Affinity and Specificity for Binding to Glycosaminoglycans Can Be Tuned by Adapting Peptide Length and Sequence

Although glycosaminoglycan (GAG)–protein interactions are important in many physiological and pathological processes, the structural requirements for binding are poorly defined. Starting with GAG-binding peptide CXCL9(74-103), peptides were designed to elucidate the contribution to the GAG-binding affinity of different: (1) GAG-binding motifs (i.e., BBXB and BBBXXB); (2) amino acids in GAG-binding motifs and linker sequences; and (3) numbers of GAG-binding motifs. The affinity of eight chemically synthesized peptides for various GAGs was determined by isothermal fluorescence titration (IFT). Moreover, the binding of peptides to cellular GAGs on Chinese hamster ovary (CHO) cells was assessed using flow cytometry with and without soluble GAGs. The repetition of GAG-binding motifs in the peptides contributed to a higher affinity for heparan sulfate (HS) in the IFT measurements. Furthermore, the presence of Gln residues in both GAG-binding motifs and linker sequences increased the affinity of trimer peptides for low-molecular-weight heparin (LMWH), partially desulfated (ds)LMWH and HS, but not for hyaluronic acid. In addition, the peptides bound to cellular GAGs with differential affinity, and the addition of soluble HS or heparin reduced the binding of CXCL9(74-103) to cellular GAGs. These results indicate that the affinity and specificity of peptides for GAGs can be tuned by adapting their amino acid sequence and their number of GAG-binding motifs.

Structure of the RZZ complex and molecular basis of Spindly-driven corona assembly at human kinetochores

In metazoans, a ≍1 megadalton (MDa) super-complex comprising the Dynein-Dynactin adaptor Spindly and the ROD-Zwilch-ZW10 (RZZ) complex is the building block of a fibrous biopolymer, the kinetochore fibrous corona. The corona assembles on mitotic kinetochores to promote microtubule capture and spindle assembly checkpoint (SAC) signaling. We report here a high-resolution cryo-EM structure that captures the essential features of the RZZ complex, including a farnesyl binding site required for Spindly binding. Using a highly predictive in vitro assay, we demonstrate that the SAC kinase MPS1 is necessary and sufficient for corona assembly at supercritical concentrations of the RZZ-Spindly (RZZS) complex, and describe the molecular mechanism of phosphorylation-dependent filament nucleation. We identify several structural requirements for RZZS polymerization in rings and sheets. Finally, we identify determinants of kinetochore localization and corona assembly of Spindly. Our results describe a framework for the long-sought-for molecular basis of corona assembly on metazoan kinetochores.

Computational Analysis of Flight Deck Structural Behaviour under Variable Loadings

The flight deck of an aircraft carrier is subjected to various loads. In addition, the operation of fixed-wing aircraft presents unique structural requirements for the deck. This paper, therefore, compares the structural behaviour of a flight deck which was designed following the guidelines of three classification societies: Lloyd’s Register (LR), Det Norske Veritas Germanischer Lloyd (DNV), and Registro Italiano Navale (RINA). The loading scenarios considered in this work represent the operation of an F-35B Lightning jet from a Queen Elizabeth-class (QEC) aircraft carrier. A commercial finite element analysis (FEA) software ANSYS was also used to investigate the deflection, stress and strain on the deck plates. The analysis identified that only the calculated deck thickness values based on the LR regulations would meet the requirement for the class. This finding was further supported by the FEA.

Structural Requirements of 1-(2-Pyridinyl)-5-pyrazolones for Disproportionation of Boronic Acids

We observed an unusual formation of four-coordinate boron(III) complexes from the reaction of 1-(2-pyridinyl)-5-pyrazolone derivatives with arylboronic acids in the basic media. The exact mechanism is not clear; however, the use of unprotected boronic acid and the presence of a bidentate ligand appeared to be the key structural requirements for the transformation. The results suggest that base-promoted disproportionation of arylboronic acid with the assistance of the [N,O]-bidentate ligation of 1-(2-pyridinyl)-5-pyrazolone should take place and facilitate the formation of pyrazole diarylborinate. Experiments to obtain a deeper understanding of its mechanism are currently underway.

Coherence as Competence

Being incoherent is often viewed as a paradigm kind of irrationality. Numerous authors attempt to explain the distinct-seeming failure of incoherence by positing a set of requirements of structural rationality. I argue that the notion of coherence that structural requirements are meant to capture is very slippery, and that intuitive judgments – in particular, a charge of a distinct, blatant kind of irrationality – are very imperfectly correlated with respecting the canon of structural requirements. I outline an alternative strategy for explaining our patterns of normative disapproval, one appealing to feasible dispositions to conform to substantive, non-structural norms. A wide range of paradigmatic cases of incoherence, I will argue, involve manifesting problematic dispositions, dispositions that manifest across a range of cases as blatant-seeming normative failures.

The relation between crystal structure and the occurrence of quantum-rotor induced polarization

Among hyperpolarization techniques, quantum-rotor induced polarization (QRIP), also known as Haupt effect, is a peculiar one. It is on one hand rather simple to apply by cooling and heating of a sample. On the other hand, only the methyl groups of a few substances seem to allow for the effect, which strongly limits the applicability of QRIP. While it is known, that a high tunnel frequency is favorable, the structural requirements for the effect to occur are not exhaustively studied yet. Here we report on our efforts to heuristically recognize structural motifs in molecular crystals able to allow to produce QRIP.

Redefining the specificity of phosphoinositide-binding by human PH domain-containing proteins

Pleckstrin homology (PH) domains are presumed to bind phosphoinositides (PIPs), but specific interaction with and regulation by PIPs for most PH domain-containing proteins are unclear. Here we employ a single-molecule pulldown assay to study interactions of lipid vesicles with full-length proteins in mammalian whole cell lysates. Of 67 human PH domain-containing proteins initially examined, 36 (54%) are found to have affinity for PIPs with various specificity, the majority of which have not been reported before. Further investigation of ARHGEF3 reveals distinct structural requirements for its binding to PI(4,5)P2 and PI(3,5)P2, and functional relevance of its PI(4,5)P2 binding. We generate a recursive-learning algorithm based on the assay results to analyze the sequences of 242 human PH domains, predicting that 49% of them bind PIPs. Twenty predicted binders and 11 predicted non-binders are assayed, yielding results highly consistent with the prediction. Taken together, our findings reveal unexpected lipid-binding specificity of PH domain-containing proteins.