Quarternary structure amplification of protein folding differences observed in ‘native’ platelet factor-4

AbstractPlatelet factor 4 (PF4), a protein of 70 residues, exists in solution as a distribution of monomer, dimer and tetramer (M-D-T) states in slow exchange on a 600 MHz 1H-NMR chemical shift time scale. Well-resolved Y60 ring proton resonances in each aggregate state allow derivation of M-D-T populations. Under the same set of solution conditions, M-D-T aggregate state distributions vary from prep to prep or with repeated freeze-drying or raising/lowering the solution pH. These variations are not the result of chemical modifications and reflect differences in the strength of subunit associations and therefore folding. Variations are greatest at pH values at or below the pKas of carboxylate groups, supporting the idea that electrostatic interactions modulate PF4 subunit interactions. Treating these distributions as true equilibria results in free energy differences of about 0.5 kcal/mol subunit. Quarternary structure amplifies free energy differences among various folded states of monomer PF4

Synthesis-Oriented Situational Analysis in User Interface Design

Analytic evaluation is a term describing a class of techniques for examining a representation of a user interface design, discovering design flaws and/or predicting user task performance. In our work with analytic evaluation, we have observed limitations on the effectiveness and efficiency of analytic techniques for formative evaluation supporting the iterative design and re-design cycle. Here we support those observations with arguments based on theoretical limitations of the models underlying these techniques. By way of comparison we discuss desirable characteristics for an alternative approach. In our search for such an alternative, we have developed the Task Mapping Model, a substantively different approach to analysis for supporting the user interface design. We briefly describe the Task Mapping Model and give some examples illustrating its desirable characteristics

The marriage of chemokines and galectins as functional heterodimers

Trafficking of leukocytes and their local activity profile are of pivotal importance for many (patho)physiological processes. Fittingly, microenvironments are complex by nature, with multiple mediators originating from diverse cell types and playing roles in an intimately regulated manner. To dissect aspects of this complexity, effectors are initially identified and structurally characterized, thus prompting familial classification and establishing foci of research activity. In this regard, chemokines present themselves as role models to illustrate the diversification and fine-tuning of inflammatory processes. This in turn discloses the interplay among chemokines, their cell receptors and cognate glycosaminoglycans, as well as their capacity to engage in new molecular interactions that form hetero-oligomers between themselves and other classes of effector molecules. The growing realization of versatility of adhesion/growth-regulatory galectins that bind to glycans and proteins and their presence at sites of inflammation led to testing the hypothesis that chemokines and galectins can interact with each other by protein-protein interactions. In this review, we present some background on chemokines and galectins, as well as experimental validation of this chemokine-galectin heterodimer concept exemplified with CXCL12 and galectin-3 as proof-of-principle, as well as sketch out some emerging perspectives in this arena

Molecular basis for cellular retinoic acid-binding protein 1 in modulating CaMKII activation

Introduction: Cellular retinoic acid (RA)-binding protein 1 (CRABP1) is a highly conserved protein comprised of an anti-parallel, beta-barrel, and a helix-turn-helix segment outside this barrel. Functionally, CRABP1 is thought to bind and sequester cytosolic RA. Recently, CRABP1 has been established as a major mediator of rapid, non-genomic activity of RA in the cytosol, referred to as “non-canonical” activity. Previously, we have reported that CRABP1 interacts with and dampens the activation of calcium-calmodulin (Ca2+-CaM)-dependent kinase 2 (CaMKII), a major effector of Ca2+ signaling. Through biophysical, molecular, and cellular assays, we, herein, elucidate the molecular and structural mechanisms underlying the action of CRABP1 in dampening CaMKII activation.Results: We identify an interaction surface on CRABP1 for CaMKII binding, located on the beta-sheet surface of the barrel, and an allosteric region within the helix segment outside the barrel, where both are important for interacting with CaMKII. Molecular studies reveal that CRABP1 preferentially associates with the inactive form of CaMKII, thereby dampening CaMKII activation. Alanine mutagenesis of residues implicated in the CaMKII interaction results in either a loss of this preference or a shift of CRABP1 from associating with the inactive CaMKII to associating with the active CaMKII, which corresponds to changes in CRABP1’s effect in modulating CaMKII activation.Conclusions: This is the first study to elucidate the molecular and structural basis for CRABP1’s function in modulating CaMKII activation. These results further shed insights into CRABP1’s functional involvement in multiple signaling pathways, as well as its extremely high sequence conservation across species and over evolution

Lactose binding to human galectin-7 (p53-induced gene 1) induces long-range effects through the protein resulting in increased dimer stability and evidence for positive cooperativity.

16 pags, 11 figs, 3 tabs. -- Supplementary data for this article are available online at: http://glycob.oxfordjournals.org/lookup/suppl/doi:10.1093/glycob/cwt005/-/DC1The product of p53-induced gene 1 is a member of the galectin family, i.e., galectin-7 (Gal-7). To move beyond structural data by X-ray diffraction, we initiated the study of the lectin by nuclear magnetic resonance (NMR) and circular dichroism spectroscopies, and molecular dynamics (MD) simulations. In concert, our results indicate that lactose binding to human Gal-7 induces long-range effects (minor conformational shifts and changes in structural dynamics) throughout the protein that result in stabilization of the dimer state, with evidence for positive cooperativity. Monte Carlo fits of 15N-Gal-7 HSQC titrations with lactose using a two-site model yield K1 = 0.9 ± 0.6 × 103 M−1 and K2 = 3.4 ± 0.8 × 103 M−1. Ligand binding-induced stabilization of the Gal-7 dimer was supported by several lines of evidence: MD-based calculations of interaction energies between ligand-loaded and ligand-free states, gel filtration data and hetero-FRET spectroscopy that indicate a highly reduced tendency for dimer dissociation in the presence of lactose, CD-based thermal denaturation showing that the transition temperature of the lectin is significantly increased in the presence of lactose, and saturation transfer difference (STD) NMR using a molecular probe of the monomer state whose presence is diminished in the presence of lactose. MD simulations with the half-loaded ligand-bound state also provided insight into how allosteric signaling may occur. Overall, our results reveal long-range effects on Gal-7 structure and dynamics, which factor into entropic contributions to ligand binding and allow further comparisons with other members of the galectin family.This work was supported by a research grant from the National Institutes of Health (CA 096090 to K.H.M.), the RAS program “Molecular and Cellular Biology” RFBR grant (No. 12 04 31360 to E.E.), the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no 260600 (“GlycoHIT”), grants CTQ2009-08536 and BFU2009-10052 and a FPI PhD fellowship to M.A.B. from the Spanish Ministry of Science and Innovation and the CIBER of Respiratory Diseases (CIBERES), an initiative from the Spanish Institute of Health Carlos III (ISCIII). E.E. was supported by a Travel Grant from the Minnesota Supercomputing Institute (University of Minnesota) during her stay in the research lab of Prof. K.H. Mayo. I.N. was supported in the Mayo lab by National Institutes of Health Hematology Training Grant (HL 07062

Binary-induced collapse of a compact, collisionless cluster

We improve and extend Shapiro's model of a relativistic, compact object which is stable in isolation but is driven dynamically unstable by the tidal field of a binary companion. Our compact object consists of a dense swarm of test particles moving in randomly-oriented, initially circular, relativistic orbits about a nonrotating black hole. The binary companion is a distant, slowly inspiraling point mass. The tidal field of the companion is treated as a small perturbation on the background Schwarzschild geometry near the hole; the resulting metric is determined by solving the perturbation equations of Regge and Wheeler and Zerilli in the quasi-static limit. The perturbed spacetime supports Bekenstein's conjecture that the horizon area of a near-equilibrium black hole is an adiabatic invariant. We follow the evolution of the system and confirm that gravitational collapse can be induced in a compact collisionless cluster by the tidal field of a binary companion.Comment: 9 Latex pages, 14 postscript figure

What is the sugar code?

54 p.-11 fig.A code is defined by the nature of the symbols, which are used to generate information-storing combinations (e.g. oligo- and polymers). Like nucleic acids and proteins, oligo- and polysac-charides are ubiquitous, and they are a biochemical platform for establishing molecular mes-sages. Of note, the letters of the sugar code system (third alphabet of life) excel in coding ca-pacity by making an unsurpassed versatility for isomer (code word) formation possible by var-iability in anomery and linkage position of the glycosidic bond, ring size and branching. The enzymatic machinery for glycan biosynthesis (writers) realizes this enormous potential for building a large vocabulary. It includes possibilities for dynamic editing/erasing as known from nucleic acids and proteins. Matching the glycome diversity, a large panel of sugar receptors (lectins) has developed based on more than a dozen folds. Lectins ‘read’ the glycan-encoded information. Hydrogen/coordination bonding and ionic pairing together with stacking and C-H/- interactions as well as modes of spatial glycan presentation underlie the selectivity and specificity of glycan-lectin recognition. Modular design of lectins together with glycan display and the nature of the cognate glycoconjugate account for the large number of post-binding events. They give an entry to the glycan vocabulary its functional, often context-dependent meaning(s), hereby building the dictionary of the sugar codeFunding by the NIH grant CA242351 (to M.C.), the SFI Investigator Programme Awards 16/IA/4419 (to P.V.M.) and 13/IA/1959 & 16/RC/3889 (to S.O.) as well as by the grant BFU 2016-77835-R of the Spanish Ministry of Economy, Industry and Competitiveness (to A.R.).Peer reviewe

The carbohydrate-binding domain on galectin-1 is more extensive for a complex glycan than for simple saccharides: implications for galectin–glycan interactions at the cell surface

gal-1 (galectin-1) mediates cell–cell and cell–extracellular matrix adhesion, essentially by interacting with β-galactoside-containing glycans of cell-surface glycoconjugates. Although most structural studies with gal-1 have investigated its binding to simple carbohydrates, in particular lactose and N-acetyl-lactosamine, this view is limited, because gal-1 functions at the cell surface by interacting with more complex glycans that are heterogeneous in size and composition. In the present study we used NMR spectroscopy to investigate the interaction of human gal-1 with a large (120 kDa) complex glycan, GRG (galactorhamnogalacturonate glycan), that contains non-randomly distributed mostly terminal β(1→4)-linked galactose side chains. We used 15N–1H-HSQC (heteronuclear single quantum coherence) NMR experiments with 15N-enriched gal-1 to identify the GRG-binding region on gal-1 and found that this region covers a large surface area on gal-1 that includes the quintessential lactose-binding site and runs from that site through a broad valley or cleft towards the dimer interface. HSQC and pulsed-field-gradient NMR diffusion experiments also show that gal-1 binds GRG with a gal-1:GRG stoichiometry of about 5:1 (or 6:1) and with average macroscopic and microscopic equilibrium dissociation constants (Kd) of 8×10−6 M and 40×10−6 M (or 48×10−6 M) respectively, indicating stronger binding than to lactose (Kd=520×10−6 M). Although gal-1 may bind GRG in various ways, the glycan can be competed for by lactose, suggesting that there is one major mode of interaction. Furthermore, even though terminal motifs on GRG are Gal-β(1→4)-Gal rather than the traditional Gal-β(1→4)-Glc/GlcNAc (where GlcNAc is N-acetylglucosamine), we show that the disaccharide Gal-β(1→4)-Gal can bind gal-1 at the lactose-binding domain. In addition, gal-1 binding to GRG disrupts inter-glycan interactions and decreases glycan-mediated solution viscosity, a glycan decongestion effect that may help explain why gal-1 promotes membrane fluidity and lateral diffusion of glycoconjugates within cell membranes. Overall, our results provide an insight into the function of galectin in situ and have potential significant biological consequences

Chemokines and galectins form heterodimers to modulate inflammation

Chemokines and galectins are simultaneously upregulated and mediate leukocyte recruitment during inflammation. Until now, these effector molecules have been considered to function independently. Here, we tested the hypothesis that they form molecular hybrids. By systematically screening chemokines for their ability to bind galectin‐1 and galectin‐3, we identified several interacting pairs, such as CXCL12 and galectin‐3. Based on NMR and MD studies of the CXCL12/galectin‐3 heterodimer, we identified contact sites between CXCL12 β‐strand 1 and Gal‐3 F‐face residues. Mutagenesis of galectin‐3 residues involved in heterodimer formation resulted in reduced binding to CXCL12, enabling testing of functional activity comparatively. Galectin‐3, but not its mutants, inhibited CXCL12‐induced chemotaxis of leukocytes and their recruitment into the mouse peritoneum. Moreover, galectin‐3 attenuated CXCL12‐stimulated signaling via its receptor CXCR4 in a ternary complex with the chemokine and receptor, consistent with our structural model. This first report of heterodimerization between chemokines and galectins reveals a new type of interaction between inflammatory mediators that can underlie a novel immunoregulatory mechanism in inflammation. Thus, further exploration of the chemokine/galectin interactome is warranted