Long-Lived, High-Strength States of ICAM-1 Bonds to β2 Integrin, I: Lifetimes of Bonds to Recombinant αLβ2 Under Force

Using single-molecule force spectroscopy to probe ICAM-1 interactions with recombinant α_Lβ₂ immobilized on microspheres and β₂ integrin on neutrophils, we quantified an impressive hierarchy of long-lived, high-strength states of the integrin bond, which start from basal levels with integrin activation in solutions of divalent cations and shift dramatically upward to hyperactivated states with cell signaling in leukocytes. Taking advantage of very rare events, we used repeated measurements of bond lifetimes under steady ramps of force to achieve a direct assay for the off-rates of ICAM-1 from β₂ integrin in each experiment. Of fundamental importance, the assay for off-rates does not depend on how the force is applied over time, and remains valid when the rates of dissociation change with different levels of force. In this first article, we present results from tests of a monovalent ICAM-1 probe against immobilized α_Lβ₂ in environments of divalent cations (Ca²⁺, Mg²⁺, and Mn²⁺) and demonstrate in detail the method for assay of off-rates. When extrapolated to zero force, the force-free values for the off-rates are found to be consistent with published solution-based assays of soluble ICAM-1 dissociation from immobilized LFA-1, i.e., ∼10⁻²/s in Mg²⁺ or Mn²⁺ and ∼1/s in Ca²⁺. At the same time, as expected for adhesive function, we find that the β₂ integrin bonds activated in Mn²⁺ or Mg²⁺ possess significant and persistent mechanical strength (e.g., >20 pN for >1 s) even when subjected to slow force ramps (<10 pN/s). As discussed in our companion article, using the same assay, we find that although the rates of dissociation for diICAM-1fc bonds to LFA-1 on neutrophils in Mn²⁺ are similar to those for mICAM-1 bonds to recombinant α_Lβ₂ on microspheres, they appear to represent a dimeric attachment to a pair of tightly clustered integrin heterodimers. The mechanical strengths and lifetimes of the dimeric interactions increase dramatically when the neutrophils are stimulated by the chemokine IL-8 or are bound with an allosterically activating (anti-CD18) monoclonal antibody, demonstrating the major impact of cell signaling on LFA-1.     

Two Barriers or not? Dynamic Force Spectroscopy on the Integrin α7β1 Invasin Complex

Dynamic force spectroscopy was used to test force-induced dissociation of the complex between the integrin α₇β₁ and the bacterial protein invasin. Both proteins were used in truncated forms comprising the respective binding sites. Using the biomembrane force-probe, the bond system was exposed to 14 different loading rates ranging from 18 pN/s to 5.3 nN/s. At each rate, bond rupture spectra were collected. Median forces ranged from 8 to 72 pN. These showed two linear regimes when plotted against the logarithm of the force-loading rate. However, a statistical analysis of the full rupture force spectra including the detection limits of the setup showed that all measured data are well described by dissociation over a single barrier.     

Thermodynamic Analyses of Nucleotide Binding to an Isolated Monomeric β Subunit and the α3β3γ Subcomplex of F1-ATPase

Rotation of the γ subunit of the F₁-ATPase plays an essential role in energy transduction by F₁-ATPase. Hydrolysis of an ATP molecule induces a 120° step rotation that consists of an 80° substep and 40° substep. ATP binding together with ADP release causes the first 80° step rotation. Thus, nucleotide binding is very important for rotation and energy transduction by F₁-ATPase. In this study, we introduced a βY341W mutation as an optical probe for nucleotide binding to catalytic sites, and a βE190Q mutation that suppresses the hydrolysis of nucleoside triphosphate (NTP). Using a mutant monomeric βY341W subunit and a mutant α₃β₃γ subcomplex containing the βY341W mutation with or without an additional βE190Q mutation, we examined the binding of various NTPs (i.e., ATP, GTP, and ITP) and nucleoside diphosphates (NDPs, i.e., ADP, GDP, and IDP). The affinity (1/K_d) of the nucleotides for the isolated β subunit and third catalytic site in the subcomplex was in the order ATP/ADP > GTP/GDP > ITP/IDP. We performed van’t Hoff analyses to obtain the thermodynamic parameters of nucleotide binding. For the isolated β subunit, NDPs and NTPs with the same base moiety exhibited similar ΔH⁰ and ΔG⁰ values at 25°C. The binding of nucleotides with different bases to the isolated β subunit resulted in different entropy changes. Interestingly, NDP binding to the α₃β(Y341W)₃γ subcomplex had similar K_d and ΔG⁰ values as binding to the isolated β(Y341W) subunit, but the contributions of the enthalpy term and the entropy term were very different. We discuss these results in terms of the change in the tightness of the subunit packing, which reduces the excluded volume between subunits and increases water entropy.     

Synthesis and characterization of tetrairon-tungsten cluster complex containing a μ4, η-carbonyl ligand

Reaction of [(PPh₂C₅H₄)Cp₃Fe₄(CO)₄] (1) with (CO)₄W(CH₃CN)₂ at ambient temperature affords [(CO)₄W(PPh₂C₅H₄)Cp₃Fe₄(CO)₄] (2) as the major product, together with a small amount of [(CO)₅W(PPh₂C₅H₄)Cp₃Fe₄(CO)₄] (3). Compound 3 can be obtained in good yield by treating (CO)₅W(CH₃CN) with equal molar of 1, and reaction of 3 with Me₃NO in acetonitrile solvent produces 2 exclusively. The crystal structures of 2 and 3 have been determined by an X-ray diffraction study. Compound 2 contains an interesting μ₄, η²-CO ligand, where two electrons donated by the carbon atom are involved to bridge a Fe₃ face and two electrons from oxygen are donated to the tungsten(0) atom.     

The amino acid sequences of two large glycopeptides derived from the carbohydrate-carrying region of α1-acid glycoprotein

Specific fragmentation with cyanogen bromide and subsequent reduction and carboxymethylation of α₁-acid glycoprotein, a normal human plasma globulin, permitted isolation of a large fragment which was shown to represent the amino-terminal half and to contain the total carbohydrate moiety of this protein. The amino acid sequences of two large glycopeptides derived from this fragment were established. One glycopeptide was composed of 22 amino acid residues and one carbohydrate unit, and the other consisted of 65 amino acid residues and carried four carbohydrate units.     

Suppression of type II collagen-induced arthritis by the intravenous administration of type II collagen or its constituent peptide α1, (II) CB10

Intravenous administration of Type II collagen to rats prior to immunization with Type II collagen suppresses hind paw inflammation, humoral response to Type II collagen, and the severity of the arthritic lesion. Suppression of inflammation and its severity as well as the humoral response can also be induced by the prior intravenous administration of α₁ (II) CB₁₀ a cyanogen bromide peptide derived from Type II collagen. Suppression of arthritis is disease specific; intravenous administration of either Type II collagen or α₁ (II) CB₁₀ does not have an effect on adjuvant-induced arthritis. These studies indicate that structural determinants of α₁ (II) CB₁₀ (M_r, 30,000), a peptide located near the carboxy terminus of the collagen molecule, can induce suppression and suggest that these determinants may be responsible for the suppression of arthritis when Type II collagen is administered intravenously.     

The effect of α1-acid glycoprotein (orosomucoid) on triglyceride metabolism in the nephrotic syndrome

We have identified α₁-acid glycoprotein as a new co-factor in the lipoprotein lipase reaction. We isolated an active form of the compound from nephrotic urine that is effective both $\text{in vitro}$ and $\text{in vivo}$. α₁-acid glycoprotein increased lipolysis 100% in the presence of C-II apolipoprotein in a lipoprotein lipase assay system. Rats with induced nephrotic syndrome showed a decrease in triglyceride clearance. $\text{T}\text{1}\text{2}$ was increased from 14 min to 43 min. The injection of α₁-acid glycoprotein restored the lipid clearance to normal. These findings suggest that elevated plasma triglycerides in human nephrotic patients is the direct result of excessive loss of α₁-acid glycoprotein from plasma into urine. We propose that replacement therapy may be possible.     

Functional properties of hemoglobin leiden (α2Aβ26 or7 Glu deleted)

Hemoglobin Leiden is an abnormal human hemoglobin in which a glutamic acid residue has been deleted from the β-chain at position 6 or 7. The α-amino groups of the β-chain N-termini in tetrameric hemoglobin A are thought to be directly involved in the binding of simple anions and organic phosphates (1). The deletion of the 4th or 5th residue of the A helix in hemoglobin Leiden shortens the N-terminus of the β-chain, and the results reported here show that the anion binding site has been affected. Hemoglobin Leiden shows a decreased response to inorganic phosphate, chloride, 2,3-diphosphoglycerate, and inositol hexaphosphate, both in equilibria and kinetics of ligand binding. Although hemoglobin Leiden shows an altered response to anions, neither the cooperativity of ligand binding nor the Bohr effect are significantly altered by the deletion. The decreased effect of cofactors seems to be due to a decrease in the strength of anion binding which may be attributed to the altered geometry of the anion binding site.     

Structural Insights into Conformational Stability of Wild-Type and Mutant β1-Adrenergic Receptor

Recent experiments to derive a thermally stable mutant of turkey beta-1-adrenergic receptor (β₁AR) have shown that a combination of six single point mutations resulted in a 20°C increase in thermal stability in mutant β₁AR. Here we have used the all-atom force-field energy function to calculate a stability score to detect stabilizing point mutations in G-protein coupled receptors. The calculated stability score shows good correlation with the measured thermal stability for 76 single point mutations and 22 multiple mutants in β₁AR. We have demonstrated that conformational sampling of the receptor for various mutants improve the prediction of thermal stability by 50%. Point mutations Y227A^5.58, V230A^5.61, and F338M^7.48 in the thermally stable mutant m23-β₁AR stabilizes key microdomains of the receptor in the inactive conformation. The Y227A^5.58 and V230A^5.61 mutations stabilize the ionic lock between R139^3.50 on transmembrane helix3 and E285^6.30 on transmembrane helix6. The mutation F338M^7.48 on TM7 alters the interaction of the conserved motif NPxxY(x)_5,6F with helix8 and hence modulates the interaction of TM2-TM7-helix8 microdomain. The D186-R317 salt bridge (in extracellular loops 2 and 3) is stabilized in the cyanopindolol-bound wild-type β₁AR, whereas the salt bridge between D184-R317 is preferred in the mutant m23. We propose that this could be the surrogate to a similar salt bridge found between the extracellular loop 2 and TM7 in β₂AR reported recently. We show that the binding energy difference between the inactive and active states is less in m23 compared to the wild-type, which explains the activation of m23 at higher norepinephrine concentration compared to the wild-type. Results from this work throw light into the mechanism behind stabilizing mutations. The computational scheme proposed in this work could be used to design stabilizing mutations for other G-protein coupled receptors.     

The Interaction of αB-Crystallin with Mature α-Synuclein Amyloid Fibrils Inhibits Their Elongation

αB-Crystallin is a small heat-shock protein (sHsp) that is colocalized with α-synuclein (αSyn) in Lewy bodies—the pathological hallmarks of Parkinson's disease—and is an inhibitor of αSyn amyloid fibril formation in an ATP-independent manner in vitro. We have investigated the mechanism underlying the inhibitory action of sHsps, and here we establish, by means of a variety of biophysical techniques including immunogold labeling and nuclear magnetic resonance spectroscopy, that αB-crystallin interacts with αSyn, binding along the length of mature amyloid fibrils. By measurement of seeded fibril elongation kinetics, both in solution and on a surface using a quartz crystal microbalance, this binding is shown to strongly inhibit further growth of the fibrils. The binding is also demonstrated to shift the monomer-fibril equilibrium in favor of dissociation. We believe that this mechanism, by which a sHsp interacts with mature amyloid fibrils, could represent an additional and potentially generic means by which at least some chaperones protect against amyloid aggregation and limit the onset of misfolding diseases.     

Synthesis, structural and electrochemical features of alicyclic and aromatic α,α′-N2- and-S2-dioximate macrobicyclic cobalt(II,III) and ruthenium(II) tris-complexes

The triribbed-functionalized cobalt(II,III) and ruthenium(II) clathrochelate derivatives of the vic-dioximes with two nitrogen or sulfur atoms in α-positions to π-conjugated diazomethine chelate fragments of a macrobicyclic framework were obtained in moderate yields under mild and high dilution conditions by nucleophilic substitution of six reactive chlorine atoms of the boron-capped macrobicyclic cobalt and ruthenium(II) precursors with N₂- and S₂-dinucleophiles (ethylenediamine and the corresponding α-dithiols in the presence of triethylamine, respectively). The complexes obtained were characterized using elemental analysis, MALDI-TOF mass spectrometry, IR, UV-Vis, ¹H and ¹³C{¹H} NMR and EPR spectroscopies, magnetochemistry and X-ray crystallography. The MN₆-coordination polyhedra of all the X-ray studied clathrochelates possess a slightly distorted trigonal prismatic geometry. The encapsulated cobalt(II) ions are shifted from the centers of the cavities formed by the macrobicyclic ligand due to the Jahn-Teller distortion, while the ruthenium and iron(II) ions in their clathrochelate analogs do occupy these centers. The main geometrical parameters of the macrobicyclic frameworks vary with Shannon radius of an encapsulated metal ion. In the case of the tris-ethylenediamine cobalt(III) clathrochelate, the field strength of the macrobicyclic amine ligand is essentially lower than those for their aromatic and aliphatic analogs because of the negative σ_para-effect of the ribbed alkylamine substituents. The magnetometry and EPR data confirmed the low-spin character of the cobalt(II) complexes synthesized. The electrochemically generated oxidized cobalt clathrochelates are stable in the CVA time scale, whereas their ruthenium- and iron-containing analogs as well as the reduced forms of all the cobalt, ruthenium and iron complexes obtained are unstable.     

Conformational heterogeneity of α-synuclein in membrane

α-Synuclein (αS) is a natively disordered protein in solution, thought to be involved in the fusion of neurotransmitter vesicles to cellular membranes during neurotransmission. Monomeric αS has been previously characterized in two distinct membrane-associated conformations: a broken-helix structure, and an extended helix. By employing atomistic molecular dynamics and a novel membrane representation with significantly enhanced lipid mobility (HMMM), we investigate the process of spontaneous membrane binding of αS and the conformational dynamics of monomeric αS in its membrane-bound form.     

Macromolecular Crowding Modulates Folding Mechanism of α/β Protein Apoflavodoxin

Protein dynamics in cells may be different from those in dilute solutions in vitro, because the environment in cells is highly concentrated with other macromolecules. This volume exclusion because of macromolecular crowding is predicted to affect both equilibrium and kinetic processes involving protein conformational changes. To quantify macromolecular crowding effects on protein folding mechanisms, we investigated the folding energy landscape of an α/β protein, apoflavodoxin, in the presence of inert macromolecular crowding agents, using in silico and in vitro approaches. By means of coarse-grained molecular simulations and topology-based potential interactions, we probed the effects of increased volume fractions of crowding agents (ϕ_c) as well as of crowding agent geometry (sphere or spherocylinder) at high ϕ_c. Parallel kinetic folding experiments with purified Desulfovibro desulfuricans apoflavodoxin in vitro were performed in the presence of Ficoll (sphere) and Dextran (spherocylinder) synthetic crowding agents. In conclusion, we identified the in silico crowding conditions that best enhance protein stability, and discovered that upon manipulation of the crowding conditions, folding routes experiencing topological frustrations can be either enhanced or relieved. Our test-tube experiments confirmed that apoflavodoxin''s time-resolved folding path is modulated by crowding agent geometry. Macromolecular crowding effects may be a tool for the manipulation of protein-folding and function in living cells.     

Novel naftopidil derivatives containing methyl phenylacetate and their blocking effects on α1D/1A-adrenoreceptor subtypes

α₁-Adrenoceptor (α₁-AR) antagonists are considered to be the most effective monotherapy agents for lower urinary tract symptoms associated with benign prostatic hyperplasia (LUTS/BPH). In this study, we synthesized compounds 2–17, which are novel piperazine derivatives that contain methyl phenylacetate. We then evaluated the vasodilatory activities of these compounds. Among them, we found that compounds 2, 7, 12, which contain 2-OCH₃, 2-CH₃ or 2, 5-CH₃, respectively, exhibited potent α₁-blocking activity similar to protype drug naftopidil (1). The antagonistic effects of 2, 7, and 12 on the (?)-noradrenaline-induced contractile response of isolated rat prostatic vas deferens (α_1A), spleen (α_1B) and thoracic aorta (α_1D) were further characterized to assess the sub receptor selectivity. Compared with naftopidil (1) and terazosin, compound 12 showed the most desirable α_1D/1A subtype selectivity, especially improved α_1A subtype selectivity, and the ratios pA₂ (α_1D)/pA₂ (α_1B) and pA₂ (α_1A)/pA₂ (α_1B) were 17.0- and 19.5-fold, respectively, indicating less cardiovascular side effects when used to treat LUTS/BPH. Finally, we investigated the chiral pharmacology of 12. We found, however, that the activity of enantiomers (R)-12 and (S)-12 are not significantly different from that of rac-12.     

Crystal structure of α/β-galactoside α2,3-sialyltransferase from a luminous marine bacterium, Photobacterium phosphoreum

α/β-Galactoside α2,3-sialyltransferase produced by Photobacterium phosphoreum JT-ISH-467 is a unique enzyme that catalyzes the transfer of N-acetylneuraminic acid residue from cytidine monophosphate N-acetylneuraminic acid to acceptor carbohydrate groups. The enzyme recognizes both mono- and di-saccharides as acceptor substrates, and can transfer Neu5Ac to both α-galactoside and β-galactoside, efficiently. To elucidate the structural basis for the broad acceptor substrate specificity, we determined the crystal structure of the α2,3-sialyltransferase in complex with CMP. The overall structure belongs to the glycosyltransferase-B structural group. We could model a reasonable active conformation structure based on the crystal structure. The predicted structure suggested that the broad substrate specificity could be attributed to the wider entrance of the acceptor substrate binding site.     

Influence of prostaglandins (PG) E2 and F2α on the inflammatory process

PGF_2^α, but not PGE₂, induces a slight pedal edema when given alone. Both compounds were equipotent in the carrageenin-induced rat paw edema. Locally administered, PGE₂ and PGF_2^α did not exacerbate, but rather inhibited inflammations induced by various agents such as 1% carrageenin or 1% egg white. The administration of PGE₂ directly into cotton pellets or into the rat's hind paw in combination with M. butyricum significantly inhibited, respectively, granuloma formation and the polyarthritis. Subcutaneously, both prostaglandins inhibited the adjuvant induced polyarthritis. Neither PGE₂ nor PGF_2^α inhibited the anti-edema properties of non-steroidal or steroidal anti-inflammatory standards. A greater anti-edema activity was observed with the combination treatment than with the anti-inflammatory standards alone. We were unable to decrease the anti-inflammatory activity of the steroidal and non-steroidal standards or increase the inflammatory potential of the phlogistic agents.     

Long-Lived, High-Strength States of ICAM-1 Bonds to β2 Integrin, II: Lifetimes of LFA-1 Bonds Under Force in Leukocyte Signaling

Using single-molecule force spectroscopy to probe ICAM-1 interactions with recombinant α_Lβ₂ immobilized on microspheres and β₂ integrin on neutrophils, we quantified an impressive hierarchy of long-lived, high-strength states of the integrin bond, which start from basal levels with activation in solutions of divalent cations and shift dramatically upward to hyperactivated states with cell signaling. Taking advantage of very rare events, we used repeated measurements of bond lifetimes under steady ramps of force to achieve a direct assay for the off-rates of ICAM-1 from β₂ integrin throughout the course of each experiment. In our companion article I, we demonstrate the assay using results from tests of a monovalent ICAM-1 probe against recombinant α_Lβ₂ on microspheres in millimolar solutions of divalent cations (Ca²⁺, Mg²⁺, Mn²⁺). In this article, we examine the impact of inside-out and outside-in signaling in neutrophils on the lifetimes and mechanical strengths of ICAM-1 bonds to β₂ integrin on the cell surface. Even though ICAM-1 bonds to recombinant α_Lβ₂ on microspheres in Mg²⁺ or Mn²⁺ can live for long periods of time under slow pulling, here we show that stimulation of neutrophils in Mg²⁺ plus the chemokine IL-8 (i.e., inside-out signaling) induces several-hundred-fold longer lifetimes for ICAM-1 attachments to LFA-1, creating strong bonds at very slow pulling speeds where none are perceived in Mg²⁺ or Mn²⁺ alone. Similar changes are observed with outside-in signaling, i.e., long lifetimes and increased bond strength also occur when neutrophils are bound with the activating (anti-CD18) monoclonal 240Q. Limiting our investigation to rare events using very dilute ICAM-1 probes, we show that although the prolonged lifetimes of cell surface attachments for both inside-out and outside-in signaling exhibit single-bond-like statistics for dissociation under force, they are consistent with a tightly coupled dimeric ICAM-1 interaction with a pair of LFA-1 heterodimers.     

Concerted Interconversion between Ionic Lock Substates of the β2 Adrenergic Receptor Revealed by Microsecond Timescale Molecular Dynamics

The recently solved crystallographic structures for the A_2A adenosine receptor and the β₁ and β₂ adrenergic receptors have shown important differences between members of the class-A G-protein-coupled receptors and their archetypal model, rhodopsin, such as the apparent breaking of the ionic lock that stabilizes the inactive structure. Here, we characterize a 1.02 μs all-atom simulation of an apo-β₂ adrenergic receptor that is missing the third intracellular loop to better understand the inactive structure. Although we find that the structure is remarkably rigid, there is a rapid influx of water into the core of the protein, as well as a slight expansion of the molecule relative to the crystal structure. In contrast to the x-ray crystal structures, the ionic lock rapidly reforms, although we see an activation-precursor-like event wherein the ionic lock opens for ∼200 ns, accompanied by movements in the transmembrane helices associated with activation. When the lock reforms, we see the structure return to its inactive conformation. We also find that the ionic lock exists in three states: closed (or locked), semi-open with a bridging water molecule, and open. The interconversion of these states involves the concerted motion of the entire protein. We characterize these states and the concerted motion underlying their interconversion. These findings may help elucidate the connection between key local events and the associated global structural changes during activation.     

A 3D Structure Model of Integrin α4β1 Complex: I. Construction of a Homology Model of β1 and Ligand Binding Analysis

It is well established that integrin α4β1 binds to the vascular cell adhesion molecule (VCAM) and fibronectin and plays an important role in signal transduction. Blocking the binding of VCAM to α4β1 is thought to be a way of controlling a number of disease processes. To better understand how various inhibitors might block the interaction of VCAM and fibronectin with α4β1, we began constructing a structure model for the integrin α4β1 complex. As the first step, we have built a homology model of the β1 subunit based on the I domain of the integrin CD11B subunit. The model, including a bound Mg²⁺ ion, was optimized through a specially designed relaxation scheme involving restrained minimization and dynamics steps. The native ligand VCAM and two highly active small molecules (TBC772 and TBC3486) shown to inhibit binding of CS-1 and VCAM to α4β1 were docked into the active site of the refined model. Results from the binding analysis fit well with a pharmacophore model that was independently derived from active analog studies. A critical examination of residues in the binding site and analysis of docked ligands that are both potent and selective led to the proposal of a mechanism for β1/β7 ligand binding selectivity.     

The Shapes of Z-α1-Antitrypsin Polymers in Solution Support the C-Terminal Domain-Swap Mechanism of Polymerization

Emphysema and liver cirrhosis can be caused by the Z mutation (Glu342Lys) in the serine protease inhibitor α1-antitrypsin (α1AT), which is found in more than 4% of the Northern European population. Homozygotes experience deficiency in the lung concomitantly with a massive accumulation of polymers within hepatocytes, causing their destruction. Recently, it was proposed that Z-α1AT polymerizes by a C-terminal domain swap. In this study, small-angle x-ray scattering (SAXS) was used to characterize Z-α1AT polymers in solution. The data show that the Z-α1AT trimer, tetramer, and pentamer all form ring-like structures in strong support of a common domain-swap polymerization mechanism that can lead to self-terminating polymers.