Structural variations in the glycosaminoglycan-protein linkage region of recombinant decorin expressed in Chinese hamster ovary cells

Decorin is a small flbroblast proteoglycan consisting of a coreprotein and a single chondroitin/dermatan sulfate chain. Thestructure of the carbohydrate-protein linkage region of therecombinant decorin expressed in Chinese hamster ovary cellswas investigated. The decorin was secreted in the culture mediumand isolated by anion-exchange chromatography. The glycosaminoglycanchain was released from the decorin by β-elimination usingalkaline NaBH₄, and then digested with chondroitinase ABC. Thesetreatments resulted in a major and a few minor hexasaccharidealditols derived from the carbohydrate-protein linkage region.Their structures were analyzed by enzymatic digestion in conjunctionwith high-performance liquid chromatography. Two of these compoundshave the conventional hexasaccharide core, HexA1-3GalNAcβ1-4GlcAβ1-3Galβ1-3Galβ1-4Xyl-ol.One is nonsulfated, and the other is monosulfated on C4 of theGalNAc residue. They represent 12% and 60% of the total linkageregion, respectively. The other compound has the hexasaccharidealditol with an internal iduronic acid residue HexA1-3GalNAc(4-sulfate)β1-4IdoA1-3GaIβ1-3Galβ1-4Xyl-ol,which was previously demonstrated in one of the five linkagehexasaccharide alditols isolated from dennatan sulfate proteoglycansof bovine aorta (Sugahara et al, J. Biol Chem., 270, 7204–7212,1995).The compound accounts for 11% of the total linkage region. Thesestructural variations in the linkage hexasaccharide region ofthe decorin strikingly contrast to the uniformity demonstratedin the linkage hexasaccharide structure of human inter--trypsininhibitor (Yamada et al, Glycobiology, 5, 335–341,1995)and urinary trypsin inhibitor (Yamada et al, Eur. J. Biochem.,233, 687–693, 1995), both of which have a single chondroi-tinsulfate chain with a uniform linkage hexasaccharide structure,HexA1-3GalNAc(4-sulfate)β1-4GlcAβ1-3Gal(4-sulfate)β1-3Galβ1-4Xyl,containing a 4-O-sulfated Gal residue. chondroitin sulfate decorin dermatan sulfate glycosaminoglycan proteoglycan     

A dynamic view of domain-motif interactions

Many protein-protein interactions are mediated by domain-motif interaction, where a domain in one protein binds a short linear motif in its interacting partner. Such interactions are often involved in key cellular processes, necessitating their tight regulation. A common strategy of the cell to control protein function and interaction is by post-translational modifications of specific residues, especially phosphorylation. Indeed, there are motifs, such as SH2-binding motifs, in which motif phosphorylation is required for the domain-motif interaction. On the contrary, there are other examples where motif phosphorylation prevents the domain-motif interaction. Here we present a large-scale integrative analysis of experimental human data of domain-motif interactions and phosphorylation events, demonstrating an intriguing coupling between the two. We report such coupling for SH3, PDZ, SH2 and WW domains, where residue phosphorylation within or next to the motif is implied to be associated with switching on or off domain binding. For domains that require motif phosphorylation for binding, such as SH2 domains, we found coupled phosphorylation events other than the ones required for domain binding. Furthermore, we show that phosphorylation might function as a double switch, concurrently enabling interaction of the motif with one domain and disabling interaction with another domain. Evolutionary analysis shows that co-evolution of the motif and the proximal residues capable of phosphorylation predominates over other evolutionary scenarios, in which the motif appeared before the potentially phosphorylated residue, or vice versa. Our findings provide strengthening evidence for coupled interaction-regulation units, defined by a domain-binding motif and a phosphorylated residue.     

Host-pathogen interactions: a proteomic view

Host-pathogen interactions reflect the balance of host defenses and pathogen virulence mechanisms. Advances in proteomic technologies now afford opportunities to compare protein content between complex biologic systems ranging from cells to animals and clinical samples. Thus, it is now possible to characterize host-pathogen interactions from a global proteomic view. Most reports to date focus on cataloging protein content of pathogens and identifying virulence-associated proteins or proteomic alterations in host response. A more in-depth understanding of host-pathogen interactions has the potential to improve our mechanistic understanding of pathogenicity and virulence, thereby defining novel therapeutic and vaccine targets. In addition, proteomic characterization of the host response can provide pathogen-specific host biomarkers for rapid pathogen detection and characterization, as well as for early and specific detection of infectious diseases. A review of host-pathogen interactions focusing on proteomic analyses of both pathogen and host will be presented. Relevant genomic studies and host model systems will be also be discussed.     

Structural basis of the enhanced stability of a mutant ribozyme domain and a detailed view of RNA--solvent interactions

BACKGROUND: The structure of P4-P6, a 160 nucleotide domain of the self-splicing Tetrahymena thermophila intron, was solved previously. Mutants of the P4-P6 RNA that form a more stable tertiary structure in solution were recently isolated by successive rounds of in vitro selection and amplification. RESULTS: We show that a single-site mutant (Delta C209) possessing greater tertiary stability than wild-type P4-P6 also crystallizes much more rapidly and under a wider variety of conditions. The crystal structure provides a satisfying explanation for the increased stability of the mutant; the deletion of C209 allows the adjacent bulged adenine to enter the P4 helix and form an A-G base pair, presumably attenuating the conformational flexibility of the helix. The structure of another mutant (Delta A210) was also solved and supports this interpretation. The crystals of Delta C209 diffract to a higher resolution limit than those of wild-type RNA (2.25 A versus 2.8 A), allowing assignment of innersphere and outersphere coordination contacts for 27 magnesium ions. Structural analysis reveals an intricate solvent scaffold with a preponderance of ordered water molecules on the inside rather than the surface of the folded RNA domain. CONCLUSIONS: In vitro evolution facilitated the identification of a highly stable, structurally homogeneous mutant RNA that was readily crystallizable. Analysis of the structure suggests that improving RNA secondary structure can stabilize tertiary structure and perhaps promote crystallization. In addition, the higher resolution model provides new details of metal ion-RNA interactions and identifies a core of ordered water molecules that may be integral to RNA tertiary structure formation.     

Structural determinants of spermidine-DNA interactions

Twelve different analogs of spermidine (SPD) and SPD itself were compared for their ability to modulate two conformational transitions of DNA; the B-to-Z conformational transition of poly(dG-me⁵dC) and the thermal melting transition of calf thymus DNA. The analogs consisted of five N-ethyl-SPD derivatives [N¹-ethyl-SPD, N⁴-ethyl-SPD, N⁸-ethyl-SPD, N¹,N⁸-bis(ethyl)SPD and N¹,N⁴,N⁸-tri(ethyl)SPD], which differed in the number and/or position of the ethyl substitution (the alkyl series); three N-acetyl-SPD derivatives (N¹-acetyl-SPD, N⁴-acetyl-SPD, and N⁸-acetyl-SPD), which were comparable to the N-ethyl-SPD derivatives but not protonated at the substituted amine (the acyl series); three aliphatic analogs [nor-SPD, homo-SPD, and N¹,N⁹-bis(ethyl)homo-SPD], which differed in the interamine carbon chain length (homolog series), and 1,8-diaminooctane, which was comparable in overall chain length to SPD but lacked a central nitrogen. By comparing the relative abilities of the various analogs and SPD to modulate DNA structural transitions, it is possible to gain insight into the relative significance of the number and location of protonated amines (acyl series), the number and location of steric groups (alkyl series), aliphatic chain length (homolog series), and the central amine (1,8-diaminooctane) as determinants of SPD–DNA interactions. The B-to-Z conformational transition was facilitated to a midpoint by 2.4 μM SPD under conditions of low (i.e., 11 mM Na⁺) ionic strength. The phenomenon was affected most significantly by the number of protonated amines followed in rank order by location of the protonated amines, number of steric groups (bulk), steric group location, and aliphatic chain length. Stabilization of DNA to thermal melting was also most affected by the number of protonated amines followed by aliphatic chain length, number of steric groups, and location of protonated amines. In general, substitutions at the central (N⁴) amine of SPD exerted a significant influence on the B-to-Z transition but not on thermal melting.     

Structural background of cyclodextrin-protein interactions

Cyclodextrins are cyclic oligosaccharides with the shape of a hollow truncated cone. Their exterior is hydrophilic and their cavity is hydrophobic, which gives cyclodextrins the ability to accommodate hydrophobic molecules/moieties in the cavity. This special molecular arrangement accounts for the variety of beneficial effects cyclodextrins have on proteins, which is widely used in pharmacological applications. We have studied the interaction between beta-cyclodextrin and four non-carbohydrate-binding model proteins: ubiquitin, chymotrypsin inhibitor 2 (CI2), S6 and insulin SerB9Asp by NMR spectroscopy at varying structural detail. We demonstrate that the interaction of beta-cyclodextrin and our model proteins takes place at specific sites on the protein surface, and that solvent accessibility of those sites is a necessary but not compelling condition for the occurrence of an interaction. If this behaviour can be generalized, it might explain the wide range of different effects of cyclodextrins on different proteins: aggregation suppression (if residues responsible for aggregation are highly solvent accessible), protection against degradation (if point of attack of a protease is sterically 'masked' by cyclodextrin), alteration of function (if residues involved in function are 'masked' by cyclodextrin). The exact effect of cyclodextrins on a given protein will always be related to the particular structure of this protein.     

Structural dynamics of alpha-actinin-vinculin interactions

Alpha-actinin and vinculin orchestrate reorganization of the actin cytoskeleton following the formation of adhesion junctions. alpha-Actinin interacts with vinculin through the binding of an alpha-helix (alphaVBS) present within the R4 spectrin repeat of its central rod domain to vinculin's N-terminal seven-helical bundle domain (Vh1). The Vh1:alphaVBS structure suggests that alphaVBS first unravels from its buried location in the triple-helical R4 repeat to allow it to bind to vinculin. alphaVBS binding then induces novel conformational changes in the N-terminal helical bundle of Vh1, which disrupt its intramolecular association with vinculin's tail domain and which differ from the alterations in Vh1 provoked by the binding of talin. Surprisingly, alphaVBS binds to Vh1 in an inverted orientation compared to the binding of talin's VBSs to vinculin. Importantly, the binding of alphaVBS and talin's VBSs to vinculin's Vh1 domain appear to also trigger distinct conformational changes in full-length vinculin, opening up distant regions that are buried in the inactive molecule. The data suggest a model where vinculin's Vh1 domain acts as a molecular switch that undergoes distinct structural changes provoked by talin and alpha-actinin binding in focal adhesions versus adherens junctions, respectively.     

Structural characterization of BRCT-tetrapeptide binding interactions

BRCT(BRCA1) plays a major role in DNA repair pathway, and does so by recognizing the conserved sequence pSXXF in its target proteins. Remarkably, tetrapeptides containing pSXXF motif bind with high specificity and micromolar affinity. Here, we have characterized the binding interactions of pSXXF tetrapeptides using NMR spectroscopy and calorimetry. We show that BRCT is dynamic and becomes structured on binding, that pSer and Phe residues dictate overall binding, and that the binding affinities of the tetrapeptides are intimately linked to structural and dynamic changes both in the BRCT(BRCA1) and tetrapeptides. These results provide critical insights for designing high-affinity BRCT(BRCA1) inhibitors.     

Structural basis of thrombin-protease-activated receptor interactions

Aggregation of platelets is an essential step in the formation of a stable blood clot during vascular injury. The trypsin-like protease thrombin acts as the dominant agonist of platelet activation on engagement of protease-activated receptors (PARs). Important details on the molecular aspects of thrombin-PAR interactions have been revealed recently by structural biology. In the case of human platelets, PAR1 engages thrombin via an extended surface of recognition encompassing the active site and exosite I. In the case of murine platelets, PAR4 binds to the active site in a conformation that leaves exosite I free for interaction with cofactors like PAR3. Human PAR4 mimics the murine receptor binding mechanism for residues upstream of the scissile bond. This information is consistent with existing functional data and provides a solid background for future structural and mutagenesis studies of PAR interaction with thrombin and related proteases.     

Structural determinants for plant annexin-membrane interactions

The interactions of two plant annexins, annexin 24(Ca32) from Capsicum annuum and annexin Gh1 from Gossypium hirsutum, with phospholipid membranes have been characterized using liposome-based assays and adsorption to monolayers. These two plant annexins show a preference for phosphatidylserine-containing membranes and display a membrane binding behavior with a half-maximum calcium concentration in the sub-millimolar range. Surprisingly, the two plant annexins also display calcium-independent membrane binding at levels of 10-20% at neutral pH. This binding is regulated by three conserved surface-exposed residues on the convex side of the proteins that play a pivotal role in membrane binding. Due to quantitative differences in the membrane binding behavior of N-terminally His-tagged and wild-type annexin 24(Ca32), we conclude that the N-terminal domain of plant annexins plays an important role, reminiscent of the findings in their mammalian counterparts. Experiments elucidating plant annexin-mediated membrane aggregation and fusion, as well as the effect of these proteins on membrane surface hydrophobicity, agree with findings from the membrane binding experiments. Results from electron microscopy reveal elongated rodlike assemblies of plant annexins in the membrane-bound state. It is possible that these structures consist of protein molecules directly interacting with the membrane surface and molecules that are membrane-associated but not in direct contact with the phospholipids. The rodlike structures would also agree with the complex data from intrinsic protein fluorescence. The tubular lipid extensions suggest a role in the membrane cytoskeleton scaffolding or exocytotic processes. Overall, this study demonstrates the importance of subtle changes in an otherwise conserved annexin fold where these two plant annexins possess distinct modalities compared to mammalian and other nonplant annexins.     

Structural determinants of cooperativity in acto-myosin interactions

Regulation of muscle contraction is a very cooperative process. The presence of tropomyosin on the thin filament is both necessary and sufficient for cooperativity to occur. Data recently obtained with various tropomyosin isoforms and mutants help us to understand better the structural requirements in the thin filament for cooperative protein interactions. Forming an end-to-end overlap between neighboring tropomyosin molecules is not necessary for the cooperativity of the thin filament activation. When direct contacts between tropomyosin molecules are disrupted, the conformational changes in the filament are most probably transmitted cooperatively through actin subunits, although the exact nature of these changes is not known. The function of tropomyosin ends, alternatively expressed in various isoforms, is to confer specific actin affinity. Tropomyosin's affinity or actin is directly related to the size of the apparent cooperative unit defined as the number of actin subunits turned into the active state by binding of one myosin head. Inner sequences of tropomyosin, particularly actin-binding periods 3 to 5, play crucial role in myosin-induced activation of the thin filament. A plausible mechanism of tropomyosin function in this process is that inner tropomyosin regions are either specifically recognized by myosin or they define the right actin conformation required for tropomyosin movement from its blocking position.     

Structural aspects of homeodomain interactions with DNA

This review is devoted to the structural aspects of interaction of homeodomains with DNA. Presented are the list of all homeodomains with known spatial structure and the alignment of their amino acid sequences. The structure of homeodomains and contacts of their amino acid residues with DNA bases and sugar-phosphate backbone are described. The role of water molecules in DNA binding is discussed. Structures of multicomponent protein complexes on DNA including homeodomains are characterized.     

Structural diversity in integrin/talin interactions

The adhesion of integrins to the extracellular matrix is regulated by binding of the cytoskeletal protein talin to the cytoplasmic tail of the β-integrin subunit. Structural studies of this interaction have hitherto largely focused on the β3-integrin, one member of the large and diverse integrin family. Here, we employ NMR to probe interactions and dynamics, revealing marked structural diversity in the contacts between β1A, β1D, and β3 tails and the Talin1 and Talin2 isoforms. Coupled with analysis of recent structures of talin/β tail complexes, these studies elucidate the thermodynamic determinants of this heterogeneity and explain why the Talin2/β1D isoforms, which are co-localized in striated muscle, form an unusually tight interaction. We also show that talin/integrin affinity can be enhanced 1000-fold by deleting two residues in the β tail. Together, these studies illustrate how the integrin/talin interaction has been fine-tuned to meet varying biological requirements.