used as a sensitive technique for improving the accuracy and correctness of HbA1c measurement.     

Structures of synthetic nanobody–SARS-CoV-2 receptor-binding domain complexes reveal distinct sites of interaction

Combating the worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of new variants demands understanding of the structural basis of the interaction of antibodies with the SARS-CoV-2 receptor-binding domain (RBD). Here, we report five X-ray crystal structures of sybodies (synthetic nanobodies) including those of binary and ternary complexes of Sb16–RBD, Sb45–RBD, Sb14–RBD–Sb68, and Sb45–RBD–Sb68, as well as unliganded Sb16. These structures reveal that Sb14, Sb16, and Sb45 bind the RBD at the angiotensin-converting enzyme 2 interface and that the Sb16 interaction is accompanied by a large conformational adjustment of complementarity-determining region 2. In contrast, Sb68 interacts at the periphery of the SARS-CoV-2 RBD–angiotensin-converting enzyme 2 interface. We also determined cryo-EM structures of Sb45 bound to the SARS-CoV-2 spike protein. Superposition of the X-ray structures of sybodies onto the trimeric spike protein cryo-EM map indicates that some sybodies may bind in both “up” and “down” configurations, but others may not. Differences in sybody recognition of several recently identified RBD variants are explained by these structures.     

Disruption of shape complementarity in the ribosomal protein L1–RNA contact region does not hinder specific recognition of the RNA target site

The formation of a specific and stable complex between two (macro)molecules implies complementary contact surface regions. We used ribosomal protein L1, which specifically binds a target site on 23S rRNA, to study the influence of surface modifications on the protein?RNA affinity. The threonine residue in the universally conserved triad Thr?Met?Gly significant for RNA recognition and binding was substituted by phenylalanine, valine and alanine, respectively. The crystal structure of the mutant Thr217Val of the isolated domain I of L1 from Thermus thermophilus (TthL1) was determined. This structure and that of two other mutants, which had been determined earlier, were analysed and compared with the structure of the wild type L1 proteins. The influence of structural changes in the mutant L1 proteins on their affinity for the specific 23S rRNA fragment was tested by kinetic experiments using surface plasmon resonance (SPR) biosensor analysis. Association rate constants undergo minor changes, whereas dissociation rate constants displayed significantly higher values in comparison with that for the wild type protein. The analysed L1 mutants recognize the specific RNA target site, but the mutant L1?23S rRNA complexes are less stable compared to the wild type complexes. Copyright © 2010 John Wiley & Sons, Ltd.     

Evidence for Two Distinct Binding Sites for Lipoprotein Lipase on Glycosylphosphatidylinositol-anchored High Density Lipoprotein-binding Protein 1 (GPIHBP1)

GPIHBP1 is an endothelial membrane protein that transports lipoprotein lipase (LPL) from the subendothelial space to the luminal side of the capillary endothelium. Here, we provide evidence that two regions of GPIHBP1, the acidic N-terminal domain and the central Ly6 domain, interact with LPL as two distinct binding sites. This conclusion is based on comparative binding studies performed with a peptide corresponding to the N-terminal domain of GPIHBP1, the Ly6 domain of GPIHBP1, wild type GPIHBP1, and the Ly6 domain mutant GPIHBP1 Q114P. Although LPL and the N-terminal domain formed a tight but short lived complex, characterized by fast on- and off-rates, the complex between LPL and the Ly6 domain formed more slowly and persisted for a longer time. Unlike the interaction of LPL with the Ly6 domain, the interaction of LPL with the N-terminal domain was significantly weakened by salt. The Q114P mutant bound LPL similarly to the N-terminal domain of GPIHBP1. Heparin dissociated LPL from the N-terminal domain, and partially from wild type GPIHBP1, but was unable to elute the enzyme from the Ly6 domain. When LPL was in complex with the acidic peptide corresponding to the N-terminal domain of GPIHBP1, the enzyme retained its affinity for the Ly6 domain. Furthermore, LPL that was bound to the N-terminal domain interacted with lipoproteins, whereas LPL bound to the Ly6 domain did not. In summary, our data suggest that the two domains of GPIHBP1 interact independently with LPL and that the functionality of LPL depends on its localization on GPIHBP1.     

The Cerebro-oculo-facio-skeletal Syndrome Point Mutation F231L in the ERCC1 DNA Repair Protein Causes Dissociation of the ERCC1-XPF Complex

The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function in the nucleotide excision repair (NER) pathway. The ERCC1 point mutation F231L, located at the hydrophobic interaction interface of ERCC1 (excision repair cross-complementation group 1) and XPF (xeroderma pigmentosum complementation group F), leads to severe NER pathway deficiencies. Here, we analyze biophysical properties and report the NMR structure of the complex of the C-terminal tandem helix-hairpin-helix domains of ERCC1-XPF that contains this mutation. The structures of wild type and the F231L mutant are very similar. The F231L mutation results in only a small disturbance of the ERCC1-XPF interface, where, in contrast to Phe²³¹, Leu²³¹ lacks interactions stabilizing the ERCC1-XPF complex. One of the two anchor points is severely distorted, and this results in a more dynamic complex, causing reduced stability and an increased dissociation rate of the mutant complex as compared with wild type. These data provide a biophysical explanation for the severe NER deficiencies caused by this mutation.     

Modulation of SHP‐1 phosphatase activity by monovalent and bivalent SH2 phosphopeptide ligands

A sequence derived from the epithelial receptor tyrosine kinase Ros (pY2267) represents a high‐affinity binding partner for protein tyrosine phosphatase SHP‐1 and was recently used as lead structure to analyze the recognition requirements for the enzyme's N‐SH2 domain. Here, we focused on a set of peptides comprising C‐terminally extended linear and conformationally constrained side chain‐bridged cyclic N‐SH2 ligands based on the consensus sequence LxpYhxh(h/b)(h/b) (x = any amino acid, h = hydrophobic, and b = basic residue). Furthermore, the bivalent peptides described were designed to modulate the activity of SHP‐1 through binding to both, the N‐SH2 domain as well as an independent binding site on the surface of the catalytic domain (PTP domain). Consistent with previous experimental findings, surface plasmon resonance experiments revealed dissociation constants of most compounds in the low micromolar range. One peptide, EGLNpYc[KVD]MFPAPEEE? NH₂, displayed favorable binding affinity, but reduced ability to stimulate SHP‐1. Docking experiments revealed that the binding of this ligand occurs in binding mode I, recently described to lead to an inhibited activation of SHP‐1. In summary, results presented in this study suggest that inhibitory N‐SH2 ligands of SHP‐1 may be obtained by designing bivalent compounds that associate with the N‐SH2 domain and simultaneously occupy a specific binding site on the PTP domain. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 102–112, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

EYA1 mutations associated with the branchio‐oto‐renal syndrome result in defective otic development in Xenopus laevis

Background information. The BOR (branchio‐oto‐renal) syndrome is a dominant disorder most commonly caused by mutations in the EYA1 (Eyes Absent 1) gene. Symptoms commonly include deafness and renal anomalies. Results. We have used the embryos of the frog Xenopus laevis as an animal model for early ear development to examine the effects of different EYA1 mutations. Four eya1 mRNAs encoding proteins correlated with congenital anomalies in human were injected into early stage embryos. We show that the expression of mutations associated with BOR, even in the presence of normal levels of endogenous eya1 mRNA, leads to morphologically abnormal ear development as measured by overall otic vesicle size, establishment of sensory tissue and otic innervation. The molecular consequences of mutant eya1 expression were assessed by QPCR (quantitative PCR) analysis and in situ hybridization. Embryos expressing mutant eya1 showed altered levels of multiple genes (six1, dach, neuroD, ngnr‐1 and nt3) important for normal ear development. Conclusions. These studies lend support to the hypothesis that dominant‐negative effects of EYA1 mutations may have a role in the pathogenesis of BOR.     

Nitric oxide‐mediated regulation of ‐amyloid clearance via alterations of MMP‐9/TIMP‐1

Fibrillar amyloid plaques are largely composed of amyloid‐beta (Aβ) peptides that are metabolized into products, including Aβ1‐16, by proteases including matrix metalloproteinase 9 (MMP‐9). The balance between production and degradation of Aβ proteins is critical to amyloid accumulation and resulting disease. Regulation of MMP‐9 and its endogenous inhibitor tissue inhibitor of metalloproteinase (TIMP)‐1 by nitric oxide (NO) has been shown. We hypothesize that nitric oxide synthase (NOS2) protects against Alzheimer's disease pathology by increasing amyloid clearance through NO regulation of MMP‐9/TIMP‐1 balance. We show NO‐mediated increased MMP‐9/TIMP‐1 ratios enhanced the degradation of fibrillar Aβ in vitro, which was abolished when silenced for MMP‐9 protein translation. The in vivo relationship between MMP‐9, NO and Aβ degradation was examined by comparing an Alzheimer's disease mouse model that expresses NOS2 with a model lacking NOS2. To quantitate MMP‐9 mediated changes, we generated an antibody recognizing the Aβ1‐16 fragment, and used mass spectrometry multi‐reaction monitoring assay for detection of immunoprecipitated Aβ1‐16 peptides. Aβ1‐16 levels decreased in brain lysates lacking NOS2 when compared with strains that express human amyloid precursor protein on the NOS2 background. TIMP‐1 increased in the APPSwDI/NOS2^?/? mice with decreased MMP activity and increased amyloid burden, thereby supporting roles for NO in the regulation of MMP/TIMP balance and plaque clearance.     

Spectrofluorimetric determination of aluminium using 2‐hydroxy‐1‐naphthylidene‐(8‐aminoquinoline)

A sensitive and selective spectrofluorimetric method has been developed for the rapid determination of aluminium. This method is based on the complex formation between aluminium and 2‐hydroxy‐1‐naphthylidene‐(8‐aminoquinoline) (HNAQ). The optimum conditions for the complex formation were a metal‐to‐ligand (M : L) stoichiometric ratio of 1:1, a pH of 5.5 and a 0.20 m acetate buffer. The fluorescence of the complex was monitored at an emission wavelength of 502 nm with excitation at 438 nm. Under these conditions, linear calibration curves were obtained in the ranges 0.05–1 and 1–5 ppm. The detection limit was 3.4 ppb for the former and 13.5 ppb for the latter. The maximum relative standard deviation of the method for an aluminium standard of 200 ppb was 1.5% (n = 5). This method was successfully applied for the determination of aluminium in drinking water, pharmaceutical antacid tablets and suspension samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Mucin-like Region of Herpes Simplex Virus Type 1 Attachment Protein Glycoprotein C (gC) Modulates the Virus-Glycosaminoglycan Interaction

Glycoprotein C (gC) mediates the attachment of HSV-1 to susceptible host cells by interacting with glycosaminoglycans (GAGs) on the cell surface. gC contains a mucin-like region located near the GAG-binding site, which may affect the binding activity. Here, we address this issue by studying a HSV-1 mutant lacking the mucin-like domain in gC and the corresponding purified mutant protein (gCΔmuc) in cell culture and GAG-binding assays, respectively. The mutant virus exhibited two functional alterations as compared with native HSV-1 (i.e. decreased sensitivity to GAG-based inhibitors of virus attachment to cells and reduced release of viral particles from the surface of infected cells). Kinetic and equilibrium binding characteristics of purified gC were assessed using surface plasmon resonance-based sensing together with a surface platform consisting of end-on immobilized GAGs. Both native gC and gCΔmuc bound via the expected binding region to chondroitin sulfate and sulfated hyaluronan but not to the non-sulfated hyaluronan, confirming binding specificity. In contrast to native gC, gCΔmuc exhibited a decreased affinity for GAGs and a slower dissociation, indicating that once formed, the gCΔmuc-GAG complex is more stable. It was also found that a larger number of gCΔmuc bound to a single GAG chain, compared with native gC. Taken together, our data suggest that the mucin-like region of HSV-1 gC is involved in the modulation of the GAG-binding activity, a feature of importance both for unrestricted virus entry into the cells and release of newly produced viral particles from infected cells.