Karl Hård

The DNA-binding domain of HIV-1 integrase has an SH3-like fold

We have determined the solution structure of the DNA-binding domain of HIV-1 integrase by nuclear magnetic resonance spectroscopy. In solution, this carboxy-terminal region of integrase forms a homodimer, consisting of two structures that closely resemble Src-homology 3 (SH3) domains. Lys 264, previously identified by mutagenesis studies to be important for DNA binding of the integrase, as well as several adjacent basic amino acids are solvent exposed. The identification of an SH3-like domain in integrase provides a new potential target for drug design.

The solution structure of the amino-terminal HHCC domain of HIV-2 integrase: a three-helix bundle stabilized by zinc

BACKGROUND Integrase mediates a crucial step in the life cycle of the human immunodeficiency virus (HIV). The enzyme cleaves the viral DNA ends in a sequence-dependent manner and couples the newly generated hydroxyl groups to phosphates in the target DNA. Three domains have been identified in HIV integrase: an amino-terminal domain, a central catalytic core and a carboxy-terminal DNA-binding domain. The amino-terminal region is the only domain with unknown structure thus far. This domain, which is known to bind zinc, contains a HHCC motif that is conserved in retroviral integrases. Although the exact function of this domain is unknown, it is required for cleavage and integration. RESULTS The three-dimensional structure of the amino-terminal domain of HIV-2 integrase has been determined using two-dimensional and three-dimensional nuclear magnetic resonance data. We obtained 20 final structures, calculated using 693 nuclear Overhauser effects, which display a backbone root-mean square deviation versus the average of 0.25 A for the well defined region. The structure consists of three alpha helices and a helical turn. The zinc is coordinated with His 12 via the N epsilon 2 atom, with His16 via the N delta 1 atom and with the sulfur atoms of Cys40 and Cys43. The alpha helices form a three-helix bundle that is stabilized by this zinc-binding unit. The helical arrangement is similar to that found in the DNA-binding domains of the trp repressor, the prd paired domain and Tc3A transposase. CONCLUSION The amino-terminal domain of HIV-2 integrase has a remarkable hybrid structure combining features of a three-helix bundle fold with a zinc-binding HHCC motif. This structure shows no similarity with any of the known zinc-finger structures. The strictly conserved residues of the HHCC motif of retroviral integrases are involved in metal coordination, whereas many other well conserved hydrophobic residues are part of the protein core.

Refined solution structure of the C-terminal DNA-binding domain of human immunovirus-1 integrase.

The structure of the C‐terminal DNA‐binding domain of human immunovirus‐1 integrase has been refined using nuclear magnetic resonance spectroscopy. The protein is a dimer in solution and shows a well‐defined dimer interface. The folding topology of the monomer consists of a five‐stranded β‐barrel that resembles that of Src homology 3 domains. Compared with our previously reported structure, the structure is now defined far better. The final 42 structures display a back‐bone root mean square deviation versus the average of 0.46 Å. Correlation of the structure with recent mutagenesis studies suggests two possible models for DNA binding. Proteins 1999;36:556–564.

NMR Studies of the Free α Subunit of Human Chorionic Gonadotropin

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone that is involved in the maintenance of the corpus luteum in early pregnancy. Glycosylation at Asn52 of its alpha subunit (alpha hCG) is essential for signal transduction, whereas the N-glycan at Asn78 stabilizes the structure of the protein. In this study, an almost complete 1H-NMR and a partial 13C-NMR spectral assignment for the amino acids and the N-glycans of alpha hCG and of an enzymatically deglycosylated form, which had a single GlcNAc residue at each of its two glycosylation sites, has been achieved. The secondary structure of alpha hCG is solution, which was determined based on NOE data, is partially similar to that of the alpha subunit in the crystal structure of hCG, but large structural differences are found for amino acid residues 33-58. In the crystal structure of hCG, residues 33-37 and 54-58 of the alpha subunit are part of an intersubunit seven-stranded beta-barrel and residues 41-47 constitute a 3(10)-helix. In contrast, in free alpha hCG in solution, amino acids 33-58 are part of a large disordered loop, indicating that in intact hCG interactions with the beta subunit of hCG stabilize the conformation of the alpha subunit. The NMR data of alpha hCG and its deglycosylated counterpart are very similar, indicating that removal of carbohydrate residues other than GlcNAc-1 does not notably affect the conformation of the protein part. However, numerous 1H-NOEs between the GlcNAc-1 residue at Asn78 and several amino acid residues show that this GlcNAc residue is tightly packed against the protein, being an integral part of the structure of the alpha subunit. 1H-NOEs across the glycosidic linkages of the glycan, resonance-line widths, and 1H and 13C chemical shifts of the other monosaccharides suggest that the remainder of the glycans at Asn78, and the glycans at Asn52 are largely extended in solution.

Rapid and simple approach for the NMR resonance assignment of the carbohydrate chains of an intact glycoprotein Application of gradient-enhanced natural abundance 1H-13C HSQC and HSQC-TOCSY to the α-subunit of human chorionic gonadotropin

The structure assessment of an intact glycoprotein in solution requires an extensive assignment of the carbohydrate NMR resonances. However, assignment of homonuelear spectra is very complicated because of the severe overlap of protein and carbohydrate signals. Application of pulsed field gradients allowed high quality natural abundance 1H‐13C HSQC and HSQC‐TOCSY spectra to be recorded of the α‐subunit of human chorionic gonadotropin. Most carbohydrate 1H‐13C correlations appear in a distinct region between the aromatic region and the protein Cα‐Hα region. The enormous reduction in overlap led to fast and unambiguous assignment of the anomeric 1H‐13C correlations. Subsequently, correlations of the monosaccharide skeleton atoms were readily assigned in the HSQC‐TOCSY spectrum.

αl-6(αl-3)-Difucosylation of the asparagine-bound N-acetylglucosamine in honeybee venom phospholipase A2

AbstractChymotryptic glycopeptides were prepared from a honeybee (Apis mellifica) venom phospholipase A2 (E.C. 3.1.1.4) fraction, with high affinity towards lentil (Lens culinaris) lectin. Treatment of the glycopeptide mixture with peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase A, followed by HPLC fractionation, yielded two oligosaccharides, which were analysed by 500 MHz1H-NMR spectroscopy to give the following structures

Structure refinement of the glucocorticoid receptor-DNA binding domain from NMR data by relaxation matrix calculations.

\begin{array}{*{20}c} {Man\alpha 1} & \backslash & {Fuc\alpha 1} & | \\ {} & 6 & {Man\beta 1 - 4GlcNAc\beta 1 - 4GlcNAc} & 6 \\ {} & 3 & {} & {} \\ {Man\alpha 1} & / & {} & {} \\ \end{array} \begin{array}{*{20}c} {Man\alpha 1} & \backslash & {Fuc\alpha 1} & | \\ {} & 6 & {Man\beta 1 - 4GlcNAc\beta 1 - 4GlcNAc} & 6 \\ {} & 3 & 3 & | \\ {Man\alpha 1} & / & {Fuc\alpha 1} & {} \\ \end{array}

O-mannosylation of recombinant human insulin-like growth factor I (IGF-I) produced in Saccharomyces cerevisiae.

This is the first report on a naturally occurring glycoprotein N-glycan with two fucose residues linked to the asparagine-boundN-acetylglucosamine.