Interaction of lipid-free apolipoprotein A-I with cholesterol revealed by molecular modeling

We report the modeling of the interaction of differently self-associated lipid-free apoA-I with cholesterol monomer and tail-to-tail (TT) or face-to-face (FF) cholesterol dimer. Cholesterol dimerization is exploited to reconcile the existing experimental data on cholesterol binding to apoA-I with extremely low critical micelle concentration of cholesterol. Two crystal structures of 1–43 N-truncated apolipoprotein Δ(1-43)A-I tetramer (PDB ID: 1AV1, structure B), 185–243 C-truncated apolipoprotein Δ(185-243)A-I dimer (PDB ID: 3R2P, structure M) were analyzed. Cholesterol monomers bind to multiple binding sites in apoA-I monomer, dimer and tetramer with low, moderate and high energy (?10 to ?28 kJ/mol with Schrödinger package), still insufficient to overcome the thermodynamic restriction by cholesterol micellization (?52.8 kJ/mol). The binding sites partially coincide with the putative cholesterol-binding motifs. However, apoA-I monomer and dimer existing in structure B, that contain nonoverlapping and non-interacting pairs of binding sites with high affinity for TT and FF cholesterol dimers, can bind in common 14 cholesterol molecules that correspond to existing values. ApoA-I monomer and dimer in structure M can bind in common 6 cholesterol molecules. The values of respective total energy of cholesterol binding up to 64.5 and 67.0 kJ/mol for both B and M structures exceed the free energy of cholesterol micellization. We hypothesize that cholesterol dimers may simultaneously interact with extracellular monomer and dimer of lipid-free apoA-I, that accumulate at acid pH in atheroma. The thermodynamically allowed apolipoprotein-cholesterol interaction outside the macrophage may represent a new mechanism of cholesterol transport by apoA-I from atheroma, in addition to ABCA1-mediated cholesterol efflux.     

Analysis of intermolecular interaction energy inputs in benzene-imidazole and imidazole-imidazole systems in parallel displaced and T-configuration

Intermolecular interactions in several dimer aromatic systems were analyzed to determine how various energy contributions (electrostatic, exchange, repulsion, and polarization) change depending on the value of monomers separation. Different contributions to the intermolecular energy interactions between imidazole-imidazole and benzene-imidazole dimers are studied using the aug-cc-pVDZ basis set in the framework of ab initio Hartree-Fock and second-order Møller-Plesset perturbation theory methods. Special attention is paid to the exchange and dispersion energy binding contributions.     

Characteristics of self-quenching of the fluorescence of lipid-conjugated rhodamine in membranes

Self- or concentration quenching of octadecylrhodamine B (C18-Rh) fluorescence increases linearly in egg phosphatidylcholine (PC) vesicles but exponentially in vesicles composed of egg PC:cholesterol, 1:1, as the probe concentration is raised to 10 mol%. Cholesterol-dependent enhancement of self-quenching also occurs when N-(lissamine-rhodamine-B-sulfonyl)dioleoylphosphatidylethanolamine is substituted for C18-Rh and resembles that in dipalmitoylphosphatidylcholine vesicles below, as opposed to above, the phase transition. These effects are not due to changes in dimer:monomer absorbance. Stern-Volmer plots indicate a dependence of quenching on nonfluorescent dimers both in the presence and absence of cholesterol. Decreases in fluorescence lifetimes with increasing probe concentration parallel decreases in residual fluorescence of C18-Rh with increasing probe concentration in PC and PC + cholesterol membranes, respectively. Decreases in the steady-state polarization of C18-Rh fluorescence as its concentration is raised to 10 mol% indicate energy transfer with emission between probe molecules in PC and to a lesser extent in PC + cholesterol membranes. The calculated R0 for 50% efficiency of energy transfer from excited state probe to monomer was 55-58 A and to dimer was 27 A. Since lateral diffusion of C18-Rh is probably too slow to permit collisional quenching during the lifetime of the probe, even if C18-Rh were concentrated in a separate phase, C18-Rh self-quenching appears to be due mainly to energy transfer without emission to nonfluorescent dimers.     

Mechanism and evolution of protein dimerization.

We have investigated the mechanism and the evolutionary pathway of protein dimerization through analysis of experimental structures of dimers. We propose that the evolution of dimers may have multiple pathways, including (1) formation of a functional dimer directly without going through an ancestor monomer, (2) formation of a stable monomer as an intermediate followed by mutations of its surface residues, and (3), a domain swapping mechanism, replacing one segment in a monomer by an equivalent segment from an identical chain in the dimer. Some of the dimers which are governed by a domain swapping mechanism may have evolved at an earlier stage of evolution via the second mechanism. Here, we follow the theory that the kinetic pathway reflects the evolutionary pathway. We analyze the structure-kinetics-evolution relationship for a collection of symmetric homodimers classified into three groups: (1) 14 dimers, which were referred to as domain swapping dimers in the literature; (2) nine 2-state dimers, which have no measurable intermediates in equilibrium denaturation; and (3), eight 3-state dimers, which have stable intermediates in equilibrium denaturation. The analysis consists of the following stages: (i) The dimer is divided into two structural units, which have twofold symmetry. Each unit contains a contiguous segment from one polypeptide chain of the dimer, and its complementary contiguous segment from the other chain. (ii) The division is repeated progressively, with different combinations of the two segments in each unit. (iii) The coefficient of compactness is calculated for the units in all divisions. The coefficients obtained for different cuttings of a dimer form a compactness profile. The profile probes the structural organization of the two chains in a dimer and the stability of the monomeric state. We describe the features of the compactness profiles in each of the three dimer groups. The profiles identify the swapping segments in domain swapping dimers, and can usually predict whether a dimer has domain swapping. The kinetics of dimerization indicates that some dimers which have been assigned in the literature as domain swapping cases, dimerize through the 2-state kinetics, rather than through swapping segments of performed monomers. The compactness profiles indicate a wide spectrum in the kinetics of dimerization: dimers having no intermediate stable monomers; dimers having an intermediate with a stable monomer structure; and dimers having an intermediate with a stable structure in part of the monomer. These correspond to the multiple evolutionary pathways for dimer formation. The evolutionary mechanisms proposed here for dimers are applicable to other oligomers as well.     

Membrane-Mediated Protein-Protein Interactions and Connection to Elastic Models: A Coarse-Grained Simulation Analysis of Gramicidin A Association

To further foster the connection between particle based and continuum mechanics models for membrane mediated biological processes, we carried out coarse-grained (CG) simulations of gramicidin A (gA) dimer association and analyzed the results based on the combination of potential of mean force (PMF) and stress field calculations. Similar to previous studies, we observe that the association of gA dimers depends critically on the degree of hydrophobic mismatch, with the estimated binding free energy of >10 kcal/mol in a distearoylphosphatidylcholine bilayer. Qualitative trends in the computed PMF can be understood based on the stress field distributions near a single gA dimer and between a pair of gA dimers. For example, the small PMF barrier, which is ∼1 kcal/mol independent of lipid type, can be captured nearly quantitatively by considering membrane deformation energy associated with the region confined by two gA dimers. However, the PMF well depth is reproduced poorly by a simple continuum model that only considers membrane deformation energy beyond the annular lipids. Analysis of lipid orientation, configuration entropy, and stress distribution suggests that the annular lipids make a significant contribution to the association of two gA dimers. These results highlight the importance of explicitly considering contributions from annular lipids when constructing approximate models to study processes that involve a significant reorganization of lipids near proteins, such as protein-protein association and protein insertion into biomembranes. Finally, large-scale CG simulations indicate that multiple gA dimers also form clusters, although the preferred topology depends on the protein concentration. Even at high protein concentrations, every gA dimer requires contact to lipid hydrocarbons to some degree, and at most three to four proteins are in contact with each gA dimer; this observation highlights another aspect of the importance of interactions between proteins and annular lipids.     

The conduction of protons in different stereoisomers of dioxolane-linked gramicidin A channels

Two different stereoisomers of the dioxolane-linked gramicidin A (gA) channels were individually synthesized (the SS and RR dimers;. Science. 244:813-817). The structural differences between these dimers arise from different chiralities within the dioxolane linker. The SS dimer mimics the helicity and the inter- and intramolecular hydrogen bonding of the monomer-monomer association of gA's. In contrast, there is a significant disruption of the helicity and hydrogen bonding pattern of the ion channel in the RR dimer. Single ion channels formed by the SS and RR dimers in planar lipid bilayers have different proton transport properties. The lipid environment in which the different dimers are reconstituted also has significant effects on single-channel proton conductance (g(H)). g(H) in the SS dimer is about 2-4 times as large as in the RR. In phospholipid bilayers with 1 M [H(+)](bulk), the current-voltage (I-V) relationship of the SS dimer is sublinear. Under identical experimental conditions, the I-V plot of the RR dimer is supralinear (S-shaped). In glycerylmonooleate bilayers with 1 M [H(+)](bulk), both the SS and RR dimers have a supralinear I-V plot. Consistent with results previously published (. Biophys. J. 73:2489-2502), the SS dimer is stable in lipid bilayers and has fast closures. In contrast, the open state of the RR channel has closed states that can last a few seconds, and the channel eventually inactivates into a closed state in either phospholipid or glycerylmonooleate bilayers. It is concluded that the water dynamics inside the pore as related to proton wire transfer is significantly different in the RR and SS dimers. Different physical mechanisms that could account for this hypothesis are discussed. The gating of the synthetic gA dimers seems to depend on the conformation of the dioxolane link between gA's. The experimental results provide an important framework for a detailed investigation at the atomic level of proton conduction in different and relatively simple ion channel structures.     

Characterization of mitogen-activated protein kinase (MAPK) dimers

Phosphorylated ERK2 has an increased capacity to form homodimers relative to unphosphorylated ERK2. We have characterized the nature of the ERK2 dimer and have mutated residues in the crystal dimer interface to examine the impact of dimerization on ERK2 activity. Analysis of the mutants by gel filtration indicates that at least five residues must be mutated simultaneously to produce an ERK2 mutant that is predominantly monomeric. Mutants, whether monomers or dimers, have specific protein kinase activities under fixed assay conditions that are roughly equivalent to wild-type ERK2. The ratio of dimers to monomers is increased as the salt concentration increases, consistent with a strong hydrophobic contribution to the energy of dimer formation. ERK2 dimerization also requires divalent cations. Sedimentation analysis indicates that the related c-Jun N-terminal kinase SAPKalphaI/JNK2 also forms dimers, but dimerization displays no dependence on phosphorylation; the unphosphorylated and phosphorylated forms of the kinase behave similarly, with low micromolar dimer dissociation constants.     

Triggering of T cell activation via CD4 dimers

The onset of activation in Th cells is triggered by localized co-engagement of TCRs and the coreceptor CD4. A CD4 crystal suggested that CD4 may form dimers in some circumstances. In this study, we use live-cell fluorescence resonance energy transfer imaging to demonstrate that CD4 dimers are present at a basal level on the cell surface and accumulate at the synapse. Mechanistically, we reveal two conditions under which dimers are highly relevant. First, CD4 dimers are more proficient in mediating prolonged cell contacts with APCs in the presence or absence of Ag. This is consistent with a model whereby the dimer functions to increase T-APC avidity. Second, we show that dimer mutations result in an increased level of an inactive lckTyr(505) bound to the CD4 molecule relative to dimer-competent CD4. We also find a consistent defect in signaling onset in these cells. This supports a role for CD4 dimerization in maintaining active signaling machinery. We suggest that modulation of the dimer/monomer ratio may permit tuning of activation thresholds during initial engagement.     

Ultra-violet light induced changes in DNA dynamics may enhance TT-dimer recognition

Short-wave ultra-violet light promotes the formation of DNA dimers between adjacent thymine bases, and if unrepaired these dimers may induce skin cancer. Living cells have a very robust repair system capable of repairing hundreds of lesions every day. Although many of the details of the dimer repair mechanism are known, it is still a mystery how the dimers are recognized. Because the dimers are hidden from repair proteins diffusing in the cell nucleus, it has been surmised that dimer recognition is indirect. In this paper, a new recognition signal is suggested by a theory of the dimer-induced large amplitude, prolonged oscillations in the motion of the two strands in double-stranded DNA molecules. These large amplitude oscillations of the two DNA strands, localized around the dimer will unveil the dimer allowing the repair proteins to bind to the dimer site. The temperature dependence of the recognition rate is correlated with the inter-strand fluctuations and must decrease with decreasing temperature according to the findings in this paper. Moreover the probability for finding a large opening is localized to the dimer neighbourhood and these large openings may play an important role in dimer-repair protein biochemistry.     

An extended hydrophobic core induces EF-hand swapping

The structure of calbindin D(9k) with two substitutions was determined by X-ray crystallography at 1.8-A resolution. Unlike wild-type calbindin D(9k), which is a monomeric protein with two EF-hands, the structure of the mutated calbindin D(9k) reveals an intertwined dimer. In the dimer, two EF-hands of the monomers have exchanged places, and thus a 3D domain-swapped dimer has been formed. EF-hand I of molecule A is packed toward EF-hand II of molecule B and vice versa. The formation of a hydrophobic cluster, in a region linking the EF-hands, promotes the conversion of monomers to 3D domain-swapped dimers. We propose a mechanism by which domain swapping takes place via the apo form of calbindin D(9k). Once formed, the calbindin D(9k) dimers are remarkably stable, as with even larger misfolded aggregates like amyloids. Thus calbindin D(9k) dimers cannot be converted to monomers by dilution. However, heating can be used for conversion, indicating high energy barriers separating monomers from dimers.     

Biophysical Characterization of the Dimer and Tetramer Interface Interactions of the Human Cytosolic Malic Enzyme

The cytosolic NADP⁺-dependent malic enzyme (c-NADP-ME) has a dimer-dimer quaternary structure in which the dimer interface associates more tightly than the tetramer interface. In this study, the urea-induced unfolding process of the c-NADP-ME interface mutants was monitored using fluorescence and circular dichroism spectroscopy, analytical ultracentrifugation and enzyme activities. Here, we demonstrate the differential protein stability between dimer and tetramer interface interactions of human c-NADP-ME. Our data clearly demonstrate that the protein stability of c-NADP-ME is affected predominantly by disruptions at the dimer interface rather than at the tetramer interface. First, during thermal stability experiments, the melting temperatures of the wild-type and tetramer interface mutants are 8–10°C higher than those of the dimer interface mutants. Second, during urea denaturation experiments, the thermodynamic parameters of the wild-type and tetramer interface mutants are almost identical. However, for the dimer interface mutants, the first transition of the urea unfolding curves shift towards a lower urea concentration, and the unfolding intermediate exist at a lower urea concentration. Third, for tetrameric WT c-NADP-ME, the enzyme is first dissociated from a tetramer to dimers before the 2 M urea treatment, and the dimers then dissociated into monomers before the 2.5 M urea treatment. With a dimeric tetramer interface mutant (H142A/D568A), the dimer completely dissociated into monomers after a 2.5 M urea treatment, while for a dimeric dimer interface mutant (H51A/D90A), the dimer completely dissociated into monomers after a 1.5 M urea treatment, indicating that the interactions of c-NADP-ME at the dimer interface are truly stronger than at the tetramer interface. Thus, this study provides a reasonable explanation for why malic enzymes need to assemble as a dimer of dimers.     

Binary and ternary aggregation within tethered protein constructs

The free energy per monomer of a protein aggregate varies with the number of participating monomers n. The change of this free energy with aggregate size, DeltaDeltaG(n), is difficult to determine by sedimentation or concentration studies. We introduce a kinetic approach to quantitate the free energy of aggregates in the presence of tethers. By linking the protein U1A into dimers and trimers, a high effective concentration of the monomers is achieved, together with exact size control of the aggregates. We found that the free energy of the aggregate relative to the native monomer reached a maximum for n = 2, and decreased by DeltaDeltaG(2) = -3.1 kT between dimer and trimer.     

Molecular-dynamics simulations of pyronine 6G and rhodamine 6G dimers in aqueous solution

We have carried out molecular-dynamics (MD) simulations on dimers of the positively charged laser dyes pyronine 6G (P6G) and rhodamine 6G (R6G) in aqueous solution, generating trajectories of 2.5 ns for various computational protocols. We discuss how the choice of atomic partial charges and the length of the trajectories affect the predicted structures of the dimers and compare our results to those of earlier MD-simulations, which were restricted to only 0.7 ns. Our results confirm that monomers of P6G easily undergo relative rotations within the dimer, but we found new conformations of the R6G dimer at longer simulation times. In addition, we analyzed in detail the energy change during the formation of dimers. With suitable corrections, the electrostatic energy from an Ewald treatment agrees with the results from an approach relying on a residue-based cutoff. For P6G, we show that the strong solvent-mediated electrostatic attraction between the monomers is counteracted by an almost equally large solvent-induced entropy contribution to yield a small driving force to dimer formation, in very good agreement with the free-energy change from a thermodynamic-integration procedure. Thus, earlier rationalizations of the dimer formation, based only on energy arguments, yield a qualitatively wrong picture.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Perfect hemihedral twinning in crystals of the γ-subunit of translation initiation factor 2 from <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis>: Cause and effect

The crystal structure of the γ-subunit of translation initiation factor 2 from the archaeon Sulfolobus solfataricus (SsoIF2γ) has been solved based on perfectly hemihedral twinned data. The protein was cocrystallized with the 10-fold molar excess of GTP analog (GDPCP) over protein. However, no nucleotide was found in the structure, and the model demonstrated the apo form of the protein. Two slightly different molecules in the asymmetric unit of the crystal are related by the non-crystallographic 2-fold axis and form a tightly associated dimer. This dimer is stabilized by an intermolecular hydrophobic core and hydrogen bonds. Lack of GDPCP in the nucleotide-binding pocket of the γ-subunit and significant excess of dimers over monomers in the crystallization solution suggest that these dimers are the building blocks of the crystal. Contrary to SsoIF2γ monomers, these dimers are able to crystallize in two oppositely oriented slightly different crystal domains, thus forming a twinned crystal. Comparison of crystallization conditions for the twinned and untwinned crystals of apo SsoIF2γ showed that stabilization of the dimers in the solution may be caused by higher sodium salt concentration. Since amino acid residues involved in intermolecular contacts in the dimer are responsible for binding of the γand α-subunits within SsoIF2, increase in sodium salt concentration may prevent functioning of SsoIF2 in the cell.     

Reaction pathway for the quaternary structure change in hemoglobin

We perform a computer simulation of the quaternary structure change during the allosteric transition of hemoglobin. The simulation is based on a docking procedure by which αβ dimers of human hemoglobin are associated into tetramers after being rotated in various orientations. The stability of tetramers thus reconstituted is estimated from the values of a simplified energy function describing nonbonded interactions and from the area of the surface buried in dimer–dimer contacts (their interface area), which we take to represent stabilizing interactions and solvent contribution. A systematic analysis of tetramers reconstituted with twofold symmetry reveals that when the dimers have the R tertiary structure, only tetramers having R-like quaternary structures are stable. When the dimers have the T tertiary structure, they may associate into T-like tetramers or a variety of quaternary structures ranging from T to near R, thus tracing a plausible reaction pathway for the allosteric transition. We subject intermediates of this pathway to energy refinement with rigid αβ dimers. The refinement demonstrates that symmetrical structures are more stable than non symmetrical ones. A detailed analysis of dimer–dimer contacts in intermediates shows how close packing is maintained over large interfaces throughout the quaternary structure change, especially in the “switch region” of contact between the C helix of α-chains and the FG corner of β-chains.     

The cyclobutane dimers of 5-methylcytosine and their deamination products.

The photochemical reactions of 5-methylcytosine (m(5)C), a minor component of mammalian DNA, have been studied at a concentration of 2 mM in frozen 10 mM aqueous NaCl solution at dry ice temperature (194.5 K). For these studies, low-pressure lamps emitting mainly UVB radiation were used. We have isolated and characterized three cyclobutane dimers, namely the cis-anti(c,a) the cis-syn(c,s) and the trans-syn(t,s) forms. While the c,a and the t,s cyclobutane dimers are relatively stable towards deamination upon standing in solution at 277 K, the c,s isomer is gradually converted into the corresponding c,s m(5)C-thymine (Thy) mixed dimer; this latter reaction occurs considerably faster at 310 K. The t,s cyclobutane dimer is converted into the corresponding m(5)C-Thy mixed dimer upon incubation at 373 K, while the c,a dimer is converted into a mixture of m(5)C and c,a mixed dimer when incubated at 310 K. Irradiation of equimolar mixtures of Thy (1 mM) and m(5)C (1 mM) under similar conditions yields each of the three m(5)C cyclobutane dimers, as well as significant amounts of c,a, c,s and t,s m(5)C-Thy mixed cyclobutane dimers. These m(5)C-Thy dimers undergo decompositions similar in nature to the processes undergone by m(5)C cyclobutane dimers. Pseudo-first order rate constants for deamination of the c,s m(5)C homodimer and c,s m(5)C-Thy heterodimer at various temperatures and at pH 7.7 have been measured and the enthalpies and entropies of activation have been evaluated for the deamination processes for these two compounds. The two dimers have half-lives of about 14 and 22 h, respectively, at 310 K; however, at 273 K, the corresponding half-lives can be evaluated as being around 30 and 36 days, respectively.     

Weak protein-protein interactions are sufficient to drive assembly of hepatitis B virus capsids

Hepatitis B virus (HBV) is an enveloped DNA virus with a spherical capsid (or core). The capsid is constructed from 120 copies of the homodimeric capsid protein arranged with T = 4 icosahedral symmetry. We examined in vitro assembly of purified E. coli expressed HBV capsid protein. After equilibration, concentrations of capsid and dimer were evaluated by size exclusion chromatography. The extent of assembly increased as temperature and ionic strength increased. The concentration dependence of capsid assembly conformed to the equilibrium expression: K(capsid) = [capsid]/[dimer](120). Given the known geometry for HBV capsids and dimers, the per capsid assembly energy was partitioned into energy per subunit-subunit contact. We were able to make three major conclusions. (i) Weak interactions (from -2.9 kcal/mol at 21 degrees C in low salt to -4.4 kcal/mol at 37 degrees C in high salt) at each intersubunit contact result in a globally stable capsid; weak intersubunit interactions may be the basis for the phenomenon of capsid breathing. (ii) HBV assembly is characterized by positive enthalpy and entropy. The reaction is entropy-driven, consistent with the largely hydrophobic contacts found in the crystal structure. (iii) Increasing NaCl concentration increases the magnitude of free energy, enthalpy, and entropy, as if ionic strength were increasing the amount of hydrophobic surface buried by assembly. This last point leads us to suggest that salt acts by inducing a conformational change in the dimer from an assembly-inactive form to an assembly-active form. This model of conformational change linked to assembly is consistent with immunological differences between dimer and capsid.     

Influence of hydrogen bonds on edge-to-face interactions between pyridine molecules

Edge-to-face interactions between two pyridine molecules and the influence of simultaneous hydrogen bonding of one or both of the pyridines to water on those interactions were studied by analyzing data from ab initio calculations. The results show that the edge-to-face interactions of pyridine dimers that are hydrogen bonded to water are generally stronger than those of non-H-bonded pyridine dimers, especially when the donor pyridine forms a hydrogen bond. The binding energy of the most stable edge-to-face interacting H-bonded pyridine dimer is ?5.05 kcal/mol, while that for the most stable edge-to-face interacting non-H-bonded pyridine dimer is ?3.64 kcal/mol. The interaction energy data obtained in this study cannot be explained solely by the differences in electrostatic potential between pyridine and the pyridine–water dimer. However, the calculated cooperative effect can be predicted using electrostatic potential maps.     

N-cadherin-mediated cell motility requires cis dimers

Cadherins are expressed on the cell surface as a dimer in the membrane of one cell (cis dimer) that interacts with a cis dimer on an adjacent cell to form an adhesive trans dimer. It is well established that both cis and trans dimers must form for the cadherin to be an effective adhesion protein. In addition to their adhesive activity cadherins also play an important role in modulating cell behavior by regulating cell motility and signal transduction. Whether or not cis or trans dimers are necessary for the nonadhesive functions of cadherins has not been addressed. Here we show that N-cadherin cis dimers are necessary to induce cell motility in epithelial cells and that N-cadherin's ability to modulate the steady state levels of activated small GTPases requires both cis and trans dimers.