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 view of glycosaminoglycan-protein interactions

The essential role of protein-glycosaminoglycan interactions in the regulation of various physiological processes has been recognized for several decades but it is only recently that the molecular basis underlying such interactions has emerged. The different methodologies to elucidate the three-dimensional features of glycosaminoglycans along with the interactions with proteins cover high resolution NMR spectroscopy, X-ray crystallography, molecular modeling, and hydrodynamic measurements. The structural results that have accumulated have been organized in databases that allow rapid searching with entries related either to the type of glycosaminoglycan or the type of protein. Finally, three selected examples enlightening the complexity of the nature of the interactions occurring between proteins and glycosaminoglycans are given. The example of interactions between heparin and antithrombin III illustrates how such a complex mechanism as the regulation of blood coagulation by a specific pentasaccharide can be dissected through the combined use of dedicated carbohydrate chemistry and structural glycobiology. The second example deals with the study of complexes between chemokines and heparin, and shows how multimolecular complexes of proteins can be organized in space throughout the action of glycosaminoglycans. Again, the synthesis of chemical mimetics offers an unexpected route to the development of novel glycotherapeutics. Finally, the area of enzymes/glycosaminoglycans complexes is briefly covered to realize the limited knowledge that we have for such an important class of biomacromolecular complexes.     

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.     

Anion-cation interactions in the pore of neuronal background chloride channels

Background Cl channels in neurons and skeletal muscle are significantly permeable for alkali cations when tested with asymmetrical concentrations of the same salt. Both anion and cation permeation were proposed to require binding of an alkali cation with the pore (Franciolini, F., and W. Nonner. 1987. Journal of General Physiology. 90:453-478). We tested this hypothesis by bilaterally substituting large alkali cations for Na and found no significant changes of unitary conductance at 300 mM symmetrical concentrations. In addition, all organic cations examined were permeant in a salt gradient test (1,000 mM internal@300 mM external), including triethanolamine, benzyltrimethylamine, and bis-tris-propane (BTP, which is divalent at the tested pH of 6.2). Inward currents were detected following substitution of internal NaCl by the Na salts of the divalent anions of phosphoric, fumaric, and malic acid. Zero-current potentials in gradients of the Na and BTP salts of varied anions (propionate, F, Br, nitrate) that have different permeabilities under bi-ionic conditions, were approximately constant, as if the permeation of either cation were coupled to the permeation of the anion. These results rule out our earlier hypothesis of anion permeation dependent on a bound alkali cation, but they are consistent with the idea that the tested anions and cations form mixed complexes while traversing the Cl channel.     

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.     

Structural systems biology: modelling protein interactions

Much of systems biology aims to predict the behaviour of biological systems on the basis of the set of molecules involved. Understanding the interactions between these molecules is therefore crucial to such efforts. Although many thousands of interactions are known, precise molecular details are available for only a tiny fraction of them. The difficulties that are involved in experimentally determining atomic structures for interacting proteins make predictive methods essential for progress. Structural details can ultimately turn abstract system representations into models that more accurately reflect biological reality.     

Structural features of the G-protein/GPCR interactions

Background

The details of the functional interaction between G proteins and the G protein coupled receptors (GPCRs) have long been subjected to extensive investigations with structural and functional assays and a large number of computational studies.

Scope of review

The nature and sites of interaction in the G-protein/GPCR complexes, and the specificities of these interactions selecting coupling partners among the large number of families of GPCRs and G protein forms, are still poorly defined.

Major conclusions

Many of the contact sites between the two proteins in specific complexes have been identified, but the three dimensional molecular architecture of a receptor-Gα interface is only known for one pair. Consequently, many fundamental questions regarding this macromolecular assembly and its mechanism remain unanswered.

General significance

In the context of current structural data we review the structural details of the interfaces and recognition sites in complexes of sub-family A GPCRs with cognate G-proteins, with special emphasis on the consequences of activation on GPCR structure, the prevalence of preassembled GPCR/G-protein complexes, the key structural determinants for selective coupling and the possible involvement of GPCR oligomerization in this process.     

Structural and functional analysis of the RANTES-glycosaminoglycans interactions

Chemokines mediate their biological activity through activation of G protein coupled receptors, but most chemokines, including RANTES, are also able to bind glycosaminoglycans (GAGs). Here, we have investigated, by site-directed mutagenesis and chemical acetylation, the role of RANTES basic residues in the interaction with GAGs using surface plasmon resonance kinetic analysis. Our results indicate that (i) RANTES exhibited selectivity in GAGs binding with highest affinity (K(d) = 32.1 nM) for heparin, (ii) RANTES uses the side chains of residues R44, K45, and R47 for heparin binding, and blocking these residues in combination abolished heparin binding. The biological relevance of RANTES-GAGs interaction was investigated in CHO-K1 cells expressing CCR5, CCR1, or CCR3 and the various GAGs that bind RANTES. Our results indicate that the heparin binding site, defined as the 40s loop, is only marginally involved in CCR5 binding and activation, but largely overlaps the CCR1 and CCR3 binding and activation domain in RANTES. In addition, enzymatic removal of cell surface GAGs by glycosidases did not affect CCR5 binding and Ca(2+) response. Furthermore, addition of soluble GAGs inhibited both CCR5 binding and functional response, with a rank of potency similar to that found in surface plasmon resonance experiments. Thus, cell surface GAGs is not a prerequisite for receptor binding or signaling, but soluble GAGs can inhibit the binding and the functional response of RANTES to CCR5 expressing cells. However, the marked selectivity of RANTES for different GAGs may serve, in vivo, to control the concentration of specific chemokines in inflammatory situations and locations.     

Structural analyses of death domains and their interactions

The death domain (DD), which is a versatle protein interaction module, is the prime mediator of the interactions necessary for apoptosis, innate immunity and the necrosis signaling pathway. Because DD mediated signaling events are associated with critical human diseases, studies in these areas are of great biological importance. Accordingly, many biochemical and structural studies of DD have been conducted in the past decade to investigate apoptotic and innate immune signaling. Evaluation of the molecular structure of DD and their interactions with partners have shown the underlying molecular basis for the assembly of DD mediated complexes and for the regulation of apoptosis and innate immunity. This review summarizes the structure and function of various DDs and DD:DD complexes involved in those signaling pathways.     

Binding interactions in a kinase active site modulate background ATP hydrolysis

Kinases play central roles in many cellular processes, transferring the terminal phosphate groups of nucleoside triphosphates (NTPs) onto substrates. In the absence of substrates, kinases can also hydrolyse NTPs producing NDPs and inorganic phosphate. Hydrolysis is usually much less efficient than the native phosphoryl transfer reaction. This may be related to the fact that NTP hydrolysis is metabolically unfavorable as it unproductively consumes the cell's energy stores. It has been suggested that substrate interactions could drive changes in NTP binding pocket, activating catalysis only when substrates are present. Structural data show substrate-induced conformational rearrangements, however there is a lack of corresponding functional information. To better understand this phenomenon, we developed a suite of isothermal titration calorimetry (ITC) kinetics methods to characterize ATP hydrolysis by the antibiotic resistance enzyme aminoglycoside-3′-phosphotransferase-IIIa (APH(3′)-IIIa). We measured K_m, k_cat, and product inhibition constants and single-turnover kinetics in the presence and absence of non-substrate aminoglycosides (nsAmgs) that are structurally similar to the native substrates. We found that the presence of an nsAmg increased the chemical step of cleaving the ATP γ-phosphate by at least 10- to 20-fold under single-turnover conditions, supporting the existence of interactions that link substrate binding to substantially enhanced catalytic rates. Our detailed kinetic data on the association and dissociation rates of nsAmgs and ADP shed light on the biophysical processes underlying the enzyme's Theorell-Chance reaction mechanism. Furthermore, they provide clues on how to design small-molecule effectors that could trigger efficient ATP hydrolysis and generate selective pressure against bacteria harboring the APH(3′)-IIIa.