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.

14N population transfers in two-dimensional 13C-14N-1H triple-resonance magic-angle spinning nuclear magnetic resonance spectroscopy.

A two-dimensional (2D) experiment has been used to show that 14N irradiation and magic-angle spinning (MAS) results in population transfers between the 14N Zeeman levels. This experiment was applied to a sample of N-acetyl-D,L-valine, a material where asymmetric doublets resulting from 13C-14N dipolar coupling are clearly resolved in the 13C spectrum at a field of 7 T for Carbon atoms directly bonded to the nitrogen atom. The 13C transverse magnetization was allowed to evolve in the F1 and F2 dimensions, and the 14N spins were irradiated during the mixing period. Cross-peaks were observed in the 2D 13C spectrum between the two peaks of the CH asymmetric doublet. Since one peak of the doublet results primarily from coupling to the [formula: see text] state and the other peak from coupling to [formula: see text] states, population changes between the 14N Zeeman levels have occurred during the mixing period. These population transfers are a consequence of the time dependence of the 14N quadrupole splitting Q under MAS conditions and 14N irradiation. Level anti-crossings of the 14N Zeeman levels occur at the zero-crossings of Q, and a continuous and slow change in Q will result in the transfer of 14N populations between the different Zeeman levels. If these passages are adiabatic, then the system returns to its original state after two zero-crossings. This is consistent with the experimental observation that the intensities of the cross-peaks for 14N irradiation are greater for half a rotor period than a full rotor period.

Refined solution structure of the dimeric N-terminal HHCC domain of HIV-2 integrase.

The solution structure of the dimeric N-terminal domain of HIV-2 integrase (residues 1-55, named IN(1-55)) has been determined using NMR spectroscopy. The structure of the monomer, which was already reported previously [Eijkelenboom et al. (1997) Curr. Biol., 7, 739-746], consists of four alpha-helices and is well defined. Helices alpha1, alpha2 and alpha3 form a three-helix bundle that is stabilized by zinc binding to His12, His16, Cys40 and Cys43. The dimer interface is formed by the N-terminal tail and the first half of helix alpha3. The orientation of the two monomeric units with respect to each other shows considerable variation. 15N relaxation studies have been used to characterize the nature of the intermonomeric disorder. Comparison of the dimer interface with that of the well-defined dimer interface of HIV-1 IN(1-55) shows that the latter is stabilized by additional hydrophobic interactions and a potential salt bridge. Similar interactions cannot be formed in HIV-2 IN(1-55) [Cai et al. (1997) Nat. Struct. Biol., 4, 567-577], where the corresponding residues are positively charged and neutral ones.

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 carboxyterminal 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.

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 beta-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 A. Correlation of the structure with recent mutagenesis studies suggests two possible models for DNA binding. Proteins 1999;36:556-564.