Energy Flux Through the Magnetopause During Flux Transfer Events in Hybrid-Vlasov 2D Simulations.

Solar wind-magnetosphere coupling drives magnetospheric dynamic phenomena by enabling energy exchange between magnetospheric and solar wind plasmas. In this study, we examine two-dimensional noon-midnight meridional plane simulation runs of the global hybrid-Vlasov code Vlasiator with southward interplanetary magnetic field driving. We compute the energy flux, which consists of the Poynting flux and hydrodynamic energy flux components, through the Earth's magnetopause during flux transfer events (FTEs). The results demonstrate the spatiotemporal variations of the energy flux along the magnetopause during an FTE, associating the FTE leading (trailing) edge with an energy injection into (escape from) the magnetosphere on the dayside. Furthermore, FTEs traveling along the magnetopause transport energy to the nightside magnetosphere. We identify the tail lobes as a primary entry region for solar wind energy into the magnetosphere, consistent with results from global magnetohydrodynamic simulations and observations.

Cystathionine ß-synthase (CBS) domains confer multiple forms of Mg2+-dependent cooperativity to family II pyrophosphatases.

Regulated family II pyrophosphatases (CBS-PPases) contain a nucleotide-binding insert comprising a pair of cystathionine ß-synthase (CBS) domains, termed a Bateman module. By binding with high affinity to the CBS domains, AMP and ADP usually inhibit the enzyme, whereas ATP activates it. Here, we demonstrate that AMP, ADP, and ATP bind in a positively cooperative manner to CBS-PPases from four bacteria: Desulfitobacterium hafniense, Clostridium novyi, Clostridium perfringens, and Eggerthella lenta. Enzyme interaction with substrate as characterized by the Michaelis constant (Km) also exhibited positive catalytic cooperativity that decreased in magnitude upon nucleotide binding. The degree of both types of cooperativity increased with increasing concentration of the cofactor Mg(2+) except for the C. novyi PPase where Mg(2+) produced the opposite effect on kinetic cooperativity. Further exceptions from these general rules were ADP binding to C. novyi PPase and AMP binding to E. lenta PPase, neither of which had any effect on activity. A genetically engineered deletion variant of D. hafniense PPase lacking the regulatory insert was fully active but differed from the wild-type enzyme in that it was insensitive to nucleotides and bound substrate non-cooperatively and with a smaller Km value. These results indicate that the regulatory insert acts as an internal inhibitor and confers dual positive cooperativity to CBS domain-containing PPases, making them highly sensitive regulators of the PPi level in response to the changes in cell energy status that control adenine nucleotide distribution. These regulatory features may be common among other CBS domain-containing proteins.

Leukocyte integrins αLß2, αMß2 and αXß2 as collagen receptors--receptor activation and recognition of GFOGER motif.

Integrins αLß2, αMß2 and αXß2 are expressed on leukocytes. Their primary ligands are counter transmembrane receptors or plasma proteins, such as intercellular cell adhesion molecule-1 (ICAM-1) or components of complement system (iC3b, iC4b), respectively. Function blocking antibodies for these integrins may also reduce cell adhesion to collagens. To make the first systematical comparison of human α(L)ß2, α(M)ß2 and α(X)ß2 as collagen receptors, we produced the corresponding integrin αI domains both in wild-type and activated form and measured their binding to collagens I-VI. In the "closed" (wild-type) conformation, the α(L)I and α(M)I domains bound with low avidity to their primary ligands, and the interaction with collagens was also very weak. Gain-of-function mutations α(L) I306G, α(L) K287C/K294C and α(M) I316G are considered to mimic "open", activated αI domains. The binding of these activated αI domains to the primary ligands was clearly stronger and they also recognized collagens with moderate avidity (K(d)400 nM). After activation, the αLI domain favored collagen I (K(d )≈ 80 nM) when compared to collagen IV. The integrin αXI domain acted in a very different manner since already in native, wild-type form it bound to collagen IV and iC3b (K(d) ≈ 200-400 nM). Antibodies against αXß2 and αMß2 blocked promyelocytic leukemia cell adhesion to the collagenous GFOGER motif, a binding site for the ß1 integrin containing collagen receptors. In brief, leukocyte ß2 integrins may act as collagen receptors in a heterodimer specific manner.

Structure of collagen receptor integrin α(1)I domain carrying the activating mutation E317A.

We have analyzed the structure and function of the integrin α(1)I domain harboring a gain-of-function mutation E317A. To promote protein crystallization, a double variant with an additional C139S mutation was used. In cell adhesion assays, the E317A mutation promoted binding to collagen. Similarly, the double mutation C139S/E317A increased adhesion compared with C139S alone. Furthermore, soluble α(1)I C139S/E317A was a higher avidity collagen binder than α(1)I C139S, indicating that the double variant represents an activated form. The crystal structure of the activated variant of α(1)I was solved at 1.9 Å resolution. The E317A mutation results in the unwinding of the αC helix, but the metal ion has moved toward loop 1, instead of loop 2 in the open α(2)I. Furthermore, unlike in the closed αI domains, the metal ion is pentacoordinated and, thus, prepared for ligand binding. Helix 7, which has moved downward in the open α(2)I structure, has not changed its position in the activated α(1)I variant. During the integrin activation, Glu(335) on helix 7 binds to the metal ion at the metal ion-dependent adhesion site (MIDAS) of the ß(1) subunit. Interestingly, in our cell adhesion assays E317A could activate collagen binding even after mutating Glu(335). This indicates that the stabilization of helix 7 into its downward position is not required if the α(1) MIDAS is already open. To conclude, the activated α(1)I domain represents a novel conformation of the αI domain, mimicking the structural state where the Arg(287)-Glu(317) ion pair has just broken during the integrin activation.

Effects of conformational activation of integrin alpha 1I and alpha 2I domains on selective recognition of laminin and collagen subtypes.

Collagen receptor integrins alpha 1 beta 1 and alpha 2 beta 1 can selectively recognize different collagen subtypes. Here we show that their alpha I domains can discriminate between laminin isoforms as well: alpha 1I and alpha 2I recognized laminin-111, -211 and -511, whereas their binding to laminin-411 was negligible. Residue Arg-218 in alpha1 was found to be instrumental in high-avidity binding. The gain-of-function mutation E318W makes the alpha 2I domain to adopt the "open" high-affinity conformation, while the wild-type alpha 2I domain favors the "closed" low-affinity conformation. The E318W mutation markedly increased alpha 2I domain binding to the laminins (-111, -211 and -511), leading us to propose that the activation state of the alpha 2 beta 1 integrin defines its role as a laminin receptor. However, neither wild-type nor alpha 2IE318W domain could bind to laminin-411. alpha 2IE318W also bound tighter to all collagens than alpha 2I wild-type, but it showed reduced ability to discriminate between collagens I, IV and IX. The corresponding mutation, E317A, in the alpha 1I domain transformed the domain into a high-avidity binder of collagens I and IV. Thus, our results indicate that conformational activation of integrin alpha 1I and alpha 2I domains leads to high-avidity binding to otherwise disfavored collagen subtypes.

Small molecule designed to target metal binding site in the alpha2I domain inhibits integrin function.

Integrin alpha2beta1 is a potential target molecule in drug development. We have established "design" criteria for molecules that bind to the "closed" conformation of alpha2I domain via Mg(2+) in MIDAS (metal ion dependent adhesion site) while simultaneously forming interactions with neighboring amino acid residues. Specific tetracyclic Streptomyces products belonging to the group of aromatic polyketides fulfill our criteria and inhibit alpha2beta1 integrin. All previously described inhibitors of alphaI domain integrins act in an allosteric manner.

Kinetic and mutational analyses of the major cytosolic exopolyphosphatase from Saccharomyces cerevisiae.

Yeast exopolyphosphatase (scPPX) processively splits off the terminal phosphate group from linear polyphosphates longer than pyrophosphate. scPPX belongs to the DHH phosphoesterase superfamily and is evolutionarily close to the well characterized family II pyrophosphatase (PPase). Here, we used steady-state kinetic and binding measurements to elucidate the metal cofactor requirement for scPPX catalysis over the pH range 4.2-9.5. A single tight binding site for Mg(2+) (K(d) of 24 microm) was detected by equilibrium dialysis. Steady-state kinetic analysis of tripolyphosphate hydrolysis revealed a second site that binds Mg(2+) in the millimolar range and modulates substrate binding. This step requires two protonated and two deprotonated enzyme groups with pK(a) values of 5.0-5.3 and 7.6-8.2, respectively. The catalytic step requiring two deprotonated groups (pK(a) of 4.6 and 5.6) is modulated by ionization of a third group (pK(a) of 8.7). Conservative mutations of Asp(127), His(148), His(149) (conserved in scPPX and PPase), and Asn(35) (His in PPase) reduced activity by a factor of 600-5000. N35H and D127E substitutions reduced the Mg(2+) affinity of the tight binding site by 25-60-fold. Contrary to expectations, the N35H variant was unable to hydrolyze pyrophosphate, but markedly altered metal cofactor specificity, displaying higher catalytic activity with Co(2+) bound to the weak binding site versus the Mg(2+)- or Mn(2+)-bound enzyme. These results provide an initial step toward understanding the dynamics of scPPX catalysis and reveal significant functional differences between structurally similar scPPX and family II PPase.