PPS96 Projects

Cristina Cantale

The Viral Integrases

The Integrase Protein

3D structure

HIV IN has been subject to various crystallization attempts, but its low solubility and the tendency to form aggregates stopped them. Deletion mutants prepared to test the functionality of different domains resulted also in producing deleted proteins with different solubility from the wild type. The soluble mutants were candidate for structural studies.
HIV-1 IN50-212 was a promising one. Its biophysical, enzymatic and spectroscopic properties were measured and found not largely altereted in comparison with full length protein. Its solubility was good, but the aggregation was retained (Hickman A.B. et al., 94). Some point mutation of IN50-212 were tested, looking for a mutation improving solubility against aggregation (Jenkins et al., 95). A single mutation F(185)K produced a soluble protein, existing as a monodispersed dimer in solution. This protein, containig the catalytical core domain, was crystallized and its structure was resolved at 2.5 Angstrom (Dyde et al., 94).
The strategy of inducing mutations to obtain soluble proteins has been applied at whole HIV-1 IN protein, and a mutant containig two mutations, F185K and C280S, has been selected. It is completely active and it exists in solution in an equilibrium between dimeric and tetrameric form (1M NaCl). It is a good candidate for solving the HIV-1 IN structure (Cannon P. et al , 96).
The structure of HIV 1 IN 220-270in solution has been determined using multidimensional NMR spectroscopy (Lodi P.J. et al., 95).
The same strategy of selecting suitable mutants was followed with ASV IN (Kulkosky J. et al., 95).
In 1995 the ASV IN structure was also solved at high resolution, using a deletion mutant ASV IN52-207, without further mutations (Bujacz et al., 95).

3D structure of HIV-1 IN catalytic domain

The structure consists of a central 5 strands beta sheet and six helices. Its overall topology resembles the E. coli ribonuclease H (RNase H). Other enzymes share this topology, like RNase H domain of HIV-1 Reverse Transcriptase, Holliday junction-specific endonuclease RuvC and the core domain of the transposase protein of bacteriophage Mu.
The structure contains a disordered region, from 140 to 154 AAs, including the E152, one of the three key AAs of the catalytic triad. D64 and D116 are ordered and superimpose well on two catalytic residues of HIV-1 RNase H.
The first six residues (from 50-55) at N-terminus and the last four residues at C-terminus are not visible.
Fig. 4 is a simple diagram of HIV IN and RNase H topologies.

The core is a dimer, with a main, large region of contact encompassing beta3, alpha1, alpha3, alpha5 and alpha6 (about 1300 square Angstroms per subunit) and contains salt bridges and hydrogen bonds.
It is not mediated through the proposed leucine zipper motif.
Here is a picture of the core domain of HIV-1 IN (12Kb)

Look also at 1HRH (123Kb) HIV-1 RNase H domain and here (18Kb)
end at 1HRJ (338Kb) Holliday junction endonuclease RuvC from E.coli and here (21Kb).

Look at this picture (24Kb) to see the remarkable residues, that is the conserved ones and/or those known to produce interesting mutants.
Look also here at the catalytic core highlighted residues (14 Kb)

3D structure of HIV-1 IN C-terminal domain

Here is a picture of the C-terminus structure.
In solution IN220-270 is a dimer. Each monomer is composed of a five stranded beta-barrel. The interface is formed by three antiparallel strands (namely 2, 3 and 4) from each monomer faced in an antiparallel fashion. The interface is mainly stabilized by hydrophobic interactions. In this picture (37Kb) the AAs interacting at the dimer interface are highligthed.

The overall topology is very similar to SH3 domains, which are found in proteins involved in signal trasduction (Eijkelenboom A. et al., 95). SH3-like folds are widely present in proteins, despite of the absence of any significant sequence identity.

Look at the structure of alpha spectrin SH3 domain here (32Kb)
The AAs probably involved in DNA recognition are evidentiated in this picture (21Kb) and are compatible with a bind to the major groove of DNA.

3D structure of ASV IN catalytic domain

Some different structures were solved, because ASV IN52-207 was crystallized using different conditions with reference to protein buffers (Hepes or citrate) and precipitants (PEG/IPR and ammonium sulphate). A selenomethionine substituted protein was also prepared and two different data collection temperatures (20C° and -165C°) were used.
The effect of different crystallization conditions are generally undetectable among the structures except for a ten residue loop between beta5 and alpha4, which changes conformation. The loop protudes from the molecule. The high temperature factors found at its tips suggest that this is a flexible region. It contains two turns, when crystals grow in PEG and one smooth turn, intramolecularly stabilized, when crystals grow in ammonium sulphate. It is interesting to note that about the same region (12 residue instead of 10) was found disordered in the structure of core domain of HIV-IN .
A comparison with 3D structure of HIV IN catalytic core reveals a similar general topology, with previously reported analogy with RNase H and RuvC.
Fig. 5 is a schematic diagram of the ASV IN catalytic core fragment topology.

Here is a picture of ASV IN (27Kb)


The ASV IN catalytic core is a dimer, with an interface located between alpha1 from a monomer and alpha5 from the other, with mainly hydrophobic interactions. Protruding from the space between helices, residues from the beta3 strand also participate. The general shape of the interface is a cavity filled by some positively charged residues and a few water molecules . Several other polar interactions between charged residues contribute to stabilize the dimer.

This picture gives just a rought idea of the catalytic core (which is a dimer).
Water molecules play a key role both in catalytic core and in stabilization of folding, together with some conserved residues. The necessity for a high conserved S85 residue (S81 in HIV-IN) has been correlated with a structural effect in folding stabilization.
The triad D64D121E157 is in such position as to form an active site. The side chains interact with each other by three water molecules, one of which (Wat324) appears to be particularly important and could be replaced by a divalent cation.
Look here at the catalytic core (30Kb).
The comparison betweeen the two 3D available structures of integrases reveals a similar architecture, but there are considerable deviations. Looking at the dimers, the corresponding elements in each monomer can be recognized, but shifted up 6A° , with a reported relative 13.3° rotation and 4.8 A° translation.
Furthermore there are large differences in the orientations of the key residues in the active site that are difficult to understand.
The extension of dimerization is less in ASV IN core fragment, 766 square Angstroms against 1395 square Angstroms in HIV-1 IN and is compatible with the weaker association observed for ASV IN with respect to HIV IN.
Very recently the structure of ASV IN catalytic domain crystallized in presence of divalent cations Mg2+ and Mn2+ has been solved (Bujacz G. et al., 96).
Here you can see a picture of the triad and the metal ion bound to asp64 and asp121.

Here you can download the respective coordinates.
1VSD (90Kb)
1VSF (87Kb)

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Last updated 25th Oct '96