Selective incorporation of 5-hydroxytryptophan blocks long range electron transfer in oxalate decarboxylase

Oxalate decarboxylase from Bacillus subtilis is a binuclear Mn-dependent acid stress response enzyme that converts the mono-anion of oxalic acid into formate and carbon dioxide in a redox neutral unimolecular disproportionation reaction. A π-stacked tryptophan dimer, W96 and W274, at the interface between two monomer subunits facilitates long-range electron transfer between the two Mn ions and plays an important role in the catalytic mechanism. Substitution of W96 with the unnatural amino acid 5-hydroxytryptophan leads to a persistent EPR signal which can be traced back to the neutral radical of 5-hydroxytryptophan with its hydroxyl proton removed. 5-Hydroxytryptophan acts as a hole sink preventing the formation of Mn(III) at the N-terminal active site and strongly suppresses enzymatic activity. The lower boundary of the standard reduction potential for the active site Mn(II)/Mn(III) couple can therefore be estimated as 740 mV against the normal hydrogen electrode at pH 4, the pH of maximum catalytic efficiency. Our results support the catalytic importance of long-range electron transfer in oxalate decarboxylase while at the same time highlighting the utility of unnatural amino acid incorporation and specifically the use of 5-hydroxytryptophan as an energetic sink for hole hopping to probe electron transfer in redox proteins.     

Interaction of cytochrome c with cytochrome oxidase: two different docking scenarios

Cytochrome c is the specific and efficient electron transfer mediator between the two last redox complexes of the mitochondrial respiratory chain. Its interaction with both partner proteins, namely cytochrome c(1) (of complex III) and the hydrophilic Cu(A) domain (of subunit II of oxidase), is transient, and known to be guided mainly by electrostatic interactions, with a set of acidic residues on the presumed docking site on the Cu(A) domain surface and a complementary region of opposite charges exposed on cytochrome c. Information from recent structure determinations of oxidases from both mitochondria and bacteria, site-directed mutagenesis approaches, kinetic data obtained from the analysis of isolated soluble modules of interacting redox partners, and computational approaches have yielded new insights into the docking and electron transfer mechanisms. Here, we summarize and discuss recent results obtained from bacterial cytochrome c oxidases from both Paracoccus denitrificans, in which the primary electrostatic encounter most closely matches the mitochondrial situation, and the Thermus thermophilus ba(3) oxidase in which docking and electron transfer is predominantly based on hydrophobic interactions.     

Specificity of the interaction between the Paracoccus denitrificans oxidase and its substrate cytochrome c: comparing the mitochondrial to the homologous bacterial cytochrome c(552), and its truncated and site-directed mutants

Under in vitro conditions, bacterial cytochrome c oxidases may accept several nonhomologous c-type electron donors, including the evolutionarily related mitochondrial cytochrome c. Several lines of evidence suggest that in intact membranes the heme aa(3) oxidase from Paracoccus denitrificans receives its electrons from the membrane-bound cytochrome c(552). Both the structures of the oxidase and of a heterologously expressed, soluble fragment of the c(552) have been determined recently, but no direct structural information about a static cocomplex is available. Here, we analyze the kinetic properties of the isolated oxidase with the full-size c(552), with two truncated soluble forms, and with a set of site-specific mutants within the presumed docking site of the cytochrome, all heterologously expressed in Escherichia coli. Our data indicate that all three forms, the wild type and both truncations, are fully competent kinetically and exhibit biphasic kinetic behavior, however, under widely different ionic strength conditions. When mutations in lysine residues clustered around the interaction domain were introduced into the smallest fragment of c(552), both kinetic parameters, K(M) and k(cat), were drastically influenced. On the other hand, when the nonmutated truncated form was used to donate electrons to a set of oxidase mutants with replacements clustered along the docking site on subunit II, we observe distinct differences when comparing the kinetic properties of the widely used horse heart cytochrome c with those of the bacterial c(552). We conclude that the specific docking sites for the two types of cytochromes differ to some extent.     

Phosphorylation of the arginine/serine repeats of lamin B receptor by SRPK1—Insights from molecular dynamics simulations

Background

Arginine/serine (RS) repeats are found in several proteins in metazoans with a wide variety of functions, many of which are regulated by SR protein kinase 1 (SRPK1)-mediated phosphorylation. Lamin B receptor (LBR) is such a protein implicated in chromatin anchorage to the nuclear envelope.

Methods

Molecular dynamics simulations were used to investigate the conformation of two LBR peptides containing four (human-) and five (turkey-orthologue) consecutive RS dipeptides, in their unphosphorylated and phosphorylated forms and of a conserved peptide, in isolation and in complex with SRPK1. GST pull-down assays were employed to study LBR interactions.

Results

Unphosphorylated RS repeats adopt short, transient helical conformations, whereas serine phosphorylation induces Arginine-claw-like structures. The SRSRSRSPGR peptide, overlapping with the LBR RS repeats, docks into the known, acidic docking groove of SRPK1, in an extended conformation. Phosphorylation by SRPK1 is necessary for the association of LBR with histone H3.

Conclusions

The C-terminal region of the LBR RS domain constitutes a recognition platform for SRPK1, which uses the same recognition mechanism for LBR as for substrates with long RS domains. This docking may promote unfolding of the RS repeats destined to be phosphorylated. Phosphorylation induces Arginine-claw-like conformations, irrespective of the RS-repeat length, that may facilitate interactions with basic partners.

General significance

Our results shed light on the conformational preferences of an important class of repeats before and after their phosphorylation and support the idea that even short RS domains may be constituents of recognition platforms for SRPK1, thus adding to knowledge towards a full understanding of their phosphorylation mechanism.     

Histones H3/H4 form a tight complex with the inner nuclear membrane protein LBR and heterochromatin protein 1

We have recently shown that heterochromatin protein 1 (HP1) interacts with the nuclear envelope in an acetylation-dependent manner. Using purified components and in vitro assays, we now demonstrate that HP1 forms a quaternary complex with the inner nuclear membrane protein LBR and a sub-set of core histones. This complex involves histone H3/H4 oligomers, which mediate binding of LBR to HP1 and cross-link these two proteins that do not interact directly with each other. Consistent with previous observations, HP1 and LBR binding to core histones is strongly inhibited when H3/H4 are modified by recombinant CREB-binding protein, revealing a new mechanism for anchoring domains of under-acetylated chromatin to the inner nuclear membrane.     

Mutations in the docking site for cytochrome c on the Paracoccus heme aa3 oxidase. Electron entry and kinetic phases of the reaction.

Introducing site-directed mutations in surface-exposed residues of subunit II of the heme aa3 cytochrome c oxidase of Paracoccus denitrificans, we analyze the kinetic parameters of electron transfer from reduced horse heart cytochrome c. Specifically we address the following issues: (a) which residues on oxidase contribute to the docking site for cytochrome c, (b) is an aromatic side chain required for electron entry from cytochrome c, and (c) what is the molecular basis for the previously observed biphasic reaction kinetics. From our data we conclude that tryptophan 121 on subunit II is the sole entry point for electrons on their way to the CuA center and that its precise spatial arrangement, but not its aromatic nature, is a prerequisite for efficient electron transfer. With different reaction partners and experimental conditions, biphasicity can always be induced and is critically dependent on the ionic strength during the reaction. For an alternative explanation to account for this phenomenon, we find no evidence for a second cytochrome c binding site on oxidase.     

Herbicides interacting with lanthanide(III) and calcium(II) ions: structural and biological similarities of Gd and Ca polymers

In this paper we report the synthesis and characterization of Ca(II), Gd(III) and Ce(III) complexes with chlorophenoxyalkanoic acids, which are commonly used as herbicides. The Gd(III) and Ca(II) complexes were characterized by the typical formulas [Gd(III)(L)(3)(H(2)O)(2).2dmf](n) and [Ca(L)(2)(MeOH)(2)](n) [L=[2,4-D=2,4-dichlorophenoxyacetic acid, 2,4,5-T=2,4,5-trichlorophenoxyacetic acid, MCPA=2-methyl-4-chlorophenoxy acetic acid and 2,4-DP=2-(2,4-dichlorophenoxy)propanoic acid]]. The crystal structure of the Gd(III) complex with 2,4-D shows that the compound is a one-dimensional polymer with a [Gd(III)(2)(2,4-D)(6)(H(2)O)(4)] dimeric repeat unit and the gadolinium atoms are in a nine-coordination environment, while the crystal structure of the Ca analog shows that it also has a polymeric structure with a [Ca(2)(2,4-D)(4)(CH(3)OH)(4)] dimeric repeat unit and the calcium atoms are in an eight-coordination environment. The gadolinium compound displays three different coordination modes for the carboxylato moiety, bidentate chelate, bidentate double bound and bidentate triple bound, while the calcium compound displays only one, bidentate triple bound. Coordination spheres are completed with oxygens of H(2)O or MeOH molecules, respectively. The complexes were tested against a few common bacteria by minimum inhibitory concentration (MIC) experiments and did not exhibit any antimicrobial action at concentrations up to 1600 microg/ml.     

Temporal association of protamine 1 with the inner nuclear membrane protein lamin B receptor during spermiogenesis

During mammalian spermiogenesis, histones are replaced by transition proteins, which are in turn replaced by protamines P1 and P2. P1 protamine contains a short arginine/serine-rich (RS) domain that is highly phosphorylated before being deposited into sperm chromatin and almost completely dephosphorylated during sperm maturation. We now demonstrate that, in elongating spermatids, this phosphorylation is required for the temporal association of P1 protamine with lamin B receptor (LBR), an inner nuclear membrane protein that also possesses a stretch of RS dipeptides at its nucleoplasmic NH(2)-terminal domain. Previous studies have shown that the cellular protein p32 also binds tightly to the unmodified RS domain of LBR. Extending those findings, we now present evidence that p32 prevents phosphorylation of LBR and furthermore that dissociation of this protein precedes P1 protamine association. Our data suggest that docking of protamine 1 to the nuclear envelope is an important intermediate step in spermiogenesis and reveal a novel role for SR protein kinases and p32.