PPS96 Projects
Cristina Cantale
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)
1ITG(77Kb)
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