Consequences of IkappaB alpha hydroxylation by the factor inhibiting HIF (FIH)

The factor inhibiting HIF-1 (FIH-1) hydroxylates many ankyrin repeat-containing proteins including IκBα. It is widely speculated that hydroxylation of IκBα has functional consequences, but the effects of hydroxylation have not been demonstrated. We prepared hydroxylated IκBα and compared it to the unhydroxylated protein. Urea denaturation and amide H/D exchange experiments showed no change in the “foldedness” upon hydroxylation. Surface plasmon resonance measurements of binding to NFκB showed no difference in the NFκB binding kinetics or thermodynamics. Ubiquitin-independent proteasomal degradation experiments showed no difference in the half-life of the protein. Thus, it appears that hydroxylation of IκBα by FIH-1 is inconsequential, at least for the functions we could assay in vitro.

Structured summary

MINT-8051494: NF-kappa-B p65 (uniprotkb:Q04207) physically interacts (MI:0915) with NF-kappa-B p50 (uniprotkb:P25799) and I-kappa-B alpha (uniprotkb:O15111) by surface plasmon resonance (MI:0107)     

Examination of a novel head-stalk protein family in Giardia lamblia characterised by the pairing of ankyrin repeats and coiled-coil domains

The intestinal pathogen Giardia lamblia possesses several unusual organelle features, including two equivalent nuclei, no mitochondria or peroxisomes, and a developmentally regulated rough endoplasmic reticulum and Golgi. Giardia also possesses a number of complex and unique cytoskeleton structures that dictate cell shape, motility and attachment. Our investigations of cytoskeletal proteins have revealed the presence of a new protein family. Proteins in this family contain both ankyrin repeats and coiled-coil domains; although these are common protein motifs, their pairing is unique, thus establishing a new class of head-stalk proteins. Examination of the G. lamblia genome shows evidence for at least 18 genes coding for proteins with a series of ankyrin repeats followed by a lengthy coiled-coil domain and at least an additional 14 genes coding for proteins with a prominent coiled-coil domain flanked by two series of ankyrin repeats. We have examined one of these proteins, Giardia Axoneme Associated Protein (GASP-180), in detail. GASP-180 is a 180 kDa protein containing five ankyrin repeats in a 200 amino acid N-terminal domain separated by a short spacer from an approximately 1375 amino acid coiled-coil domain. Using anti-peptide antibodies raised against a unique 20 amino acid sequence found at the C-terminus, we have determined that GASP-180 is present in cytoskeleton extractions of the parasite and localises to the proximal base of the anterior flagellar axonemes. The combination of the localisation and the structural and functional motifs of GASP-180 make it a strong candidate to participate in control of flagellar activity.     

Access to the complement factor B scissile bond is facilitated by association of factor B with C3b protein

Factor B is a zymogen that carries the catalytic site of the complement alternative pathway C3 convertase. During convertase assembly, factor B associates with C3b and Mg(2+) forming a pro-convertase C3bB(Mg(2+)) that is cleaved at a single factor B site by factor D. In free factor B, a pair of salt bridges binds the Arg(234) side chain to Glu(446) and to Glu(207), forming a double latch structure that sequesters the scissile bond (between Arg(234) and Lys(235)) and minimizes its unproductive cleavage. It is unknown how the double latch is released in the pro-convertase. Here, we introduce single amino acid substitutions into factor B that preclude one or both of the Arg(234) salt bridges, and we examine their impact on several different pro-convertase complexes. Our results indicate that loss of the Arg(234)-Glu(446) salt bridge partially stabilizes C3bB(Mg(2+)). Loss of the Arg(234)-Glu(207) salt bridge has lesser effects. We propose that when factor B first associates with C3b, it bears two intact Arg(234) salt bridges. The complex rapidly dissociates unless the Arg(234)-Glu(446) salt bridge is released whereupon conformational changes occur that activate the metal ion-dependent adhesion site and partially stabilize the complex. The remaining salt bridge is then released, exposing the scissile bond and permitting factor D cleavage.     

Cofactor Activity in Factor VIIIa of the Blood Clotting Pathway Is Stabilized by an Interdomain Bond between His281 and Ser524 Formed in Factor VIII

The factor VIII (FVIII) crystal structure suggests a possible bonding interaction of His²⁸¹ (A1 domain) with Ser⁵²⁴ (A2 domain), although the resolution of the structure (∼4 Å) does not firmly establish this bonding. To establish that side chains of these residues participate in an interdomain bond, we prepared and examined the functional properties of a residue swap variant (H281S/S524H) where His²⁸¹ and Ser⁵²⁴ residues were exchanged with one another and a disulfide-bridged variant (H281C/S524C) where the two residues were replaced with Cys. The latter variant showed efficient disulfide bonding of the A1 and A2 domains. The swap variant showed WT-like FVIII and FVIIIa stability, which were markedly reduced for H281A and S524A variants in an earlier study. The disulfide-bridged variant showed ∼20% increased FVIII stability, and FVIIIa did not decay during the time course measured. This variant also yielded 35% increased thrombin peak values compared with WT in a plasma-based thrombin generation assay. Binding analyses of H281S-A1/A3C1C2 dimer with S524H-A2 subunit yielded a near WT-like affinity value, whereas combining the variant dimer or A2 subunit with the WT complement yielded ∼5- and ∼10-fold reductions, respectively, in affinity. Other functional properties including thrombin generation potential, FIXa binding affinity, K_m for FX of FXase complexes, thrombin activation efficiency, and down-regulation by activated protein C showed similar results for the two variants compared with WT FVIII. These results indicate that the side chains of His²⁸¹ and Ser⁵²⁴ are in close proximity and contribute to a bonding interaction in FVIII that is retained in FVIIIa.     

Allosteric Regulation of a Protein Acetyltransferase in Micromonospora aurantiaca by the Amino Acids Cysteine and Arginine

ACT domains (amino acid-binding domains) are linked to a wide range of metabolic enzymes that are regulated by amino acid concentration. Seventy proteins with ACT-GCN5-related N-acetyltransferase (GNAT) domain organization were found in actinomycetales. In this study, we investigate the ACT-containing GNAT acetyltransferase, Micau_1670 (MaKat), from Micromonospora aurantiaca ATCC 27029. Arginine and cysteine were identified as ligands by monitoring the conformational changes that occur upon amino acids binding to the ACT domain in the MaKat protein using FRET assay. It was found that MaKat is an amino acid-regulated protein acetyltransferase, whereas arginine and cysteine stimulated the activity of MaKat with regard to acetylation of acetyl-CoA synthetase (Micau_0428). Our research reveals the biochemical characterization of a protein acetyltransferase that contains a fusion of a GNAT domain with an ACT domain and provides a novel signaling pathway for regulating cellular protein acetylation. These findings indicate that acetylation of proteins and acetyltransferase activity may be tightly linked to cellular concentrations of some amino acids in actinomycetales.     

Recognition of unmodified histone H3 by the first PHD finger of bromodomain-PHD finger protein 2 provides insights into the regulation of histone acetyltransferases monocytic leukemic zinc-finger protein (MOZ) and MOZ-related factor (MORF)

MOZ (monocytic leukemic zinc-finger protein) and MORF (MOZ-related factor) are histone acetyltransferases important for HOX gene expression as well as embryo and postnatal development. They form complexes with other regulatory subunits through the scaffold proteins BRPF1/2/3 (bromodomain-PHD (plant homeodomain) finger proteins 1, 2, or 3). BRPF proteins have multiple domains, including two PHD fingers, for potential interactions with histones. Here we show that the first PHD finger of BRPF2 specifically recognizes the N-terminal tail of unmodified histone H3 (unH3) and report the solution structures of this PHD finger both free and in complex with the unH3 peptide. Structural analysis revealed that the unH3 peptide forms a third antiparallel β-strand that pairs with the PHD1 two-stranded antiparallel β-sheet. The binding specificity was determined primarily through the recognition of arginine 2 and lysine 4 of the unH3 by conserved aspartic acids of PHD1 and of threonine 6 of the unH3 by a conserved asparagine. Isothermal titration calorimetry and NMR assays showed that post-translational modifications such as H3R2me2as, H3T3ph, H3K4me, H3K4ac, and H3T6ph antagonized the interaction between histone H3 and PHD1. Furthermore, histone binding by PHD1 was important for BRPF2 to localize to the HOXA9 locus in vivo. PHD1 is highly conserved in yeast NuA3 and other histone acetyltransferase complexes, so the results reported here also shed light on the function and regulation of these complexes.